Back to EveryPatent.com
United States Patent |
6,200,755
|
Virtanen
|
March 13, 2001
|
Optical disk-based assay devices and methods
Abstract
Optical disk-based assay devices and methods are described, in which
analyte-specific signal elements are disposed on an optical disk
substrate. In preferred embodiments, the analyte-specific signal elements
are disposed readably with the disk's tracking features. Also described
are cleavable signal elements particularly suitable for use in the assay
device and methods. Binding of the chosen analyte simultaneously to a
first and a second analyte-specific side member of the cleavable signal
element tethers the signal-responsive moiety to the signal element's
substrate-attaching end, despite subsequent cleavage at the cleavage site
that lies intermediate the first and second side members. The signal
responsive moiety reflects, absorbs, or refracts incident laser light.
Described are nucleic acid hybridization assays, nucleic acid sequencing,
immunoassays, cell counting assays, and chemical detection. Adaptation of
the assay device substrate to function as an optical waveguide permits
assay geometries suitable for continuous monitoring applications.
Inventors:
|
Virtanen; Jorma (Irvine, CA)
|
Assignee:
|
Burstein Technologies, Inc. (Irvine, CA)
|
Appl. No.:
|
120049 |
Filed:
|
July 21, 1998 |
Intern'l Class: |
C12Q 001/68; C12P 019/34; C07H 019/00; C07H 021/00; C07H 021/02 |
Field of Search: |
435/6,91.1,91.2
536/22.1,23.1,24.3,25.3
|
References Cited
U.S. Patent Documents
Re33064 | Sep., 1989 | Carter et al. | 436/34.
|
3791932 | Feb., 1974 | Schuurs et al. | 195/103.
|
3817837 | Jun., 1974 | Rubenstein et al. | 195/103.
|
3817838 | Jun., 1974 | Harris et al. | 195/103.
|
3850752 | Nov., 1974 | Schuurs et al. | 195/103.
|
3939350 | Feb., 1976 | Kronick et al. | 250/365.
|
3996345 | Dec., 1976 | Ullman et al. | 424/12.
|
4037257 | Jul., 1977 | Chari | 360/51.
|
4062733 | Dec., 1977 | Edwards et al. | 195/103.
|
4104029 | Aug., 1978 | Maier, Jr. | 23/230.
|
4160645 | Jul., 1979 | Ullman | 23/230.
|
4233402 | Nov., 1980 | Maggio et al. | 435/7.
|
4275149 | Jun., 1981 | Litman et al. | 435/7.
|
4277437 | Jul., 1981 | Maggio | 422/61.
|
4287300 | Sep., 1981 | Gibbons et al. | 435/5.
|
4366241 | Dec., 1982 | Tom et al. | 435/7.
|
4472509 | Sep., 1984 | Gansow et al. | 436/548.
|
4542102 | Sep., 1985 | Dattagupta et al. | 435/6.
|
4608344 | Aug., 1986 | Carter et al. | 436/34.
|
4756971 | Jul., 1988 | Virtanen et al. | 428/405.
|
4877745 | Oct., 1989 | Hayes et al. | 436/166.
|
5021236 | Jun., 1991 | Gries et al. | 424/9.
|
5087556 | Feb., 1992 | Ertinghausen | 435/7.
|
5112134 | May., 1992 | Chow et al. | 356/427.
|
5118605 | Jun., 1992 | Urdea | 435/6.
|
5132097 | Jul., 1992 | Van Deusen et al. | 422/82.
|
5164319 | Nov., 1992 | Hafeman et al. | 435/291.
|
5168057 | Dec., 1992 | Oh et al. | 435/174.
|
5278048 | Jan., 1994 | Parce et al. | 436/29.
|
5334837 | Aug., 1994 | Ikeda et al. | 250/339.
|
5345213 | Sep., 1994 | Semancik et al. | 338/34.
|
5384261 | Jan., 1995 | Winkler et al. | 436/518.
|
5405783 | Apr., 1995 | Pirrung et al. | 436/518.
|
5412087 | May., 1995 | McGall et al. | 536/24.
|
5424186 | Jun., 1995 | Fodor et al. | 435/6.
|
5429807 | Jul., 1995 | Matson et al. | 422/131.
|
5445934 | Aug., 1995 | Fodor et al. | 435/6.
|
5462839 | Oct., 1995 | deRooij et al. | 430/320.
|
5489678 | Feb., 1996 | Fodor et al. | 536/22.
|
5510270 | Apr., 1996 | Fodor et al. | 436/518.
|
5521289 | May., 1996 | Hainfeld et al. | 530/391.
|
5580696 | Dec., 1996 | Yashiro | 430/270.
|
5599662 | Feb., 1997 | Respess | 435/5.
|
5624711 | Apr., 1997 | Sundberg et al. | 427/261.
|
5892577 | Apr., 1999 | Gordon | 356/73.
|
6030581 | Feb., 2000 | Virtanen | 422/68.
|
B1 3646346 | Feb., 1972 | Catt | 250/83.
|
B1 3654090 | Apr., 1972 | Schuurs et al. | 435/7.
|
Foreign Patent Documents |
0 521 421 A2 | Jan., 1993 | EP.
| |
WO 96/09548 | Mar., 1996 | WO | .
|
WO 96/35940 | Nov., 1996 | WO | .
|
WO 97/21090 | Jun., 1997 | WO.
| |
WO 98/01533 | Jan., 1998 | WO.
| |
WO 98/12559 | Mar., 1998 | WO.
| |
WO 98/15356 | Apr., 1998 | WO.
| |
WO 98/37238 | Aug., 1998 | WO | .
|
WO 98/38510 | Sep., 1998 | WO | .
|
WO 99/35499 | Jul., 1999 | WO | .
|
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Oppenheimer Wolff & Donnelly LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part of Applicant's
provisional U.S. patent application No. 60/053,229, filed Jul. 21, 1997,
and of Applicant's U.S. patent application No. 08/888,935, filed Jul. 7,
1997 now abandoned, which is a continuation-in-part of provisional
application Nos. 60/030,416, filed Nov. 1, 1996 and 60/021,367, filed Jul.
8, 1996. Priority is claimed to each of the above-mentioned applications,
the disclosures of each of which are incorporated herein by reference.
Claims
What is claimed is:
1. An assay device comprising:
a) a solid support substrate;
b) a cleavable signal element comprising:
a signal responsive moiety; and
a cleavable spacer for releasably attaching said signal responsive moiety
to said substrate, said cleavable spacer having an end attached to said
substrate, an end attached to said signal responsive moiety and a cleavage
site intermediate said substrate-attaching end and said signal responsive
end;
c) a first anchoring member comprising a first end attached to said
substrate and a second end adapted to bind on a first site on a chosen
analyte;
d) a second anchoring member comprising a first end attached to said signal
responsive moiety and a second end adapted to bind on a second site on
said chosen analyte; and
wherein the signal responsive moiety remains bound to the substrate after
cleavage at the cleavage site only when said first and second anchoring
members are bound to said chosen analyte.
2. An assay device according to claim 1, wherein said signal responsive
moiety is adapted to reflect or scatter incident light.
3. An assay device according to claim 2, wherein said signal responsive
moiety is a metal microsphere.
4. An assay device according to claim 3, wherein said metal microsphere
consists essentially of a metal selected from the group consisting of
gold, silver, nickel, platinum, chromium and copper.
5. An assay device according to claim 4, wherein said metal microsphere
consists essentially of gold.
6. An assay device according to claim 3, wherein said metal microsphere is
ferromagnetic.
7. An assay device according to claim 1, wherein said first anchoring
member and said second anchoring member include oligonucleotides.
8. An assay device according to claim 7, wherein said first and second
anchoring member oligonucleotides are 5 mers-20 mers.
9. An assay device according to claim 1, wherein
said first anchoring member comprises a first antibody or antibody
fragment, and
said second anchoring member comprises a second antibody or antibody
fragment.
10. An assay device according to claim 1, wherein said solid support
substrate is a plastic selected from the group consisting of
polypropylenes, polyacrylates, polyvinyl alcohols, polyethylenes,
polymethylmethacrylates and polycarbonates.
11. An assay device according to claim 10, wherein said solid support
substrate is polycarbonate.
12. An assay device according to claim 1, wherein said solid support
substrate is fashioned as a disk.
13. An assay device according to claim 1, further comprising computer
software encoded upon the support substrate.
14. An assay device according to claim 13 wherein said support substrate
comprises a compact disc.
15. An assay device according to claim 13 wherein said support substrate
comprises a digital video disc.
Description
1. FIELD OF THE INVENTION
The present invention relates to the field of analytical instrumentation
for chemical assays and diagnostics, and to the detection of small
quantities of analytes in samples. More specifically, the invention
relates to an assay device comprising an optical disk having
analyte-specific signal elements disposed readably thereon
2. BACKGROUND OF THE INVENTION
2.1 Small Scale Clinical Assays
Until recently, most clinical diagnostic assays for the detection of small
quantities of analytes in fluids have been conducted as individual tests;
that is, as single tests conducted upon single samples to detect
individual analytes. More recently, efficiency and economy have been
obtained by designing apparatus for multi-sample preparation and automated
reagent addition, and by designing apparatus for rapid analysis of large
numbers of test samples, either in parallel or in rapid serial procession.
Often, such automated reagent preparation devices and automated multiplex
analyzers are integrated into a single apparatus.
Large clinical laboratory analyzers of this type can accurately perform
hundreds of assays automatically, or semi-automatically, in one hour.
However, these analyzers are expensive and only centralized laboratories
and large hospitals can afford them. Such centralization necessitates
sample transport, and often precludes urgent or emergent analysis of
time-critical samples.
Thus, there exists a strong need for simplified clinical assays that will
both reduce the cost of such dedicated analyzers and further their
distribution. The limit of such effort is the design of clinical tests
suitable for use at the patient bedside or in the patient's home without
dedicated detectors. Blood glucose and pregnancy tests are well known
examples.
Although useful tests of this sort have been offered for many years, a
major breakthrough was the introduction of solid phase immunoassays and
other strip tests since approximately 1980. Most notable are Advance.RTM.
test (Johnson & Johnson), RAMP.TM. hCG assay (Monoclonal Antibodies,
Inc.), Clear Blue Easy.TM. (Unipath Ltd.) and ICON (Hybritech).
Clear Blue Easy.TM. has all reagents in a laminated membrane and uses
conjugated colored latex microbeads as the signal reagent. It uses a
capillary migration immunoconcentration format. The ICON is a dual
monoclonal sandwich immunoconcentration assay. This assay has been
rendered quantitative through the use of a small reflectance instrument.
Otherwise, all these methods are only qualitative.
Migration distance can be used as a basis for quantitative assays
Commercially available are Quantab.TM. (Environmental Test Systems),
AccuLevel.RTM. (Syva), AccuMeter.RTM. (ChemTrak), Clinimeter.TM. (Crystal
Diagnostics) and Q.E.D..TM. (Enzymatics). One of the newest is a
thermometer-type assay device (Ertinghausen G., U.S. Pat. No. 5,087,556)
that is not yet commercially available. These systems can be used to assay
general chemistry analytes, such as cholesterol, as well as blood levels
of therapeutic drugs.
One disadvantage, however, of each of these formats is that only one, or a
very limited number, of assays can conveniently be performed
simultaneously.
To fill the gap between massive analyzers and strips, some small
instruments have been developed. The most notable is Eclipse ICA.TM.
(Biotope, Inc.). This device is a bench-top, random-access, automated
centrifugal immunoassay and chemistry system. Patient samples are pipetted
into cassettes that are placed into a rotor. Sixteen tests can be run in
approximately 17 minutes. The results are measured by UV/Visual
spectrometry or by fluorometry. Four different types of cassette are
needed. Each cassette has a relatively complicated structure.
Despite these developments, there still exists a need for a simple device
that can easily be used for multiple quantitative assays, and preferably
requiring no specialized detector instrumentation.
2.2 Spatially-Addressable Probe Arrays
Recently, spatially addressable arrays of different biomaterials have been
fabricated on solid supports. These probe arrays permit the simultaneous
analysis of a large number of analytes. Examples are arrays of
oligonucleotides or peptides that are fixed to a solid support and that
capture complementary analytes. One such system is described by Fodor et
al., Nature, Vol. 364, Aug. 5, 1993. Short oligonucleotide probes attached
to a solid support bind complementary sequences contained in longer
strands of DNA in liquid sample; the sequence of the sample nucleic acids
is then calculated by computer based on the hybridization data so
collected.
In the assay system described by Fodor et al., the array is inverted on a
temperature regulated flow cell against a reservoir containing the tagged
target molecules. In order to distinguish the surface bound molecules, the
system requires an extremely sensitive detector.
Accordingly, there remains a need for an economical system to fabricate
spatially addressable probe arrays in a simplified format that provides
both for ready detection and the ability to assay for large numbers of
test substances (i.e. analytes) in a fluid test sample in a single step,
or a minimum number of steps, or assay for a single test substance or
analyte in a large number of fluid test samples.
2.3 Spatially Addressable Laser-Based Detection Systems
Several devices for consumer electronic use permit spatially addressable
detection of digital information. In particular, several formats have been
developed based on the information recording potential of differential
reflectance and transmittance.
In conventional audio or CD-ROM compact disks, digital information--or
digitally encoded analog information--is encoded on a circular plastic
disk by means of indentations in the disk. Typically, such indentations
are on the order of one-eighth to one-quarter of the wavelength of the
incident beam of a laser that is used to read the information present on
the disk. The indentations on the disk cause destructive interference
within the reflected beam, which corresponds to a bit having a "zero"
value. The flat areas of the disk reflect the laser light back to a
detector and the detector gives a value of "one" to the corresponding bit.
In another convention, a change of intensity of a reflected light gets a
value of one while a constant intensity corresponds to zero.
Since the indentations have been formed in the disk in a regular pattern
from a master copy containing a pre-determined distribution of bits of
"zero" and bits of "one", the resultant signal received by the detector is
able to be processed to reproduce the same information that was encoded in
the master disk.
The standard compact disk is formed from a 12 cm polycarbonate substrate, a
reflective metalized layer, and a protective lacquer coating. The format
of current CDS and CD-ROMs is described by the ISO 9660 industry standard,
incorporated herein by reference.
The polycarbonate substrate is optical-quality clear polycarbonate. In a
standard pressed, or mass-replicated CD, the data layer is part of the
polycarbonate substrate, and the data are impressed in the form of a
series of pits by a stamper during the injection molding process. During
this process, molten polycarbonate is injected into a mold, usually under
high pressure, and then cooled so that the polycarbonate takes on the
shape of the mirror image of the mold, or "stamper" or "stamp"; pits that
represent the binary data on a disc's substrate are therefore created in
and maintained by the polycarbonate substrate as a mirror image of the
pits of the stamper created during the mastering process. The stamping
master is typically glass.
Pits are impressed in the CD substrate in a continuous spiral. The
reflective metal layer applied thereupon, typically aluminum, assumes the
shape of the solid polycarbonate substrate, and differentially reflects
the laser beam to the reading assembly depending on the presence or
absence of "pits." An acrylic lacquer is spincoated in a thin layer on top
of the metal reflective layer to protect it from abrasion and corrosion.
Although similar in concept and compatible with CD readers, the information
is recorded differently in a recordable compact disk (CD-R). In CD-R, the
data layer is separate from the polycarbonate substrate. The polycarbonate
substrate instead has impressed upon it a continuous spiral groove as an
address for guiding the incident laser. An organic dye is used to form the
data layer. Although cyanine was the first material used for these discs,
a metal-stabilized cyanine compound is generally used instead of "raw"
cyanine. An alternative material is phthalocyanine. One such
metallophthalocyanine compound is described in U.S. Pat. No. 5,580,696.
In CD-R, the organic dye layer is sandwiched between the polycarbonate
substrate and the metalized reflective layer, usually 24 carat gold, but
alternatively silver, of the media. Information is recorded by a recording
laser of appropriate preselected wavelength that selectively melts "pits"
into the dye layer--rather than burning holes in the dye, it simply melts
it slightly, causing it to become non-translucent so that the reading
laser beam is refracted rather than reflected back to the reader's
sensors, as by a physical pit in the standard pressed CD. As in a standard
CD, a lacquer coating protects the information-bearing layers.
Other physical formats for recording and storing information are being
developed based on the same concept as the compact disk: creation of
differential reflectance or transmittance on a substrate to be read by
laser.
One such format is termed Digital Video Disc (DVD). A DVD looks like
standard CD: it is a 120 mm (4.75 inch) disk that appears as a silvery
platter, with a hole in the center for engaging a rotatable drive
mechanism. Like a CD, data is recorded on the disc in a spiral trail of
tiny pits, and the discs are read using a laser beam. In contrast to a CD,
which can store approximately 680 million bytes of digital data under the
ISO 9660 standard, the DVD can store from 4.7 billion to 17 billion bytes
of digital data. The DVD's larger capacity is achieved by making the pits
smaller and the spiral tighter, that is, by reducing the pitch of the
spiral, and by recording the data in as many as four layers, two on each
side of the disc. The smaller pit size and tighter pitch require that the
reading laser wavelength be smaller. While the smaller wavelength is
backward compatible with standard pressed CDS, it is incompatible with
current versions of the dye-based CD-R.
The following table compares DVD and CD characteristics:
TABLE 1
Comparison of DVD and CD Characteristics
DVD CD
Diameter 120 mm 120 mm
Disc Thickness 1.2 mm 1.2 mm
Substrate 0.6 mm 1.2 mm
Thickness
Track pitch 0.74 .mu.m 1.6 .mu.m
Minimum pit size 0.4 .mu.m 0.83 .mu.m
Laser wavelength 635/650 nm 780 nm
Data capacity 4.7 0.68 gigabytes
gigabytes/layer/
side
Layers 1, 2, or 4 1
Thus, a single sided/single layer DVD can contain 4.7 GB of digital
information. A single sided/dual layer DVD can contain 8.5 GB of
information. A Dual sided/single layer disk can contain 9.4 GB of
information, while a dual sided, dual layer DVD contains up to 17 GB of
information.
Each of the variations consists of two 0.6 mm substrates that are bonded
together. Depending on the capacity, the disc may have one to four
information layers. In the 8.5 GB and 17 GB options, a semi-reflector is
used in order to access two information layers from one side of the disc.
For the 8.5 GB DVD and 17 GB options, the second information layer per side
may be molded into the second substrate or may be added as a photopolymer
layer. In either case, a semi-reflector layer is required to allow both
information layers to be read from one side of the disk. For the 17 GB
DVD, it is necessary to produce two dual-layer substrates, and bond them
together.
The DVD laser reader is designed to adjust its focus to either layer depth
so that both of them can be quickly and automatically accessed.
All three of the above-described formats require that the platter be spun.
The nominal constant linear velocity of a DVD system is 3.5 to 4.0 meters
per second (slightly faster for the larger pits in the dual layer
versions), which is over 3 times the speed of a standard CD, which is 1.2
mps.
Near-field optical storage disks (TeraStor, San Jose, CA) offer even higher
density information storage than DVD. In such devices, the reading head is
as close as 150 nm from the disk, and the pit size and track pitch are
also of nanometer scale.
Holographic data storage disks offer perhaps the highest known data storage
density. Holographic recording exploits three spatial dimensions.
Despite the spatial addressability and high information density of optical
media, these media have not previously been thought useful for detection
of analytes.
2.4 Waveguide Detection
Waveguides have been used for chemical detection at least since 1982,U.S.
Pat. No. 4,608,344, Re. 33,064, incorporated herein by reference.
Absorbing and nonabsorbing analytes can be observed with waveguides. The
exponential decay of the evanescent wave in uncoated waveguides is
sensitive to the absorbance and the refractive index of the surrounding
medium. This also affects the intensity of the light that is transmitted
by the waveguide. Existing applications of waveguides to detection of
analytes show poor spatial resolution.
3. SUMMARY OF THE INVENTION
The present invention solves these and other problems in the art by
providing an assay device for detecting analyte, comprising an optical
disk having analyte-specific signal elements disposed readably thereon.
The optical disk may be read, and the analyte detection thus performed,
using optical disk readers useful for reading digitally-encoded
information, such as those capable of reading audio CD disks, CD-ROM
disks, DVD disks, DIVX disks, laser disks, near-field storage disks, or
holographic data storage disks.
In preferred embodiments, the analyte-specific signal elements are disposed
readably with the optical disk's tracking features: that is, the
analyte-specific signal elements are readable by the optics used for
tracking, although modified or additional optics are not thereby
precluded.
In a preferred embodiment of the assay device, the analyte-specific signal
elements are cleavable.
In a particularly preferred embodiment, the cleavable signal element
comprises: a cleavable spacer having a substrate-attaching end, a
signal-responsive end, and a cleavage site intermediate the
substrate-attaching end and the signal-responsive end. The cleavable
signal element further includes a signal responsive moiety attached to the
cleavable spacer at its signal responsive end.
A first side member (also termed side element or side arm) adapted to bind
a first site on a chosen analyte, and a second side member adapted to bind
a second site of the same analyte, are present on the signal element. The
first and second side members confer analyte specificity upon the
cleavable signal element.
The first side member is attached to the cleavable spacer intermediate the
signal responsive end and cleavage site, and the second side member is
attached to the cleavable spacer intermediate the spacer's cleavage site
and substrate attaching end.
Binding of the chosen analyte simultaneously to the first and second side
members of a cleavable signal element tethers, or constrains, the
signal-responsive moiety to the signal element's substrate-attaching end,
despite subsequent cleavage at the cleavage site that lies intermediate
the first and second side members; conversely, failure to bind the chosen
analyte simultaneously to the first and second side members of a cleavable
signal element permits loss, through cleavage, of that signal element's
signal-responsive moiety. The presence or absence of signal after contact
with sample and contact with cleavage agent signals the presence or
absence of analyte, respectively.
Typically, the signal responsive moiety of the cleavable signal element is
adapted to reflect, scatter, or absorb incident light, particularly
incident laser light. In preferred embodiments, the signal responsive
moiety is a metal microsphere, and most preferred, a gold microsphere,
most preferably a gold microsphere of diameter between 1-3 .mu.m. These
embodiments are suitable for detection in existing optical disk readers,
such as those used to read audio CD, CD-ROM, DVD, laser disks, near-field
optical disks, or the like.
Whether cleavable or no, the analyte-specific signal elements are disposed
in or on the assay device in a spatially-addressable pattern.
In another aspect, the invention provides a method of assaying for analyte,
comprising the steps of contacting the assay device with a sample, and
then detecting, using an optical disk reader, analyte-specific signals
therefrom.
In preferred embodiments of this aspect of the invention, the method is
performed with assay devices in which the analyte-specific signal elements
are cleavable, and the method comprises: contacting the assay device with
a sample, cleaving the cleavable signal elements, and then detecting the
signal responsive moiety of analyte-constrained cleaved signal elements.
In a related aspect, the invention provides a method of using an optical
disk reader to assay for analyte. The method comprises the step of
detecting, from an optical disk, analyte-specific signal elements disposed
readably with the disk's tracking features. In preferred embodiments, the
method comprises detecting analyte-specific signals from an assay device
in which the analyte-specific signal elements are cleavable, and signal is
detected from analyte-constrained cleaved signal elements.
The invention further provides a method of making an assay device for
detecting analyte, comprising: disposing analyte-specific signal elements
on an optical disk readably with said disk's tracking features.
The signaling element, assay devices and assay methods of the present
invention are useful both for the detection of a large number of different
analytes in a test sample and the detection of a single analyte in a large
number of samples, both quantitatively and qualitatively.
Another aspect of the present invention is to adapt existing assay methods
to employ the assay devices of the invention, including the cleavable
signal element-based assay devices. Generally, an assay adapted to use the
cleavable signal element-based assay device of the present invention
comprises the steps of: contacting the assay device with a liquid sample,
contacting the assay device with a cleaving agent adapted to cleave said
plurality of attached cleavable signal elements, and detecting the
presence of the signal responsive moiety of analyte-restrained cleaved
signal elements adherent to the solid support substrate.
The spatial addressability of signal elements on the assay device permits
identification of analytes bound to distinct signal elements, including
identification of multiple analytes in a single assay.
The invention thus provides, in one preferred embodiment of this aspect,
nucleic acid hybridization assays, in which the first and second side
members of the cleavable signal elements include oligonucleotides.
Simultaneous binding of a nucleic acid present in the assay sample to the
first and second side members of the cleavable signal element prevents
loss, through cleavage, of the signal element's signal-responsive end.
In another aspect, the invention provides an assay device comprising
cleavable signal elements responsive to a plurality of nucleic acid
sequences. This aspect of the invention provides a device and method
suitable for sequencing nucleic acid through the spatial addressability of
signals generated upon contact with a sample containing nucleic acid.
The invention further provides immunoassays. In these embodiments, the
specificity-conferring side members of the cleavable signal elements
include antibodies, antibody fragments, or antibody derivatives.
Simultaneous binding of an analyte to the antibody of the first side
member and the antibody of the second side member prevents the loss,
through cleavage, of the signal element's signal-responsive end.
The invention also provides chemical detection assays, in which properly
chosen reactive groups on a first and second side member react
specifically with functional groups on the chosen analyte to secure the
signal responsive moiety to the assay device substrate.
The invention further provides means for detecting electromagnetic
radiation. Extremely high resolution X-ray pictures can be exposed and
stored on the disk in a format suitable for direct reading on an optical
disk reader, such as a CD-ROM or DVD reader, or the like. Other
wavelengths of the electromagnetic spectrum are analogously detectable.
The invention also provides means for the detection and counting of cells,
and for measuring their dimensions and shapes. In these embodiments,
specificity-conferring recognition molecules are disposed upon the assay
device substrate. The cells adhere thereto, and are detectable upon
binding of signal responsive moieties conjugated to a second cellular
recognition molecule Cell recognition molecules include antibodies,
receptors, ligands, and adhesion molecules.
In another aspect, the invention provides assay devices that further
comprise encoded digital information in the form of computer software.
Another aspect of the present invention provides a monitoring device,
comprising an optical disk having a plurality of analyte-specific signal
elements, wherein the optical disk is adapted to function as an optical
waveguide and the analyte-specific signal elements are so disposed that
specific binding of analyte detectably alters the light-transmitting
properties of said optical waveguide. This device is suitable for
continuous, or repeated, monitoring for presence of analyte. In preferred
embodiments of this aspect of the invention, the analyte-specific signal
elements are cleavable.
The invention further provides a method of monitoring for presence of
analyte, comprising: contacting the monitoring device with a sample, and
then detecting alterations in the light-transmitting properties of said
monitoring device's optical waveguide. In a related aspect, the invention
provides a method of monitoring for presence of analyte, comprising:
contacting the monitoring device having cleavable signal elements with a
sample, detecting alterations in the light-transmitting properties of said
monitoring device's optical waveguide, cleaving the signal elements, and
then detecting the signal responsive moiety of analyte-restrained cleaved
signal elements.
The invention further provides assay devices in which the analyte-specific
signal elements are disposed on a solid support substrate fashioned other
than in a disk. In preferred embodiments of this aspect of the invention,
readable by a laser-based optical reader, the signal elements are disposed
readably with the support substrate's tracking and/or addressing features.
Additionally, the assay device substrates may be fashioned as strips,
cuvettes, test tubes, well plates, slides, gels, magnetic disks, silicon
and other chips.
It is another aspect of the present invention to provide a multiwell sample
application plate suitable for applying liquid samples in parallel to the
assay devices of the present invention. In one embodiment, the sample
application device provides a multiwell plate with a renewable surface
film.
The invention further provides instrumentation to ensure correct
registration of a sample application device and the assay device. The
instrument may optionally comprise magnets to facilitate interaction of
the sample with the assay site and/or to remove unbound molecules or
particles.
4. BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be better understood by reference to the following
drawings, in which:
FIG. 1A is a schematic representation of a plurality of cleavable spacers
covalently attached at their surface-attaching end to a derivatized site
on the assay device substrate.
FIG. 1B illustrates the attachment of a reflective signaling means, a metal
microsphere, to the signal-responsive ends of the plurality of cleavable
spacers, creating cleavable reflective signal elements;
FIG. 2A is a schematic representation of a nucleic acid hybridization assay
adapted to use the cleavable reflective signal elements of the present
invention, shortly after introduction of a sample containing nucleic
acids;
FIG. 2B is a schematic representation of a later stage of the assay
procedure of FIG. 2A, in which oligonucleotides present in the sample have
bound to complementary oligonucleotide side members of a first cleavable
signal element, but have not bound to a second, different, set of
oligonucleotide side members of a second cleavable signal element;
FIG. 2C is a schematic representation of a later stage of the assay
procedure of FIGS. 2A and 2B, following cleavage of the spacer molecules.
The reflective gold microsphere that is not tethered by the specific
hybridization of complementary oligonucleotides from the test sample is
removed from the surface of the assay device, providing a
spatially-addressable, differentially reflective signal;
FIGS. 2D-2E are schematic representations of one aspect of the invention in
which a soluble oligonucleotide added to the test sample increases
sensitivity in a nucleic acid hybridization assay;
FIG. 2F is a schematic representation, in a nucleic acid detection assay
adapted to use the cleavable reflective signal elements of the present
invention, of the use of DNA ligase to increase the strength with which
analyte-specific binding adheres the signal responsive end of the
cleavable spacer to the derivatized substrate of the assay device, thus
permitting increased stringency of wash and increased specificity of the
assay;
FIG. 3A schematically represents an immunoassay adapted to use the
cleavable reflective signal element of the present invention. FIG. 3A
illustrates antibodies, adapted to bind to an epitopic site of an antigen
suspected to be in a test sample, attached to the side members of the
cleavable spacers of a plurality of signal elements;
FIG. 3B is a schematic representation of a later stage in the assay process
represented in FIG. 3A and illustrates binding of antigen from the sample
to two antibodies of one cleavable signal element, but failure of antigen
from the sample to bind to a second set of antibody side members attached
to a second cleavable signal element;
FIG. 3C is a schematic representation of the assay of FIGS. 3A and 3B at a
still later stage in the assay process, following cleaving of the signal
element spacers. The reflective gold microsphere that is not tethered by
the specific bridging association of antigen from the sample to signal
element antibodies is removed from the surface of the assay device,
providing a spatially-addressable, differentially reflective signal;
FIGS. 4A through 4G illustrate schematically the preparation of the solid
support substrate upon which cleavable reflective signal elements are
deposited in predetermined patterns to create the spatially addressable
assay device of this invention;
FIG. 5 is a schematic representation of the chemical structure of an
exemplary cleavable spacer molecule of the cleavable reflective signal
element of this invention, subsequent to its attachment to the derivatized
plastic substrate surface of the assay device but prior to derivatization
with oligonucleotide side members, in which piv denotes a pivaloyl
protective group, MMT denotes monomethoxytrityl, and n and m each
independently represents an integer greater than or equal to one;
FIG. 6 is a further schematic representation of a cleavable spacer
molecule, particularly illustrating the site on the spacer molecule that
is susceptible to cleaving, and further indicating the sites for
attachment of side members, shown protected by Piv and MMT groups;
FIGS. 7A through 7C illustrate in schematic a means for attaching the
cleavable spacer molecules to the activated surface of the assay device
substrate. In the example illustrated, the aminated surface of the
substrate shown in FIG. 7A is converted to active esters as shown in FIG.
7B. The cleavable spacer molecules are attached via the activated esters
to the solid support as shown in FIG. 7C;
FIGS. 8A and 8B illustrate intermediate steps during the attachment of a
first oligonucleotide side member on the surface-attaching side of the
cleavage site of a plurality of cleavable spacer molecules;
FIGS. 9A and 9B are schematic representations illustrating the intermediate
steps in the attachment of a second oligonucleotide member on the signal
responsive side of the cleavage site of a plurality of cleavable spacer
molecules;
FIG. 10A is a schematic representation illustrating the substantially
complete cleavable spacer molecule of the cleavable reflective signal
element of the present invention, as attached to the solid substrate of
the assay device, and prior to the attachment of the microspheres to the
signal-responsive end of the cleavable spacer molecules;
FIG. 10B illustrates the attachment of a single reflective particle to the
signal responsive end of the cleavable spacers of FIG. 10A, completing the
cleavable reflective signal element of the present invention;
FIGS. 11A through 11G illustrate various patterns of spatially addressable
deposition of cleavable reflective signal elements on circular, planar
disk substrates, in which:
FIG. 11A particularly identifies an address line, encodable on the disk
substrate, from which the location of the cleavable spacers may be
measured. In FIG. 11A, the cleavable spacer molecules are deposited in
annular tracks;
FIG. 11B demonstrates spiral deposition of cleavable signal elements, and
particularly identifies a central void of the disk annulus particularly
adapted to engage rotational drive means;
FIG. 11C demonstrates deposition of cleavable signal elements in a pattern
suitable for assay of multiple samples in parallel, with concurrent
encoding of interpretive software on central tracks;
FIG. 11D schematically represents an embodiment in which the assay device
substrate has further been microfabricated to segregate the individual
assay sectors, thereby permitting rotation of the assay device during
sample addition without sample mixing;
FIG. 11E schematically represents an embodiment in which the assay device
substrate has further been microfabricated to compel unidirectional sample
flow during rotation of the assay device;
FIG. 11F demonstrates deposition of cleavable signal elements in a spatial
organization suitable for assaying 20 samples for 50 different analytes
each;
FIG. 11G demonstrates the orthogonally intersecting pattern created by
superimposition of spiral patterns with spiral arms of opposite direction
or chirality;
FIG. 12 is a schematic representation of detection of analyte-specific
signals generated by the assay device of FIG. 11A;
FIGS. 13A-F are a schematic example of a stamp for use in printing
oligonucleotide side members onto cleavable spacers previously attached to
a solid substrate. The stamp as shown is made of two pieces, a stamp piece
and a feeding piece. The stamp piece contains holes, which are filled by
the required chemicals through a feeding piece containing channels. The
channels in turn are connected to a glass capillary array. In this
arrangement, one row of holes is filled with the same chemical Different
hole and channel patterns can be used as needed;
FIGS. 14A and B are a schematic representation of the pattern of
oligonucleotide side member deposition resulting from a two-stage
orthogonal printing using the stamp depicted in FIGS. 13A-F. Numbers 1, 2,
3 and 4 represent different phosphoramidite sequences used in the
synthesis. In oligonucleotide synthesis using trimers, for example, number
1 can be AAA, number 2 AAC, number 3 AAG and number 4 AAT. The first
number in each spot gives the oligonucleotides building block that is most
proximal to the cleavable spacer backbone; the second number (if any)
represents the next building block. Orthogonal printing is particularly
advantageous when depositing the cleavable reflective signal elements of
the present invention on a substrate shaped as a disk;
FIGS. 15A and B are a schematic representation of a complementary concave
printing process for printing large numbers of oligonucleotide side
members simultaneously onto cleavable spacers previously attached to a
solid substrate. The cleavable spacers are not themselves shown;
FIG. 16 demonstrates one geometry in which a single sample is channeled in
parallel into four distinct sectors of the assay device. If either the
density of biobits or affinity of the biobits in the four sectors differs,
a large dynamic range of concentration may be determined by detecting the
position in each sector of the positive cleavable signal element most
distal from the sample application site;
FIGS. 17A-C demonstrate an alternative assay device geometry that dispenses
with cleavable spacers, in which a first analyte-specific side member is
attached directly to the assay device substrate, while a second
analyte-specific side member is attached directly to the signal responsive
moiety, shown here as a plastic microsphere;
FIGS. 18A-C demonstrate a further alternative geometry dispensing with
cleavable spacers, in which a first side member is attached directly to
the assay device substrate, a second side member is attached directly to
the signal responsive moiety, and analyte causes agglutination of signal
responsive moieties;
FIG. 19 shows a top view of an assay device adapted for continuous
monitoring, in which a radially disposed mirror directs incident light
into the plane of the assay device substrate which functions as an optical
waveguide. Also shown are circumferentially disposed sample application
inlets for each of 20 independent assay sectors;
FIGS. 20A-B show further detail of the continuous monitoring assay device
of FIG. 19, with FIG. 20A showing a top view of a single assay sector and
FIG. 20B showing a side view of a single assay sector;
FIGS. 21A-F show side views of an assay site during continuous monitoring
for analytes;
FIGS. 22A-C show the assay device of FIG. 21 after sample application, with
subsequent cleavage of cleavable spacers for detection using reflectance
of incident light;
FIGS. 23A-C show continuous monitoring of solid support particles;
FIG. 24 shows synthesis of dimers;
FIG. 25 shows screening of hexapeptides;
FIG. 26 demonstrates the alternative use of a diffraction grating for
directing incident light into the assay device substrate adapted for use
as an optical waveguide;
FIGS. 27A-C shows a cleavable ester moiety, the ease of hydrolysis of which
is modified by the addition of an n-pthalimidomethyl group on the alcohol
side, shown in FIG. 27A, by the addition of an .alpha., .alpha.
difluoroacid moiety on the carboxylic acid side, shown FIG. 27B, or by
addition of both, shown in FIG. 27C;
FIGS. 28A-C show an alternative geometry for nucleic acid hybridization
assays that increases the fidelity of sequence detection, useful in assays
for defined sequences, as in assays for detection of in vitro amplified
nucleic acids, and also useful in nucleic acid sequencing. FIG. 28A shows
signal responsive moieties, shown as spheres, maintained by noncovalent
sequence-specific hybridization in a storage area of the assay device.
FIG. 28B shows the presence of a single-stranded nucleic acid analyte, and
further identifies three subsequences therein. FIG. 28C shows recognition
of subsequence "a" of the analyte, causing detachment from the storage
area of the signal responsive moiety, transfer of the detached
signal-responsive moiety and transfer to a capture area, and recognition
and binding of the signal responsive moiety mediated by subsequence "c" of
the analyte;
FIG. 29 shows the adaptation of the cleavable spacer invention for
detection of a small organic molecule, norepinephrine;
FIG. 30 demonstrates the adaptation of the cleavable spacer invention for
detection of amino acids in a sample;
FIGS. 31A-B demonstrate the adaptation of the cleavable spacer invention
for detection of ethanol, using alcohol oxidase and catalase;
FIGS. 32A-C show the use of photoactivatable groups on the side members of
a cleavable spacer, for detection of incident radiation;
FIGS. 33A-C show an alternative assay geometry for for cell counting and
cell shape detection, using an optical disk without cleavable spacers.
FIG. 33A shows a plurality of first cell type-specific recognition
elements disposed on the substrate surface of an assay device, shown
schematically FIG. 33B shows binding of the cell to the cell type-specific
recognition elements. FIG. 33C shows signal responsive moieties, added
subsequently, decorating the surface of the cell, rendering it suitable
for detection;
FIGS. 34A-C present a classification of assay geometries that may be
practiced using the detection methods and assay devices of the present
invention, without the need for cleavable spacers. FIG. 34A shows
analyte-mediated binding of signal-responsive moieties in a sandwich
assay. FIG. 34B shows an analyte-mediated displacement of signal
responsive moieties, a replacement assay. FIG. 34C shows a competitive
assay;
FIGS. 35A-C present a classification of assay geometries that may be
practiced using the detection methods and assay devices of the present
invention, additionally using the cleavable spacers of the present
invention. FIG. 35A shows analyte-mediated binding of first and second
side members of a cleavable spacer in a sandwich assay. FIG. 35B shows an
analyte-mediated displacement of connected first and second side members,
a replacement assay. FIG. 35C shows a competitive assay;
FIG. 36 shows a top view and side view of a sample application plate, in
which wells suitable for holding liquid samples are disposed in a spatial
orientation suitable for applying in parallel a plurality of individual
samples to the assay sites of an assay device of the present invention;
FIGS. 37A-F show sample application using the sample application plate of
FIG. 36. FIG. 37A shows a side view of the sample application plate. FIG.
37B shows addition of samples to the wells of the sample application plate
using a robotic pipetting station with multiple pipettes. FIG. 37C shows
the assay device oriented for sample addition, with assay areas disposed
upon the assay device in registration with the wells of the sample
application plate. FIG. 37D shows direct approximation of the assay device
to the sample application plate. FIG. 37E shows gravity driven application
of samples to the assay device through inversion of the approximated
sample application plate and assay device. FIG. 37F shows further
processing of the assay device to which multiple samples have been applied
and shows disposal of the sample application plate;
FIG. 38 shows an alternative geometry for a sample application plate, in
which full-thickness air holes, suitable for application of vacuum, are
interpolated between sample application wells to prevent sample spread
between wells;
FIG. 39 shows an alternative geometry for a sample application plate,
suitable for small samples. The cross-sectional view shows hydrophobic
channels exiting the sample well to prevent air bubbles from displacing
sample;
FIG. 40 shows a sample application plate in which the hydrophobic channels
of individual sample wells communicate with a channel to which a vacuum
line, controlled by a stopcock, is attached;
FIGS. 41A-L show the use of the sample application plate of FIG. 40. FIG.
41A shows a cross-sectional view of the sample application plate. FIG. 41B
shows the application of a disposable thin plastic film. FIG. 41C
demonstrates molding of the disposable film to the sample wells upon
application of vacuum. FIG. 41D shows retention of shape due to air
pressure differences after closing of the vacuum stopcock. FIG. 41E shows
sample addition. FIG. 41F shows approximation of the assay device to the
sample application plate. FIG. 41G shows contact, in correct registration,
of the assay device to the sample application plate. FIG. 41H shows
inversion of the approximated devices, permitting gravity-fed application
of samples. FIG. 41I shows inversion to the original orientation after
sufficient time for sample application. FIG. 41J shows removal of the
assay device, addition of washing buffer to the sample application plate,
and application in correct registration to the assay device. FIG. 41K
shows removal of the assay device, further addition of water to the sample
application plate, and application thereof in correct registration to the
assay device. FIG. 41L shows disposal of the plastic film upon release of
vacuum, permitting reuse of the sample application device;
FIGS. 42A-E show a sample application plate similar to that shown in FIG.
41, in which a stamp, shown in FIG. 41C, is used to mold the disposable
film to the application plate wells instead of vacuum as in FIG. 41;
FIGS. 43A-E show sequential addition to the assay device, here termed a
bio-compact disk, of washing solution and sample, by application of
centrifugal force through rotation of the assay device and sample
applicator. The assay area is shown as a thick line;
FIG. 44 shows a clinical laboratory embodiment for applying sample.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an assay device for detecting analyte,
comprising an optical disk having analyte-specific signal elements that
are disposed readably thereon. The optical disk may be read, and the
analyte detection thus performed, using optical disk readers, including
those capable of reading audio CD disks, CD-ROM disks, DVD disks, DiVX
disks, laser disks, or by readers for other optical disk formats that are
similarly useful for digitally-encoding information. In some embodiments
the signal elements are readable with the optical disk's tracking
features: that is, the analyte-specific signal elements are readable by
the optics that read the tracking features, although modified or
additional optics are not thereby precluded
Unless otherwise specified, terms used herein have their usual and
customary meaning, as appropriate to the optical disk and assay arts.
In particular, "analyte", for purposes of this invention, includes any
substance, chemical or biological, that one wishes to detect. Thus,
"analyte" is intended to include cells when the assay device is adapted
for use in cell counting or cell shape detection, to include nucleic acids
when the device is adapted for nucleic acid probe detection or nucleic
acid sequencing, small organic or inorganic molecules when the device is
adapted for chemical assay. The term "analyte" is also intended to cover
radiation when the device is adapted, as for example by the use of
photactivatable groups, to detect incident radiation.
In preferred embodiments, the assay device and assay methods of this
invention utilize a cleavable signal element for detection of analytes in
test samples. Binding of the analyte preselected for detection prevents
the loss--through cleavage--of the signal element's signal responsive
moiety. Generation of a signal from the signal responsive moiety of the
constrained signal element is then used to signal the presence of analyte
in the sample.
In a preferred embodiment, the signal responsive moiety reflects or
scatters incident light, or is otherwise light addressable. Binding of the
analyte preselected for detection prevents the loss--through cleavage--of
the signal element's light responsive moiety. Reflection or scattering of
incident light, preferably incident laser light, from the reflective
moiety of the constrained signal element is then used to signal the
presence of analyte in the sample.
The cleavable reflective signal elements of the present invention are
particularly adapted for detection using existing laser reflectance-based
detectors, including audio compact disk (CD) readers, CD-ROM (compact disk
read-only memory) readers, laser disk readers, DVD (digital video disk)
readers, and the like. The use of the cleavable reflective signal elements
of the present invention thus permits the ready adaptation of existing
assay chemistries and existing assay schemes to detection using the large
installed base of existing laser reflectance-based detectors. This leads
to substantial cost savings per assay over standard assays using dedicated
detectors.
Furthermore, the wide and ecumenical distribution of laser-reflection based
detection equipment further permits assays--as adapted to use the
cleavable reflective signal element of the present invention--to be
distributed for point-of-service use, assays that must currently be
performed at locations determined by the presence of a dedicated detector.
Among these assays are immunoassays, cell counting, genetic detection
assays based upon hybridization, genetic detection assays based upon
nucleic acid sequencing, nucleic acid sequencing itself, chemical assays,
assays for incident radiation, and the like. The current invention thus
allows distribution of assay devices to research laboratories, physician's
offices, and individual homes that must currently be performed at
centralized locations.
Each of the laser-reflectance based detectors mentioned
hereinabove--including CD-ROM readers, DVD readers and the like--is
adapted for detecting, discriminating, and interpreting spatially
addressable digital information on their respective media: audio CD
readers are capable of specifically and separately addressing individual
digitally encoded audio tracks; CD-ROM readers are capable of specifically
and separately addressing multiple binary files, including binary files
encoding computer programs (ISO 9660, incorporated herein by reference,
defines a common addressable file structure); so too DVD readers are
capable of specifically and separately addressing binary files and
MPEG-encoded digital video signals.
The spatially addressable capabilities of the laser reflectance-based
detectors currently used to detect and interpret information encoded on
CDs and the like confer particular advantages on assays adapted to use the
cleavable reflective signal elements of the present invention.
Thus, patterned deposition of multiple signal elements on a single
supporting member or substrate, coupled with use of a detector capable of
addressing the spatial location of these individual signal elements,
permits the concurrent assay of a single sample for multiple different
analytes. The present invention is thus further directed to assay devices,
commonly referred to herein as disks, bio-compact disks, bio-CDs, BCDs,
and bio-DVDs, comprising spatially addressable combinations of cleavable
reflective signal elements of different analyte specificity. Among such
useful combinations are those that increase the predictive value or
specificity of each of the individual assays, combinations that inculpate
or exculpate particular diagnoses in a differential diagnosis,
combinations that provide broad general screening tools, and the like.
Patterned deposition of multiple signal elements with identical specificity
further permits the detection, using a single assay device, of large
concentration ranges of a single analyte. It is thus another aspect of the
present invention to provide assay devices comprising spatially
addressable cleavable reflective signal elements of identical specificity,
the physical location of which is capable of conveying concentration
information.
The spatially addressable capabilities of the laser reflectance-based
digital detectors further permits the combination of interpretive software
and the assay elements themselves on a single assay device. Another aspect
of the current invention, therefore, is an assay device upon which
software is encoded in an area spatially distinct from the patterned
deposition of cleavable-reflective signal elements. The software may
include information important for correct tracking by the incident laser,
assay interpretive algorithms, standard control values, self-diagnostics,
and the like. The software may include device drivers and software capable
of uploading the diagnostic information to remote locations. The software
may include patient education information for clinical assays, and may be
adapted for chosen audiences.
The substantially binary nature of assay data signaled by the cleavable
reflective signal elements of the present invention presents the further
advantage of rendering assays adapted to their use substantially resistant
to instrumental noise. For example, small variations in light
reflection--as from small variations in light intensity provided by the
laser source and small variation in reflective particle size--generally do
not affect the assay result because the detectors only register a signal
when light reflection reaches a threshold. Similarly, electronic noise of
the detection device itself and noise associated with an analog to digital
conversion do not affect assay results. This advantage is particularly
appreciated in designing and manufacturing robust detection instruments
useful for field testing or for performing assays under difficult
environmental operating conditions.
Furthermore, the substantially binary nature of assay data signaled by the
cleavable reflective signal elements of the present invention permits
digital correction of imperfections in signal element spatial deposition:
the assay device (disk) is read before analysis, the software stores the
signal pattern, which pattern is later subtracted from that read after
sample application and development of the assay disk.
5.1 Assays with Spatially Addressable, Cleavable Reflective Signal Elements
5.1.1 Spacer and Cleavable Site
The general operation of the cleavable reflective signal element of this
invention, also termed a bio-bit or Biobit, can be understood more
particularly by reference to FIGS. 1-3, which schematize two embodiments
of the present invention. With reference to FIG. 1, a substrate 20 is
provided with a derivatized surface 21 to which is attached cleavable
spacer molecules 30, each cleavable spacer having, in addition to a
surface-attaching end, a signal responsive end, shown proximal to metal
microsphere 40. The substrate, which may be porous or solid, although
solid is presently preferred, can be selected from a variety of materials
such as plastics, glass, mica, silicon, and the like. However, plastics
are preferred for reasons of economy, ease of derivatization for attaching
the spacer molecules to the surface, and compatibility with existing laser
reflectance-based detectors, such as CD-ROM and DVD readers. Typical
plastics that can be used are polypropylenes, polyacrylates, polyvinyl
alcohols, polyethylenes, polymethylmethacrylates and polycarbonates.
Presently preferred are polypropylene and polycarbonate, and most
preferred polycarbonate.
The surface 21 of the substrate 20 can be conveniently derivatized to
provide covalent bonding to each of the cleavable spacer molecules 30. The
metal spheres provide a convenient reflective signal-generating means for
detecting the presence of a spacer molecule bound to the assay device
substrate 20. Typical materials are gold, silver, nickel, chromium,
platinum, copper, and the like, with gold being presently preferred for
its ability readily and tightly to bind e.g. via dative binding to a free
SH group at the signal responsive end of the cleavable spacer. The metal
spheres may be solid metal or may be formed of plastic, or glass beads or
the like, on which a coating of metal has been deposited. Also, other
reflective materials can be used instead of metal. The presently preferred
gold spheres bind 51 directly to the thio group of the signal responsive
end of the cleavable spacer.
Each of the cleavable spacer molecules is attached at one end 31 to support
surface 21, e.g. via an amide linkage, and at the other end 32 to a signal
generating means (also termed a signal-responsive moiety), e.g. via a thio
radical to a reflective metal microsphere 40. The spacer molecule has a
cleavage site 33 that is susceptible to cleavage during the assay
procedure, by chemical or enzymatic means, heat, light or the like,
depending on the nature of the cleavage site. Chemical means are presently
preferred with a siloxane cleavage group, and a solution of sodium
fluoride or ammonium fluoride, exemplary, respectively, of a chemical
cleavage site and chemical cleaving agent. Other groups susceptible to
cleaving, such as ester groups or dithio groups, can also be used. Dithio
groups are especially advantageous if gold spheres are added after
cleaving the spacer.
Cleavage site 33 is between the first, surface-attaching end 31 of
cleavable spacer molecule and the second, signal-responsive end 32 of
cleavable spacer molecule 30. Spacers may contain two or more cleavage
sites to optimize the complete cleavage of all spacers.
Analyte specificity is conferred upon the cleavable spacer by side members
34a and 34b, also termed side arms, positioned on opposite sides of the
cleavage site 33; that is, positioned proximal to the surface-attaching
end and proximal to the signal-responsive end of cleavable spacer molecule
30, respectively. Side members 34a and 34b in their typical configuration
include an oligonucleotide, typically 5- to 20-mers, preferably 8- to
17-mers, most preferably 8- to 12-mers, although longer oligonucleotides
can be used. The side members may also include, without limitation and as
required, peptides, organic linkers to peptides or proteins, or the like.
A large number of cleavable spacer molecules 30 will be present at any
particular derivatized site on the solid surface 21 of the assay device,
also termed a disk, a blo-compatible disk, or BCD.
5.1.2 Nucleic Acid Assays
In one aspect of the invention, the oligonucleotide side members are
adapted to bind complementary single strands of nucleic acids that may be
present in a test sample. The complementary oligonucleotides comprise
members of a specific binding pair, i.e., one oligonucleotide will bind to
a second complementary oligonucleotide.
As is described more particularly in FIGS. 2A through 2C, schematizing one
embodiment of the invention, cleavable spacer molecules 30 at different
sites on the surface of the assay device will have different
oligonucleotide side members. As shown in FIG. 2A, one such cleavable
signal element has oligonucleotide side members 34a and 34b, whereas the
second cleavable signal element has oligonucleotide side members 35a and
35b.
As further depicted in FIGS. 2A through 2C, when contacted with a test
sample containing an oligonucleotide 36, the complementary oligonucleotide
side members 34a and 34b will bind with the oligonucleotide present in the
sample to form a double helix as is shown in FIG. 2B. Since there is no
complementarity between oligonucleotide 36 and oligonucleotide side
members 35a and 35b, there is no binding between those groups as is
further illustrated in FIG. 2B.
When the cleavage site 33 is cleaved, but for the binding by the double
helix coupled oligonucleotides, the metal microspheres 40 will be free of
the surface and removed therefrom. This is illustrated more fully in FIG.
2C. If it is desired to assay multiple samples for a single
oligonucleotide, the spacer molecules at different sites will generally
have the same oligonucleotide side members. Presence and absence of the
metal microsphere 40 may then be detected as reflectance or absence of
reflectance of incident light, particularly incident laser light.
FIG. 2F is a schematic representation of the use of DNA ligase in a further
embodiment of the nucleic acid detection embodiment of the present
invention to increase the strength with which analyte-specific binding
adheres the signal responsive end of the cleavable spacer to the
derivatized substrate of the assay device, thus permitting in this
embodiment increased stringency of wash, affording increased specificity
of the assay.
It will be appreciated by those skilled in nucleic acid detection that the
cleavable reflective signal elements of the present invention are
particularly well suited for detecting amplified nucleic acids of defined
size, particularly nucleic acids amplified using the various forms of
polymerase chain reaction (PCR), ligase chain reaction (LCR),
amplification schemes using T7 and SP6 RNA polymerase, and the like.
5.1.3 Immunoassays
In a further embodiment of the invention described in FIGS. 3A through 3C,
the oligonucleotide side members 34a, 34b, 35a, and 35b are coupled
noncovalently to modified antibodies 38a, 38b, 38c, and 38d to permit an
immunoassay. The noncovalent attachment of modified antibodies to side
members is mediated through complementarity of cleavable spacer side
member oligonucleotides and oligonucleotides that are covalently attached
to the antibodies. Use of complementary nucleic acid molecules to
effectuate noncovalent, combinatorial assembly of supramolecular
structures is described in further detail in co-owned and copending U.S.
patent applications Ser. No. 08/332,514, filed Oct. 31, 1994, 08/424,874,
filed Apr. 19, 1995, and 08/627,695, filed Mar. 29, 1996, incorporated
herein by reference. In another embodiment, antibodies can be attached
covalently to the cleavable spacer using conventional cross-linking
agents, either directly or through linkers.
The antibodies comprise a first member of a first specific binding pair and
a first member of a second specific binding pair. The second member of the
first specific binding pair and the second member of the second specific
binding pair will be different epitopic sites of an antigen of interest.
More specifically, oligonucleotide side member 35a is attached to the
antibody-oligonucleotide 38c and oligonucleotide side member 35b is
attached to antibody-oligonucleotide 38d. The antibodies 38c and 38d are
adapted to bind different epitopic sites on an antigen that may be present
in the test sample. By different epitopic sites on an antigen is intended
different, spatially separated, occurrences of the same epitope or
different epitopes present at distinct sites. At a second assay element,
the oligonucleotide side members 34a and 34b are attached to different
antibodies 38a and 38b, again each of such antibodies being adapted to
attach to a different epitopic site of an antigen.
With further reference to the immunoassay schematized in FIGS. 3A-3C, upon
application of the test solution containing antigen 39 to the collection
of cleavable reflective signal elements illustrated in FIG. 3A, antigen 39
binds antibodies 34a and 34b, thus preventing decoupling of the metal
sphere 40 from the assay device surface 20 when the cleavage site 33 is
cleaved, such as, for example, by contact with a chemical cleaving agent.
In contrast, the second cleavable signal element, which was not bound by
antigen 39 because the lack of binding affinity of the antibodies 35a and
35b to the antigen 39, allow the metal microsphere 40 to separate from the
solid surface and be removed from the sample.
Presence and absence of the metal microsphere 40 may then be detected as
reflectance or absence of reflectance of incident light, particularly
incident laser light.
As should be apparent, coupling of antibodies as depicted permits the ready
adaptation of standard immunoassay chemistries and immunoassay geometries
for use with the cleavable reflective signal elements of the present
invention. Some of these classical immunoassay geometries are further
described in U.S. Pat. No. 5,168,057, issued Dec. 1, 1992, incorporated
herein by reference. Other immunoassay geometries and techniques that may
usefully be adapted to the present invention are disclosed in Diamandis et
al. (eds.), Immunoassay, AACC Press (July 1997); Gosling et al. (eds.),
Immunoassay : Laboratory Analysis and Clinical Applications,
Butterworth-Heinemann (June 1994); and Law (ed.), Immunoassay : A
Practical Guide, Taylor & Francis (October 1996), the disclosures of which
are incorporated herein by reference. Thus, it should be apparent that the
direct detection of analyte (a capture assay) schematized in FIG. 3 is but
one of the immunoassay geometries adaptable to the cleavable reflective
signal elements and assay device of the present invention.
For example, replacement immunoassays can readily be adapted. In this
geometry, a first side member of the cleavable spacer contains an antibody
specific for an epitopic site of the analyte, as in the geometry shown in
FIG. 3. In contrast to the geometry shown in FIG. 3, however, the second
side member has a moiety that displays the determinant recognized by the
antibody on the first side member. The default state of the side members,
therefore, is a direct binding of the first side member to the second side
member, mediated by recognition of the second by the antibody of the
first. All signal responsive moieties are thus tethered to the assay
device substrate, and addition of cleavage agent releases none of the
signal responsive moieties. a more generalized depiction of such a
geometry is give in FIG. 35B.
Antigen present in the sample and displaying the appropriate epitopic
determinant will displace the immobilized antigen and cut the
antigen-antibody loop. As a result, the signal responsive moiety will be
liberated after addition of cleavage agent. To increase sensitivity, the
immobilized antigen, in this example part of the second side member,
should have lower affinity for the immobilized antibody than does the
antigen in the sample. For many antibodies a series of antigens having a
range of affinities is well known.
Competitive immunoassay is also amenable to adaptation for use with the
cleavable spacer and optical disk of the present invention. This geometry
is particularly well suited for detection of analytes that are either too
small to bridge the gap between first and second side members, or that
present a single antigenic epitope.
In this geometry, the first and second side member antibodies are tethered
in the default state by a multimeric synthetic antigen. Univalent analyte
in the sample displaces one or both antibodies, permitting subsequent loss
of the signal responsive moiety after cleavage.
When sample is flowing across the detection surface of the assay device,
for instance, through radial flow incident to rotation of the disk, it is
possible to combine replacement and capture. In the default state, signal
responsive moieties are bound by antigen-antibody interaction to the
surface of the assay device. When a sample flows over this area, the
antigen or antibody present in this sample serves to detach the signal
responsive moieties. These signal responsive moieties, for example metal
microspheres, will be captured again in an area that is coated with the
corresponding antigen or antibody. The number of spheres reports the
concentration of the analyte. The pattern of sphere deposition reports
information on the binding kinetics and is characteristic for each
analyte. Thus, the binding pattern can be used, e.g., to report the purity
of the analyte.
The cleavable signal element embodiments of the present invention present
particular advantages for immunoassays. Because the first and second side
member antibodies are spatially constrained and in close proximity, the
immunoassay is expected to be both fast and sensitive; diffusion of
antibodies through a fluid phase is obviated. Moreover, because neither
antibody may diffuse from its original site, transient dissociation of
analyte from one or the other need not lead to permanent dissociation of
the complex: the components will almost certainly recombine before the
antigen dissociates from the second antibody. This will increase
sensitivity as compared with traditional fluid phase, or semi-solid,
immunoassays.
The present invention will prove particularly valuable in immunoassays
screening for human immunodeficiency viruses, hepatitis a virus, hepatitis
B virus, hepatitis C virus, and human herpes viruses.
It will further be appreciated that antibodies are exemplary of the broader
concept of specific binding pairs, wherein the antibody may be considered
the first member of the specific binding pair, and the antigen to which it
binds the second member of the specific binding pair. In general, a
specific binding pair may be defined as two molecules the mutual affinity
of which is of sufficient avidity and specificity to permit the practice
of the present invention. Thus, the reflective cleavable signal elements
of the present invention may include other specific binding pair members
as side members. In such embodiments, the first side member of the
cleavable signal element includes a first member of a first specific
binding pair, the second side member of the cleavable spacer includes a
first member of a second specific binding pair, wherein said second member
of said first specific binding pair and said second member of said second
specific binding pair are connectably attached to one another, permitting
the formation of a tethering loop of the general formula: first member of
first specific binding pair-second member of first specific binding
pair-second member of second specific binding pair-first member of second
specific binding pair.
Among the specific binding pairs well known in the art are biologic
receptors and their natural agonist and antagonist ligands, proteins and
cofactors, biotin and either avidin or streptavidin, alpha spectrin and
beta spectrin monomers, and antibody Fc portions and Fc receptors.
5.1.4 Chemical Assays
In yet another embodiment of the present invention, the analyte-specific
side members are chosen to react with specific functional groups presented
by an analyte, as exemplified in FIGS. 29, 30 and 31.
In general, functional groups that are present in small organic or
biological molecules, such as amino, aldehydo, keto, carboxylic and thiol
groups can readily be detected using the cleavable spacer embodiment of
the present invention, so long as the molecule contains at least two such
functional groups and is large enough to form a bridge between recognition
molecules, thus tethering the signal responsive moiety to the assay device
substrate.
The bridge need not necessarily lead to formation of a covalent bond.
Acid-base interaction, hydrogen bonding, coordinate bonding and even van
der Walls interaction can be used to secure the signal responsive moiety
to the disk assay substrate. For example, both side-elements can contain
alkylamine diacetic acid unit, i.e., half of EDTA. These side-elements
will bind strongly to divalent cations, such as calcium and magnesium
ions. To confer greater analyte specificity, crown ethers and cryptands
can be used.
Furthermore, if the analyte is too small to bridge the space between first
and second side members, a competitive assay geometry may usefully be
employed, the analyte serving, either directly or indirectly, to displace
the binding of the signal responsive moiety, as further exemplified in
Example IV, below. And as further discussed below with respect to spacer
cleavage chemistries, it should be appreciated that in certain
circumstances the analyte specificity may be conferred directly by the
cleavage site, or by the cleavage site in association with auxiliary
recognition molecules, without the need for spacer side members or further
addition of a cleavage agent.
Turning, then, to the figures, FIG. 29 presents cleavable spacers that
contain a first and second side member that permit selective detection of
norepinephrine.
The first side member, proximal to the solid support substrate, here an
optical disk, contains a phenyl boronic acid moiety, which will react with
a molecule presenting two hydroxyl molecules in close proximity. The
second side member, proximal to the signal responsive moiety, here a gold
sphere, contains a pthalaldehyde group, which will react with a primary
amine.
Upon contact with norepinephrine under reducing conditions the two side
members react, thus forming a covalent bridge between the side members.
Upon cleavage, the signal responsive moiety is securely tethered to the
disk substrate, giving a positive signal indicative of the presence of
norepinephrine in the sample.
FIG. 30 depicts cleavable spacers adapted to detect amino acids using the
ninhydrin reaction. Traditionally, the ninhydrin reaction has been adapted
to generate a colored end product that can be detected visually or
spectrophotometrically. Here, the reaction is adapted to permit detection
on an optical disk.
Many such existing analytic reactions may be adapted to the optical
disk-based devices and methods of the present invention.
Although it is the spacer side members that confer analyte specificity in
the two examples given above, analyte specificity may also be conferred by
auxiliary molecules distinct from the spacer side members. In particular,
analyte specificity may be enhanced by coupling the high substrate
specificity of enzymes to the chemical reactivity of the side members, as
exemplified in FIG. 31.
FIG. 31 presents an example of adapting existing enzymatic chemistries to
the detection of ethanol using the cleavable spacer embodiment of the
present invention. In FIG. 31A, the assay device solid support substrate
is shown above, with the cleavable spacers depending below. Each signal
responsive moiety is attached in this example by two identical cleavable
spacers, the first and second side members of which contain the terminal
hydroxyl of polyethylene glycol and a primary amine, respectively. In
addition to the cleavable spacers with their signal responsive moieties,
two enzymes are also attached to the assay device substrate surface. One
is alcohol oxidase, the other catalase.
As shown in FIG. 31A, ethanol in the sample serves as a substrate for
alcohol oxidase present on the substrate surface, producing acetic acid
and hydrogen peroxide. As shown in FIG. 31B, the hydrogen peroxide, in the
presence of catalase, oxidizes the terminal hydroxyl group of the first
side member, coupling the first side member to the second, thus tethering
the signal responsive moiety to the assay device substrate
It will be appreciated that in this example it is the enzyme, alcohol
oxidase, that provides the analyte specificity. Conversely, the same
chemistries may equally be adapted to detect the presence of the enzyme
itself in the sample. In the assay given in FIG. 31, for example, omitting
the enzyme alcohol oxidase from the substrate surface allows assay for
alcohol oxidase in the applied sample. In this altered geometry, ethanol
is added to the sample to drive formation of peroxide in those samples in
which ethanol oxidase is present.
It will also be appreciated that the specificity of enzymes for biological
substrates serves as the basis for many existing assays, all of which may
be adapted, as exemplified here, for detection in optical disk-based
assays.
5.1.5 Assays for Electromagnetic and Ionizing Radiation
In yet another embodiment, the cleavable spacer of the present invention
can be used to detect electromagnetic radiation (FIG. 32). High resolution
imaging applications will particularly benefit from the nanometer scale
resolution that can be obtained by this method.
As with chemical detection, two distinguishable geometries are readily
suggested: (1) the first and second side members are coupled by
electromagnetic radiation, or (2) the spacer is directly cleaved by
electromagnetic radiation. In the first case, it is the retention of the
signal responsive moieties in a spatially-identified area after addition
of cleavage agent that reports the location of electromagnetic signal; in
the second case, it is the loss of signal responsive moieties from a
spatially-identified area, without further addition of a cleavage agent,
that reports the electromagnetic signal. Both detection methods can be
made sensitive for particular wavelengths by using chromophores.
Examples of functional groups that are sensitive to UV and/or visible
wavelengths include diacetylenes and azido groups. If both members of a
binding pair are diacetylenes, they can dimerize and even polymerize,
provided that the spacer side members contain a sufficiency of
diacetylenes, or the spacer side members are close enough so that
interspacer reaction is possible. As for azido groups, upon receipt of a
photon they generate a free radical, which will couple with almost
anything.
X-ray or .gamma.-radiation as well as ionizing or free radical forming
radiation will couple many kinds of binding pairs or, alternatively,
cleave the spacers. Scintillation compounds may be used to control the
process so that the high energy is transformed either to UV or visible
radiation
Regular film, such as IR-, visible, or X-ray film, can be applied directly
to the substrate surface of the assay device, either before the exposure
or after the development of the film. In this case the assay device will
has a reflective metal coating. The laser light will be absorbed according
to the darkness of the film and the reflection is reduced. The film can be
visualized and processed on the computer screen.
5.1.6 Modifications of Cleavable Spacer Assays
While the above-exemplified embodiments of assays using the cleavable
reflective signal elements of the present invention--detection of nucleic
acid analytes, immunoassay, assay for functional groups on small organic
molecules, and detection of radiation--have been described with signal
responsive moieties, such as reflective metal spheres, attached to the
cleavable spacer molecules prior to conducting the assay, it is
contemplated in these and other embodiments further described herein that
cleavable spacer molecules lacking a signal generating means can first be
exposed to sample, then cleaved, and the metal spheres added later so as
to attach to only those spacer molecules remaining on the surface. After
addition of the metal spheres, the surface can then be read with an
appropriate detector to identify the bound spacer molecules and analytes.
In yet another modification, the spacer cleavage site may contain, instead
of a chemically-cleavable functional group such as siloxane, a specific
binding pair that is dissociated by binding of the analyte. One such
geometry is shown in FIG. 35B, and is further discussed below in section
5.9.
Furthermore, the cleavable spacer of the present invention, which in
preferred embodiments of the present invention are particularly adapted
for detection in optical disk readers, may also usefully be employed on
other substrates. These include, but are not limited to, paper and plastic
strips, multiwell plates, magnetic disks (floppy disks), and silicon
chips. For example, gating by a field effect transistor depends upon the
local electric field; the field, in turn, may usefully be modified by the
analyte-specific binding of signal responsive moieties such as metal,
salts, such as strontium titanate, or polymers, such as polyacetylene,
polyaniline, polyphenylene, or carbon nanotubes.
5.1.7 Sample Application, Wash, and Cleavage
In each of the assay method embodiments of the invention, a sample to be
tested must be introduced. Devices particularly designed to facilitate
sample application are further described in a section below. General
aspects of sample addition will be discussed here.
In one aspect, the assay device is rotated and a fluid sample, preferably
diluted, is applied near the center of the circular assay device
substrate. The centrifugal forces associated with the rotation of the
assay device disk distribute the fluid sample across the planar face of
the solid substrate. In this manner the surface of the substrate is
uniformly covered with a constant and uniformly distributed fluid sample.
In this method of sample application, the test sample, initially about 100
.mu.l, is diluted for processing to about 1 ml. This solution is added
dropwise near the center of the rotating disk. The assay sites and
possibly the surface of the disk are hydrophilic and a fluid will form a
very thin layer on the rotating assay device disk. The thickness of the
fluid layer can be regulated by the frequency of drop addition and
frequency of disk rotation. a preferred thickness is less than 10 .mu.m,
because all molecules in the sample can then interact with the stationary
molecules bound by the spacers. About 100 .mu.l of the sample solution is
needed to cover the disk.
Other methods of sample application may be used with the cleavable
reflective signal element and assay device of the present invention. In
particular, it should be appreciated that the rotational application
above-described is suitable principally for application of a single sample
per assay device. In other aspects of the present invention, separate
samples may be applied to discrete areas of a stationary disk. In this
aspect, the assay system can assay approximately one thousand different
samples. Approximately one million gold spheres, which are applied onto a
predetermined areas on the disk, can be dedicated for each sample.
FIG. 11D shows an assay device of the present invention having 16 separate
assay sectors. FIG. 11E shows a possible direction for sample flow, with
barriers to fluid flow shown as lines.
Thus, in one embodiment of the invention, the assay device is designed to
assay, for example, 1024 patient samples simultaneously, one analyte per
assay device (i.e., per disk, each disk comprising a plurality of
cleavable spacers with identical side members conferring identical analyte
specificity). In such an embodiment, each of the spacer molecules on the
disk may be identical, so as to assay for the same analyte; spacer
molecules at particular locations on the disk will be identical to spacer
molecules at other locations on the disk. This application is particularly
useful in mass analysis conducted in clinical laboratories where a large
number of patient samples are analyzed at the same time for the presence
or absence of a single analyte.
It will also be appreciated that multiple samples may be assayed for
multiple analytes on a single assay device comprising cleavable reflective
signal elements with various analyte specificities. FIG. 11F shows an
assay device that can be used to screen 20 samples for 50 different
biomolecules.
In the latter case, it is possible to assay for a limited number of the
same analytes in a multiplicity of test samples. Patient samples may be
applied to the disk at specific locations by known methods such as ink jet
printing and micropipet arrays with disposable tips, or a combination
thereof. For large through-put operations, the assay disks may be loaded
into a cassette and test samples loaded hermetically either directly onto
the disk or into the wells in a circular plate.
After an appropriate incubation period, which may only be a few seconds to
allow the sample to traverse the surface of the support, a wash step may
be, but in some embodiments need not be, performed to remove unbound
sample. Wash stringency may be adjusted as in conventional assays to
adjust sensitivity and specificity. For example, in nucleic acid detection
embodiments, the salt concentration of the wash solution may be decreased
to increase the stringency of wash--thus reducing mismatch as between
analyte and specificity-conferring side members--or increased, to decrease
the stringency of wash, thereby permitting mismatch to occur. Adjusting
the stringency of wash in the nucleic acid hybridization and immunoassay
embodiments of the present invention is well within the skill in the art.
In one aspect, the surface of the circular disk is washed, when necessary,
by adding a wash solution near the center of the rotating disk. The sample
solution is removed as it pushes out from the periphery of the disk and is
collected. Because of the rotation of the disk, the wash step may be
eliminated if the fluid sample is adequately removed from the disk by
normal centrifugal forces and no adjustment to stringency is required.
After the wash step, if any, a solution including a cleaving agent is added
and again distributed over the surface of the disk. With reference to
FIGS. 1-3, the spacer molecule has a cleavage site 33 that is susceptible
to cleavage during the assay procedure, by chemical or enzymatic means,
heat, light or the like, depending on the nature of the cleavage site.
Chemical means are presently preferred with the siloxane cleavage group,
and a solution of sodium fluoride is exemplary as a chemical cleaving
agent for the siloxane group. Other groups susceptible to cleaving, such
as ester groups or dithio groups, can be used. Dithio groups are
especially advantageous if gold spheres are added after cleaving the
spacer.
In the case of the cleavage site being a siloxane moiety, which can be made
stable against spontaneous hydrolysis but is easily cleaved under mild
conditions by a fluoride ion, a solution of sodium or ammonium fluoride is
introduced, with concentration of 1 mM to 1 M, preferably 50 mM to 500 mM,
most preferably 100 mM (0.1 M). The cleavage step will last only a few
seconds. Although all spacers are cleaved during this step, the amide bond
between the cleavable spacer and the derivatized substrate of the assay
device remains stable to these conditions.
After application of sample and cleavage of the spacers, the detached
signal-generating moieties, preferably a reflective moiety, more
preferably a metal sphere, most preferably a gold sphere, must be removed
to provide differential signal during detection. The removal step may
include a second wash step, which may include introduction of wash
solutions.
Several means exist by which differential wash stringencies may be
developed at this stage of the assay, thereby permitting variation in the
specificity and sensitivity of the various assay methods.
In one aspect, the detached reflective moieties may be removed by rotating
the assay device, with or without addition of wash solution. In this
aspect, three parameters may be varied to provide differential stringency:
gold particle size, rotational speed, and the valency of spacer
attachment.
Gold spheres suitable for use in the cleavable reflective signal element
and assay device of the present invention are readily available in varying
diameters from Aldrich Chemical Company, British BioCell International,
Nanoprobes, Inc., and others, ranging from 1nm to and including 0.5-5
micrometers in diameter. It is within the skill in the art to create gold
spheres of lesser or greater diameter as needed in the present invention.
At a given rotational speed, the largest gold spheres experience larger
centrifugal (relative to r.sup.3) and drag forces (relative to r) and are
removed before smaller spheres with equal bonding. This provides a basis
for differential stringency of wash, and also of quantitative analysis.
The centrifugal force affecting the gold spheres may also be adjusted by
rotation frequency so that the loose and weakly bound gold spheres are
removed. Only the spacers which have bound to a complementary molecule
from the sample will continue to bind the gold spheres to the substrate
Furthermore, while the above embodiments of the invention have been
described with a single metal sphere attached to the signal-responsive end
of a single cleavable spacer, it should be appreciated that when gold is
used in a preferred embodiment of the invention, thousands of spacers may
bind one gold sphere, depending upon its diameter. Thus, the stringency of
the assay wash may be adjusted, at any given rotational speed, by varying
the diameter of the gold sphere, and by varying additionally the relative
density of cleavable spacers to gold spheres.
Thus, if virtually all spacers under a certain gold sphere are connected by
complementary molecules, the binding is very strong. If the spacers are
fixated only partially under a certain gold sphere, the sphere may remain
or be removed depending on the radius of the sphere and the frequency of
the rotation.
In extreme cases all spheres are either fixed or are removed. These are
expected alternatives for DNA analysis. In immunoassays the intermediary
cases are preferred Accordingly, the system should be optimized so that
the normal control level corresponds to 50% fixation of the gold spheres.
Higher or lower fixation corresponds to higher or lower concentrations of
the analyte, respectively, when using two antibodies for binding as
illustrated in FIG. 3.
a strong centrifugal force can be used to remove weakly bound gold spheres.
The centrifugal force pulling one gold sphere will be in the order of 0.1
nN, although this force can vary within large limits depending on the mass
of the gold sphere and the frequency of the rotation of the disk. The
force is strong enough to rupture nonspecific binding of antibodies and to
mechanically denature mismatching oligonucleotides. This is a very strong
factor for increasing the specificity of the interaction between analyte
and the cleavable signal elements of the present invention.
In embodiments of the present invention in which the reflective moiety of
the cleavable spacer is ferromagnetic, as, for example, in which the
reflective moiety is a gold-coated iron bead or an iron alloy, those
reflective moieties detached through cleavage and not secured to the assay
device substrate by analyte may be removed through application of a
magnetic field. In such embodiments, those signal elements that remain
attached to the assay device (disk) substrate will also be responsive to
the magnetic field, but their motion will be constrained by the length and
flexibility of the loop formed by the first side member-analyte-second
side member. The ability to shift the position of all attached signal
elements through application of an external magnetic field, even though
that shift will necessarily be constrained by the length and flexibility
of the first side member-analyte-second side member loop, may add, in this
embodiment, additional information. In particular, brief application of a
magnetic field will facilitate discrimination of analyte-induced signal
from random noise, the noise being unresponsive to the application of an
external magnetic field.
After removal of cleaved reflective signal moieties that are not protected
by the specific binding of analyte, the disk may be read directly.
Alternatively, the disk may first be disinfected before reading. In yet
another embodiment, the disk may be covered by an optically clear plastic
coating to prevent the further removal of the gold spheres through spin
coating with a polymerizable lacquer that is polymerized with UV-light.
Spin coating of compact disks is well established in the art. The assay
disk is expected to have a shelf-life of well over ten years.
Subsequently, the disk can be scanned by a laser reader which will detect,
through reflection, the presence of a microsphere or other reflective
element at the various spatially predetermined locations. Based on the
distance of the microsphere from the axis of rotation of the disk and the
angular distance from an address line forming a radial line on the disk,
the location of a particular metal sphere can be specifically determined.
Based on that specific location and the predetermined locations of
specific binding pairs as compared to a master distribution map, the
identity of the bound material can be identified. Thus, in the foregoing
manner it is possible in one fluid sample to analyze for thousands, or
even greater numbers, of analytes simultaneously.
5.2 Derivatization of Substrate
FIGS. 4A through 4G illustrate schematically one way in which the solid
support substrate is prepared for deposition of cleavable reflective
signal elements to create an assay device of this invention a portion of a
generally planar solid support is illustrated in FIG. 4A. As illustrated
in FIG. 4B, the surface of the support is coated with a resist 22, e.g., a
high melting point wax or the like. Next a pattern of indentations or
holes 25 in the resist is created by stamping with stamp 23 containing
protrusions 24, as illustrated in FIG. 4C. The pattern is highly regular
and indentations are made in all sites at which cleavable spacer molecules
will desirably be located on the surface of the support. Any resist
remaining at the bottom of the indentations, as illustrated in FIG. 4D, is
removed, as shown in FIG. 4E. The exposed areas of the substrate 21, as
illustrated in FIG. 4E, are activated or derivatized to provide for the
attachment of bonding groups (e.g., amino groups) to the surface of the
substrate and to any remaining resist 22, as represented in FIG. 4F.
Finally, the remaining resist is removed to expose the original surface of
the substrate to which amino groups are coupled at certain predetermined
sites as illustrated in FIG. 4G.
Blank disks are available from Disc Manufacturing, Inc. (Wilmington, Del.).
Amino derivatization may be performed by ammonia plasma using a radio
frequency plasma generator (ENI, Rochester, N.Y.).
More generally, when the assay device substrate is plastic, as in many of
the optical disk embodiments of the present invention, the plastic
substrate surface onto which spacers are to be deposited should contain
enough reactive groups, such as amino, thiol, carboxyl, aldehydo, or keto,
to enable the covalent attachment of spacers, biomolecules, and coating
agents. These active groups may be introduced in any of a number of ways
well known in the art, e.g., by mixing of surface active compounds, such
as polyethylene glycol ammonium halogenide, with the plastic polymer
during synthesis of the assay device substrate; by ammonia, oxygen,
halogen or other reactive plasma etching; or by wet chemical reaction,
such as acid or alkaline hydrolysis, nitration and subsequent reduction,
etc. It should be kept in mind that on some occasions, some of the
structures to be applied to the device surface can be attached by van der
Waals and other nonspecific or noncovalent forces.
Other physical and chemical properties of the assay device detection
surface (that is, the solid support substrate to which analyte-specific
signal elements are attached) can be modified, for purposes additional to
facilitating the bonding of signal elements.
For instance, wettability can be adjusted.
Hydrophilicity may be achieved by the amination of the surface, which also
facilitates binding of signal elements, and may also be achieved by
attaching hydrophilic molecules to the device surface. These molecules
include detergents, carbohydrates, oligonucleotides, peptides, proteins,
synthetic polymers, such as polyvinyl alcohol, polylactic acid,
polyethylene glycol, and polyethyleneimine. Similarly, hydrophobic areas
can be created by molecules that contain aliphatic alkyl groups or
perfluorinated alkyl groups. For binding to the solid support substrate,
these molecules can have carboxyl, hydroxyl, amino, carbonyl, or another
group that can be easily coupled with a surface. Coupling can be covalent
or based on weaker bonding, such as van der Waals interaction.
The surface may also be modified to reduce nonspecific binding. One general
method is silylation (Virtanen J. A. et al., "Organosilanes and their
hydrolytic polymers as surface treatment agents for use in chromatography
and electronics," U.S. Pat. No. 4,756,971, incorporated herein by
reference).
Alternatively, it is known that polyethyleneglycol (PEG)-coated particles
have much less interaction with biomolecules than do uncoated particles.
However, direct PEG-coating of the elements that confer analyte
specificity will also significantly reduce specific binding. For this
reason, binding molecules, such as antibodies, may be tethered with PEG
onto supporting surfaces. The PEG serves to prevent nonspecific binding to
the surface; specific binding by the recognition molecules, displayed away
from the surface, is unaffected.
The cleavable spacers of the present invention, the backbone of which
consists, in preferred embodiments, of PEG, are themselves an example of
this principle: the reduction in nonspecific binding, with concomitant
increase in specificity, occasioned by removing the recognition moieties
from the device substrate to a PEG spacer, is a significant advantage of
the present invention, and further argues for adapting existing nucleic
acid detection and immunoassays to the cleavable spacers of the present
invention.
To reduce nonspecific binding of sample components, the assay device
detection surface, and/or other surfaces of the assay device that contact
sample, may also be coated with soluble proteins that do not have any
specific interaction with other proteins or large biomolecules. Examples
of these are albumin, ovalbumin, prionex, avidin, streptavidin, gelatin,
casein, neutral IgG, .alpha.1-acid glycoprotein, and hemocyanin. Thus,
albumin is a very good coating material for all assays, but especially for
the immunoassays.
For nucleic acid assay devices, the surfaces can be made negatively charged
by carboxylate, sulfonate or phosphate groups, to reduce nonspecific
binding. Phosphorylated soluble proteins, such as casein and its
fragments, can be immobilized to provide a negatively-charged surface. To
effect the immobilization, the proteins can first be thiolated, for
example, by 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester
(SPDP) and then attached either on gold or on a plastic surface via thiol
group. Alternatively, proteins can simply be adsorbed on surfaces due to
hydrophobic interaction. Adsorption is best done at the isoelectric point
(for human IgG, pH=7.8) or slightly higher pH of the protein. In order to
mask charges during adsorption, the salt concentration should be at least
100 mM NaCl. Increased temperature and mixing favors adsorption. If the
protein being adsorbed is to function not only to reduce nonspecific
binding, but also for other purposes, such is the case when primary or
auxiliary recognition molecules are adsorbed, too high a temperature is of
course detrimental, as it may lead to denaturation. For similar reasons,
high detergent concentration should be avoided, because they solubilize
proteins. However, for the same reason, detergents are favored during the
assay, because they diminish nonspecific binding. For this reason the
covalent binding of proteins is preferred so that detergents can be used
in the actual assay.
Coating the assay device surface, or portions thereof, with proteins offers
the additional advantage of presenting, via the protein's many functional
groups, further opportunities for coupling molecules to the surface of the
device. Thus, proteins often have several reactive aliphatic amino groups
that are amenable to cross-linking. Similarly, carboxylic or thiol groups
can be further derivatized. The carbohydrates presented by glycoproteins
can be oxidized and the aldehydo groups coupled with amino groups in the
presence of reducing agent. Several other coupling chemistries are well
known in the art. Avidin-biotin or streptavidin-biotin interaction is very
well known and routinely used in immuno- and other assays.
In yet another approach, adsorption or coupling of specific antibodies onto
the assay device signal detection surface allows specific localization of
other molecules onto these sites by using antigen conjugates.
Detergents can be used as surface-modifying agents. In particular,
detergents originally designed and tested for their ability to solubilize
biomolecules may be used. Examples of detergent classes and detergents
that can be used for the surface treatment and solubilization include, but
are not limited to
Anionic Linear alkylbenzene sulfonate
Alkyl sulfates
.alpha.-Olefin sulfonates
Alcohol ether sulfates
Sulfosuccinates
Phosphate esters
Fatty acid salts
Perfluorocarboxylic acid salts
Abietic acid
Cationic Cetyl trimethylammonium bromide
Alkylated pyridium salts
Zwitterionic Alkyl betaine
Neutral Alkyl phenol PEG
Alkyl PEG
Alkanolamides
Glycol and Glycerol esters
Propylene glycol esters
Sorbitan and PEG sorbitan esters
Polydimethylsiloxan PEG
Amphoteric Dodecyl dimethyl amine oxide
Polymeric Polyacrylic acid
Particularly useful are nonionic Tween 20 and Triton X-100.
Other methods for the derivatization of the surface of the assay device
include spreading of liquid-crystals and deposition of Langmuir-Blodgett
(LB) films. LB-films can consist of only one monolayer or hundreds of
layers. The surface layer can be hydrophobic or hydrophilic depending on
the deposition cycle.
5.3 Synthesis of Cleavable Spacers
The two essential features of the cleavable spacers used in the cleavable
signal element embodiments of the present invention are (1) a water
soluble backbone, typically polymeric, and (2) at least one cleavage site.
As noted at several places herein, analyte-specific side members are often
present, but may be unnecessary in some embodiments.
The water soluble backbone typically will consist of a polymer, such as
polyethylene glycol, polylactic acid, polyvinylalcohol, dextran,
oligonucleotide, or polypeptide. The backbone polymer may contain side
groups, such as hydroxyls, amino groups, carboxylates, sulfonates, or
phosphates to increase the solubility, or may include such charged groups
within the backbone itself, as, for example, in the phosphodiester bonds
of an oligonucleotide.
A wide variety of cleavage sites may be used. One common class, set forth
below in Table 2, are sites subject to hydrolytic cleavage.
TABLE 2
Hydrolytically cleavable sites
Hydrolysis pH
Cleavable site Acidic Basic
Alcohols, Ethers
Alkoxymethyl ether 2-4
Bis(2-chloroethoxy)methyl ether 2-6
Tetrahydropyranyl ether 2-6
Tetrahydrothiopyranyl ether 2-4
4-Methoxytetrahydropyranyl 2-6
ether
4-Methoxytetrahydrothiopyranyl 2-6
ether
Tetrahydrofuranyl ether 4-6
Triphenylmethyl ether 2-4
Methoxytriphenylmethyl ether 2-6
Dimethoxytriphenylmethyl ether 2-6
Trimethoxytriphenylmethyl ether 4-6
.alpha.-Naphtyldiphenylmethyl ether 2-4
Trimethylsilyl ether 1-7 7-12
Isopropyldimethylsilyl ether 2-6 12
t-Butyldimethylsilyl ether 2-4 12
Tribenzylsilyl ether 2-4 12
Triisopropylsilyl ether 2-4 12
Alcohols, Esters
Acyl ester 12
.alpha.,.alpha.-Dichloroacyl esters 10-12
.alpha.,.alpha.-Difluoroacyl esters 8.5-11
Phenoxyacetate ester 8.5-11
Benzoyl ester 10-12
Carbonate 10-12
Bis (.alpha.,.alpha.-dichloroalkyl)carbonate 8.5-11
Bis (.alpha.,.alpha.-difluoroalkyl)carbonate 8.5-10
p-Nitrophenyl carbonate 8.5-10
Benzyl carbonate 10-12
p-Nitrobenzyl carbonate 10-12
S-Benzyl thiocarbonate 10-12
2,4-Dinitrophenylsulfenate 1 10-12
ester
1,2- and 1,3-Diols
Ethylidene acetal 1-4
Acetonide 1-4
Benzylidene acetal 2-4
p-Methoxybenzylidene acetal 2-6
Alkoxymethylene acetal 4-6
Alkylmethoxymethylenedioxy 4-6
derivative
Cyclic boronates 1-7 7-12
Phenols and Catechols
Methoxymethyl ether 1-4
Methylthiomethyl ether 1-4
t-Butyl ether 1
t-Butyldimethyl silyl ether 2-6
Aryl alkyl ester 1 10-12
Aryl benzoate 1 10-12
Aryl 9-fluorene carboxylate 10-12
Aryl alkyl carbonate 2-4 10-12
Aryl .alpha.,.alpha.-dichloroalkyl 8.5-11
carbonate
Aryl .alpha.,.alpha.-difluoroalkyl 8.5-10
carbonate
Aryl vinyl carbonate 10-12
Aryl benzyl carbonate 10-12
Acetonide 1-4
Diphenylmethylenedioxy 2-4
derivative
Cyclic borate 1 12
Carbonyl groups
Dimethyl acetal 1
Dimethyl ketal 1
Bis(.alpha.,.alpha.-dichloroalkyl) acetal 1
Bis(.alpha.,.alpha.-dichloroalkyl) ketal 1
Bis(.alpha.,.alpha.-difluoroalkyl) acetal 1
Bis(.alpha.,.alpha.-difluoroalkyl) ketal 1
1,3-Dioxane 1
5-Methylene-1,3-dioxane 1
5,5-Dibromo-1,3-dioxane 1 10-12
1,3-Dioxolane 1-4
4-Bromomethyl-1,3-dioxolane 1-4
4-o-Nitrophenyl-1,3-dioxolane 1-4
1,3-Oxathiolane 2-4
O-Trimethylsilyl cyanohydrin 1-7 7-12
O-Phenylthiomethyl oxime 0-1
Bismethylenedioxy derivatives 0-4
Carboxyl group
Alkoxymethyl ester 1-4
Tetrahydropyranyl ester 2-4 10-12
Benzyloxymethyl ester 1-4 12
Phenacyl ester 10-12
N-Phthalimidomethyl ester 8.5-10
.alpha.,.alpha.-Dichloroalkyl ester 8.5-11
.alpha.,.alpha.-Difluoroalkyl ester 8.5-10
.alpha.-Haloalkyl ester 0-1 10-12
2-(p-Toluenesulfonyl) ethyl 8.5-11
ester
.alpha.,.alpha.-Dimethylalkyl ester 2-4
Cinnamyl ester 1 10-12
Benzyl ester 10-12
Triphenylmethyl ester 2-6 10-12
Bis(o-nitrophenyl)methyl ester 10-12
9-Anthrylmethyl ester 0-1
2-(9,10-Dioxo)anthrylmethyl 10-12
ester
Piperonyl ester 1
t-Butyldimethylsilyl ester 4-6 8.5-10
S-t-Bytyl ester 0-1 13
2-Alkyl-1,3-oxazoline 0-1 13
N-7-Nitroindoylamide 10-12
Alkylhydrazide 0-1
N-Phenylhydrazide 0-1
Thiol group
S-p-Alkoxybenzyl thioether 0-1
S-2-Picolyl N-oxide thioether 0-1
S-Triphenylmethyl thioether 0-1
S-2,4-Dinitrophenyl thioether 7 8.5-10
S-.alpha.-Cyanoalkyl thioether 10-12
S-2-Nitro-1-phenylethyl 8.5-10
thioether
S-Benzoyl thioester 8.5-11
S-Ethyl disulfide 7 8.5-10
Amino groups
2-(.alpha.,.alpha.-Dimethylalkylsilyl)ethyl 1-4
carbamate
.alpha.,.alpha.-Dimethylalkynyl carbamate 1
.alpha.-Methyl-.alpha.-phenylethyl 0-1
carbamate
.alpha.-Methyl-.alpha.-(4-biphenylyl)ethyl 1
carbamate
.alpha., .alpha.(-Dimethyl-.beta.-haloalkyl 0-1
carbamate
.alpha.,.alpha.-Dimethyl-.beta.-cyanoalkyl 8.5-11
carbamate
.alpha.,.alpha.-Dimethylalkyl carbamate 0-4
Cyclobutyl carbamate 0-1
1-Methylcyclobutyl carbamate 1-4
1-Adamantyl carbamate 1-4
Vinyl carbamate 2-6
Allyl carbamate 0-4
Cinnamyl carbamate 0-4
8-Quinolyl carbamate 0-4 12
5-Benzisoxazolylmethyl 0-1
carbamate
Diphenylmethyl carbamate 1-4
S-Benzyl carbamate 12
N-(N'-Phenylaminothiocarbonyl) 0-1 12
derivative
.alpha.,.alpha.-Dichloroacetyl amide 8.5-11
.alpha.,.alpha.-Difluoroacetyl amide 8.5-10
N-Benzoyl amide 1 12
N-Dithiasuccinoyl amide 10-12
The chemical groups set forth in Table 2 are Cleavable, at the indicated pH
ranges, by reagents such as 1 M HCl (pH 1), 0.01 M HCl and 0.01-1 M AcOH
(pH 2-4), 0.1 N H.sub.3 BO.sub.3 and phosphate buffer (pH 4-6), 0.1 N
NaHCO.sub.3 and 0.1 M AcONa (pH 8.5-10), 0.1 N Na.sub.2 CO.sub.3 and
Ca(OH).sub.2 (pH 10-12) and 0.1-1 M NaOH (pH>12).
Table 3 sets forth another class of cleavage sites that will prove useful
in the cleavable signal element embodiments of the present invention.
TABLE 3
Other chemically-cleavable moieties
Type of cleavage Cleavage agent
Oxidative cleavage
Tetrahydrofuranyl ether Organic peracids
Methoxytriphenylmethyl ether Organic peracids
Hydroquinone diether AgNO.sub.3
Allyl carbonate KMnO.sub.4
Alkylmethyl hydrazones H.sub.2 O.sub.2 ; Organic
peracids
S-2,4-Dinitrophenyl thioether Organic peracids
4,5-Diphenyl-3-oxazolin-2-one Organic peracids
S-Benzyl carbamate H.sub.2 O.sub.2 ; Organic
peracids
Boronates H.sub.2 O.sub.2 ; Organic
peracids
Carbon-carbon double bond OsO.sub.4 + HIO.sub.4
1,2-Diol HIO.sub.4
Reductive cleavage
Tetrahydrofuranyl ether NaBH.sub.3 CN
2,4-Dinitrophenylsulfenate NaBH.sub.3 CN
ester
Boronates NaBH.sub.3 CN
Oxygen-oxygen bond Electrochemical
cleavage; NaBH.sub.3 CN
Sulfur-sulfur bond Electrochemical
cleavage; Thiols
Azobenzene Electrochemical
cleavage; NaBH.sub.3 CN;
Zn + HCl
Ferrocene Electrochemical
cleavage
Photochemical cleavage
Dinitrophenyl ether
Ion bond dissociation
Alkyl ammonium carboxylate HCl; Formic acid;
Citric acid; Na.sub.2 CO.sub.3 ;
Polyamines
Calsium di- or polycarboxylate HCl; Formic acid;
EDTA
Hydrogen bond dissociation
Hybridized oligonucleotides Urea; Chaotropic
salts; Heat
Carboxylic dimer pH > 6-7; Carboxylic
acids
Coordination bond dissociation
Histidine-Copper-Histidine Alkyl amines; HCl;
Organic acids
As shown in table 3, a variety of reagents can be used to effect oxidative
cleavage. These include osmium tetroxide, potassium permanganate, silver
nitrate, sodium periodate, peracids, iodine and hydrogenperoxide.
Furthermore, where the assay device substrate, such as an optical disk, is
metal coated, electrochemical oxidation can be used. In this latter case,
the cleavable group is positioned close to the metal surface. At the
completion of incubation of the assay device with the sample, the metal is
used as an anode.
Reductive cleavage can be accomplished chemically by (substituted)
hydroquinone, sodiumcyanoborohydride, zinc, magnesium, or aluminium.
Sodiumcyanoborohydride is often preferred, because it dissolves in water,
has high reduction potential, and is relatively stable in water.
Electrochemical reduction can be used analogously to electrochemical
oxidation.
In some assay geometries, cleavage of the cleavable moiety may itself be
used directly to signal presence of the desired analyte. In these cases,
first and second side members are not required on the cleavable spacer, as
specificity for analyte is conferred directly by the cleavage moiety
itself. For example, a boronate group in the cleavable spacer may be used
directly to signal the presence of hydrogen peroxide. If there is no
hydrogen peroxide present in the sample, the spacers will remain intact.
In the presence of the hydrogen peroxide, the spacers will be cleaved in a
concentration dependent manner.
Because hydrogen peroxide is a side product of many enzymatic reactions,
hydrogen peroxide-cleavable spacers find use in many assay geometries in
which the analyte is the enzyme substrate. As further discussed elsewhere
herein, FIG. 31 demonstrates an assay for ethanol in which hydrogen
peroxide is used to signal ethanol presence.
Although Tables 2 and 3 present the cleavable moieties individually,
several different cleavable groups may usefully be employed in one spacer.
Furthermore, different areas on the assay device can have different
cleavable groups that can be cleaved orthogonally. This allows independent
cleavage of the spacers.
Tables 2 and 3 are exemplary, not exhaustive. The pH ranges and
reactivities given in the tables refer specifically to the case in which
the identified cleavage site or moiety is incorporated within a saturated
aliphatic straight chain compound, for instance, an alkoxymethoxy group
with aliphatic alcohol, such as decanol. The skilled artisan would
understand that cleavage conditions will change predictably with changes
in the backbone structure.
Furthermore, the reactivities can be adjusted, and the range of cleavage
conditions expanded or altered, by addition of chemical moieties that
affect the cleavage site. For example, the reactivity of an ester may be
adjusted using chemical moieties on either its alcohol or carboxylic acid
sides, or both, as shown in FIG. 27.
FIG. 27A shows an aliphatic spacer containing an ester group. On the
alcohol side, between R, indicating further backbone, and the ester
itself, is an n-pthalimidomethyl group. This group renders the ester
readily cleaved. FIG. 27B shows the same spacer, but with an .alpha.,
.alpha. difluoroacid moiety between R', indicating further backbone, and
the ester itself. This acid also renders the ester more readily cleavable.
The n-pthalimidomethyl .alpha.,.alpha.-difluoroalkanoate of FIG. 27C
combines the two. Accordingly, while separately these groups would give
derivatives that are hydrolyzed between pH 8.5-10 (albeit slowly at pH
8.5), the combination will be hydrolyzed rapidly at pH 8.5.
Thus, tens of thousands, if not hundreds of thousands, of combinations that
are useful in the cleavable signal element embodiments of the present
invention can be created from the moieties described in Tables 2 and 3.
It will also be appreciated that the spacers may contain moieties that are
hydrolytically cleavable by enzymes, rather than by inorganic chemical
agents. Table 4 provides a nonexhaustive list of such moieties and their
cleavage enzymes.
TABLE 4
Hydrolytic enzymes and their substrates
Hydrolytic enzyme Substrate
Lipases
Lipase (pancreas) Primary acyl bond in
triglycerides (micelle or
monolayer, pH 8.0, Ca.sup.2+)
Lipase (castor oil) pH 4.7
Lipoprotein lipase
Phospholipases
Phospholipase A.sub.2 sn-2-Acyl bond in
phospholipids (pH 8.9, Ca.sup.2+)
Phospholipase C Bond between glycerol and
phosphate (pH 7.3, Ca.sup.2+)
Phospholipase D
Proteases
Chymotrypsin(ogen) Amides and esters of leucine,
methionine, asparagine,
glutamine, etc.
Clostripain Arginine carbonyl
Collagenase Collagen
(Pro)Elastase Elastin, N-acyl-L-alanine 3-
p-nitroanilide (pH 8.5)
Papain Proteins, amides and esters
(pH 6.5)
Lipases
Pepsin(ogen) Proteins, esters (pH 1.6)
Protease S Aspartic or glutamic moieties
in proteins (pH 6)
Protease K Proteins, amides (pH 9)
Trypsin(ogen) Lysine or arginine moieties
in proteins (pH 8.1, Ca.sup.2+)
Nucleases
DNase I Single chain and double
stranded DNA (pH 5, Mg.sup.2+)
DNase II Single chain and double
stranded DNA (pH 4.6, Mg.sup.2+),
p-nitrophenyl phosphodiesters
(pH 5.7)
Rnase RNA (pH 7.2)
RNase T1 RNA between 3'-guanylic and
adjacent nucleotides (pH 7.5)
Nuclease S1 Single stranded DNA and RNA
(pH 4.6)
Glycosidases
.beta.-Agarase 1,3-linked .beta.-D-
galactopyranose and
1,4-linked 3,6-anhydro-.alpha.-L-
galactopyranose (pH 6.0)
.alpha.-Amylase (pancreas) .alpha.-1,4-linked D-glucose units
(pH 6.8)
.alpha.-Amylase (malt) .alpha.-1,4-Linked D-glucose units
(pH 4.9)
Lipases
.beta.-Amylase (pancreas) .alpha.-1,4-Linked D-glucose units
(pH 4.8)
Cellulase .beta.-1,4-Linked D-glucose units
(pH 5.0)
Dextranase 1,6-.alpha.-glucosidic linkages (pH
6, optional activators Co.sup.2+,
Cu.sup.2+, Mn.sup.2-)
.beta.-Galctosidase .beta.-D-Glactosides (pH 7.5,
Mg.sup.2+)
Mannosidase
.alpha.-Glucosidase .alpha.-D-Glucosides (pH 6.7)
.beta.-Glucosidase .beta.-D-Glucosides (pH 5.0)
.beta.-Glucuronidase Glucuronides (pH 4.8)
Hyaluronidase 1,4-linkages between
2-acetamido-2-deocy-.beta.-D-
glucose and D-glucose
moieties (pH 5.3)
Lysozyme .beta.-1,4 bond between N-acetyl
muramic acid and N-
acetylglucosamine (pH 7.0)
Neuraminidase Sialoyl glycoproteins (pH
5.0)
Esterases
Cholesterol esterase Sterol esters (pH 6.8,
cholate)
Enzymes can be used as a cleavage reagents by incorporating into the spacer
a moiety that serves as the substrate for the given enzyme. For instance,
a spacer can contain a single-stranded oligonucleotide segment, a suitable
substrate for S1 nuclease. After incubation of an assay device containing
such cleavable spacers with sample, S1 nuclease is added under conditions
optimal to cleavage of single-stranded nucleic acid, thus cleaving the
cleavable spacers.
If, in such circumstances, the cleavable spacer side members are also
oligonucleotides, they too may be cleaved if not rendered double-stranded
by contact with fully complementary nucleic acids in the sample itself.
For cleavage of spacers containing, as the cleavable moiety, the substrate
for an enzyme, zymogens or proenzymes can be used instead of the active
enzyme itself. Such zymogens or proenzymes may be covalently bound with
the spacers or onto the assay device surface. After incubation with
sample, an activator is added that activates the zymogen or the proenzyme,
which then rapidly cleaves the cleavable spacer. Alternatively, active
enzymes can be coupled with the spacer or the substrate in the presence of
a reversible inhibitor. During the assay the inhibitor is washed away and
the spacer will be cleaved.
In yet another alternative, the cleavable spacer may be used directly to
detect enzymes in a sample. In this geometry, both the cleavage agent and
analyte-specific side members are unnecessary: enzyme that is present in
the sample will cleave all spacers that contain the enzyme's substrate.
Optionally, the local concentration of enzyme may be increased near the
spacer to facilitate cleavage: this may be done by disposing, adjacent to
the relevant spacers, a structure that recognizes the desired enzyme, such
as an antibody. The recognition molecule so positioned must not, of
course, interfere with the enzymatic activity of the analyte.
Taking into account all possible variations in the spacer backbone and in
the cleavable group, millions of different spacers can be designed and
prepared according this invention. Such preparation is within the skill in
the art.
FIGS. 5 and 6 present a representative cleavable spacer molecule with a
siloxane cleavage site. Most of the spacer, termed the backbone, is
poly(alkyleneglycol), e.g., polyethyleneglycol, having a molecular weight
of 400-10,000, preferably 400-2000. Making reference to the nomenclature
in FIG. 1, the backbone of the spacer has a first end 31 that is adapted
to couple to a derivatized amine group present on surface 21 of substrate
20, and a second end 32, which is adapted to couple with surface 41 of
metal microsphere 40 via a thio-linkage 51. The backbone includes a
cleavage site 33 between the first end 31 and the second end 32 of spacer
molecule 30. In addition, between end 31 and cleavage site 33 is a side
member 34a, commonly constructed from an oligonucleotide, and between
cleavage site 33 and end 32 is another side member 34b commonly
constructed from an oligonucleotide. Alternatively, such side members may
be peptides or other organic molecules. More than two side members can be
provided, but it is only necessary that two members are capable of forming
a connective, molecular loop around the cleavage site to bind the spacer
molecule to the surface of the substrate after cleavage at the cleavage
site. These side members can be attached to the spacer backbone by
linkers, such as polyethylene glycol.
One mode of synthesis of the representative cleavable spacer molecule 30
illustrated in FIG. 5 is substantially and generally as follows.
Chlorodimethylsilane is coupled unto both ends of a polyethyleneglycol
molecule. The silane group incorporated into the molecule reacts in the
presence of catalytic amounts of chloroplatinic acid within N-acryloyl
serine. The hydroxyl groups of both serine moieties are to be used later
in the synthesis for the construction of oligonucleotide side members. One
hydroxyl group is first protected by a monomethoxytriphenylmethyl group
and the product is purified by liquid chromatography. The other hydroxyl
group is then protected with a pivaloyl or fluorenylmethyloxycarbonyl
(FMOC) group. The serine carboxyl groups are coupled with amino terminated
poly(ethyleneglycol). The amino group at the other end is further
derivatized by 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide
ester. The other amino group is not reacted but is free to react later
with the derivatized substrate.
An alternative, but substantially similar, and more detailed description of
the spacer molecule synthesis, is provided below in Example I.
Spontaneous hydrolysis of siloxane can be made slower by substituting one
or more methylgroups with i-propyl or t-butyl groups. Several functional
groups can be used to attach spacer side-elements. These include, but are
not limited to: amino, thiol, aldehydo, keto, carboxylic, maleimido, and
.alpha.-halogenoketo groups. Many of these must be protected during
synthesis and fabrication by techniques well known in the art.
5.4 Attachment of Cleavable Spacers and Auxiliary Recognition Molecules to
Substrate
Each of the spacer molecules is attached at one end 31 to support surface
21, e.g. via an amide linkage. In order to attach the spacer molecules to
the amino-activated substrate, glutaric anhydride is reacted with the
amino groups to expose a carboxylate group, shown more particularly in
FIGS. 7A and 7B. The carboxylate groups can be esterified with
pentafluorophenol. The free amino group on the spacer molecule will couple
with this active ester. The spacer molecules and their attachment at the
discrete sites to the solid support surface 21 are shown particularly in
FIG. 7C. At this stage in the fabrication, the hydroxyl groups remain
protected. While the oligonucleotide side members could be pre-synthesized
on the spacers prior to the attachment to the solid surface support 21, it
is preferable that they be attached after the spacer molecule 30 is
attached on the solid support.
The chemistry described above for coupling a spacer to the assay device
substrate is but one example of the chemistries that may usefully be
employed; there are innumerable modifications that would be within the
skill in the art. Virtually any reaction that can serve unidirectionally
to bond the spacer to the solid support substrate of the assay device can
be used. The substrate surface may itself be chemically active, or it can
be activated or made otherwise amenable for coupling chemistry by adsorbed
molecules or particles, as is well known in the art.
Although the coupling of signal elements to the solid-support substrate of
an assay device, especially the coupling of cleavable spacers, is
particularly described, it should be recognized that other molecules may
additionally be attached to the substrate surface to facilitate particular
assays.
As mentioned above, for example, auxiliary recognition molecules may be
disposed on the assay device in proximity to the signal elements, such as
cleavable signal elements, in order to increase the Local concentration of
analyte. The coupling chemistries are identical to those used to attach
the spacer to these surfaces.
As would be recognized, any such disposition of auxiliary recognition
molecules on the solid support substrate of the assay device must be done
with attention to the location and concentration of analyte-specific
signal elements. Generally, less than 20% of the surface of an assay
device will be covered by the spheres. Were the auxiliary recognition
molecules attached in a uniform density across the surface of the device,
almost 80% of the recognition molecules on the substrate would be useless.
In fact, such molecules would, by capturing analyte in locations where
recognition cannot be signalled, would interfere with detection. The
latter problem can be alleviated by patterning the surface as is described
separately.
Auxiliary recognition molecules may also be attached, for analogous
purposes, to the surface of the signal responsive moiety of the spacer. As
with attachment of such auxiliary recognition molecules to the solid
support substrate of the assay disk, attention must be paid to the spatial
pattern in which these molecules are disposed. In the case in which the
signal responsive moiety is a gold sphere, for example, attachment of
auxiliary recognition molecules on the surface distal to the attachment to
the spacer would sequester recognized analyte away from the
analyte-specific side members of the spacer.
To avoid unnecessary coverage on the spheres, plastic spheres may be used
that are partially coated with gold. The auxiliary recognition molecules
may be attached to the gold-coated surface using dative bonding of thiols,
compelling the attachment of the auxiliary recognition molecules proximal
to the attachment of the spacers themselves. Alternatively, these
auxiliary recognition molecules can be attached to the uncoated plastic
surface using several coupling chemistries, such as amino-carboxylate,
amino-iodoacetyl, or biotin-avidin. In any case, the spacers and
recognition molecules will be attached onto the same hemisphere as is
desirable.
Yet another alternative method for attaching auxiliary recognition
molecules allows the random patterning of the substrate and use of
symmetrical signal responsive moieties, such as uniform microspheres, yet
avoids disposing the auxiliary recognition molecules so as to frustrate
productive binding of analyte. In this latter method, the auxiliary
recognition molecules are attached to the substrate and/or signal
responsive moieties with a photocleavable spacer. For example, the
recognition molecule's spacer may contain a dinitrophenyl ether grouping.
In this method, the entire solid support substrate and all signal
responsive moieties are randomly coated, either in one step or more, with
photo-cleavable auxiliary recognition molecules. Next the surface of the
assay device is illuminated by UV-light in such orientation that the
photoreactive spacers will be cleaved in places except beneath the
spheres. There is no need for a complete cleavage. The purpose is only
substantially to reduce the number of spacers in open areas that are not
useful for the assay.
As further described below, the assay device substrate may be adapted to
function as an optical waveguide in embodiments suitable for continuous
monitoring. For such embodiments, plastic is presently preferred as a
device substrate, with polycarbonate most preferred, but glass may also be
used. If glass is used as substrate, signal elements may be attached as
follows. The glass surface is first activated, i.e., silicon oxygen bonds
are hydrolyzed by hot hydrochloric acid. Three building blocks are needed
to create the spacer molecules directly on the surface of a glass
waveguide substrate. First is 11-(chlorodimethylsilyl) undecanoic acid
methylester that is coupled directly onto the surface by silicon oxygen
bond. The methyl ester is hydrolyzed by a dilute base after the coupling
to release the carboxylic group. Second is diamino polyethylene glycol
(DAPEG) that is connected with the free carboxylic group on the surface by
forming an amide bond. The excess of DAPEG will be washed away, and the
free amino group will be allowed to react with 3(2-pyridyldithio)propionic
acid N-hydroxysuccinimide ester ("SPDP") which is the third building
block. Before attachment of the gold spheres the dithio group will be
reduced with dithiothreitol. SPDP is commercially available. The length of
DAPEG can be varied between 10 nm and 1000 nm.
5.5 Design and Attachment of Signal Responsive Moieties
One feature of the current invention is the detection of analyte-specific
signals from analyte-specific signal elements disposed in a
spatially-addressable fashion on an assay device substrate. In preferred
embodiments, the signal elements are cleavable and the substrate is an
optical disk. Accordingly, this invention provides methods, compositions
and devices for attaching signal responsive moieties to spacer molecules,
particularly cleavable spacer molecules, disposed in predetermined,
spatially-addressable patterns on the surface of the assay device.
5.5.1 Gold Particles as Signal Responsive Moieties
In some preferred embodiments of the present invention, particles that
reflect or scatter light are used as signal responsive moieties. a light
reflecting and/or scattering particle is a molecule or a material that
causes incident light to be reflected or scattered elastically, i.e.,
substantially without absorbing the light energy. Such light reflecting
and/or scattering particles include, for example, metal particles,
colloidal metal such as colloidal gold, colloidal non-metal labels such as
colloidal selenium, dyed plastic particles made of latex, polystyrene,
polymethylacrylate, polycarbonate or similar materials.
The size of such particles ranges from 1nm to 10 .mu.m, preferably from 500
nm to 5 .mu.m, and most preferably from 1 to 3 .mu.m. The larger the
particle, the greater the light scattering effect. As this will be true of
both bound and bulk solution particles, however, background may also
increase with particle size used for scatter signals.
Metal microspheres 1 nm to 10 .mu.m (micrometers) in diameter, preferably
0.5-5 .mu.m, most preferably 1-3.mu.m in diameter, are presently preferred
in the light reflecting/light scattering embodiment of the present
invention. Metal spheres provide a convenient signal responsive moiety for
detection of the presence of a cleaved, yet analyte-restrained, spacer
molecule bound to the disk. Typical materials are gold, silver, nickel,
chromium, platinum, copper, and the like, or alloys thereof, with gold
being presently preferred. The metal spheres may be solid metal or may be
formed of plastic, or glass beads or the like, upon which a coating of
metal has been deposited. Similarly, the light-reflective metal surface
may be deposited on a metal microsphere of different composition. Metal
spheres may also be alloys or aggregates.
Gold spheres suitable for use in the cleavable reflective signal element
and assay device of the present invention are readily available in varying
diameters from Aldrich Chemical Company, British BioCell International,
Nanoprobes, Inc., and others, ranging from 1 .mu.m to and including 0.5
.mu.m (500 nm)-5 .mu.m in diameter. It is within the skill in the art to
create gold spheres of lesser or greater diameter as needed in the present
invention.
Much smaller spheres can be used advantageously when reading is performed
with near field optical microscopy, UV-light, electron beam or scanning
probe microscopy. Smaller spheres are preferred in these latter
embodiments because more cleavable spacers can be discriminated in a given
area of a substrate.
Although spherical particles are presently preferred, nonspherical
particles are also useful for some embodiments.
In biological applications, the signal responsive moiety--particularly gold
or latex microspheres--will preferably be coated with detergents or
derivatized so that they have a surface charge. This is done to prevent
the attachment of these particles nonspecifically with surfaces or with
each other.
The presently preferred gold spheres bind directly to the thio group of the
signal responsive end of the cleavable spacer, yielding a very strong
bond.
After the oligonucleotide side arm synthesis is completed, as further
described below, the pyridyldithio group present at the signal-responsive
end of the spacer molecule 30 is reduced with dithioerythritol or the
like. The reaction is very fast and quantitative, and the resulting
reduced thio groups have a high affinity for gold. Thiol groups bind gold
virtually irreversibly; the gold-sulfur bonding energy is 160 kJ/mole.
Halo groups similarly have high affinity for gold. Accordingly, gold
spheres are spread as a suspension in a liquid (e.g., distilled water) by
adding the suspension to the surface of the solid support 21. The gold
spheres will attach only to the sites covered by thio terminated spacers
and will not attach to the remaining surface of the substrate.
Furthermore, while the above embodiments of the invention have been
described with a single metal sphere attached to the signal-responsive end
of a single cleavable spacer, it should be appreciated that when gold is
used in a preferred embodiment of the invention, thousands of spacers may
bind one gold sphere, depending upon its diameter. It is estimated that
one sphere of 1-3 .mu.m may be bound by approximately 1,000-10,000
cleavable spacers.
As a result, the stringency of the assay wash may be adjusted, at any given
rotational speed, by varying not only the diameter of the gold sphere, but
also the relative density of cleavable spacers to gold spheres.
Accordingly, if virtually all spacers under a certain gold sphere are
connected by complementary molecules, the binding is very strong. If the
spacers are fixated only partially under a certain gold sphere, the sphere
may remain or be removed depending on the radius of the sphere and the
frequency of the rotation.
5.5.2 Other Light-Responsive Signal Responsive Moieties
In some other embodiments of the cleavable signal element and assay device
of the present invention, a light-absorbing rather than light-reflective
material can be used as a signal responsive moiety. In this embodiment,
the absence of reflected light from an addressed location, rather than its
presence, indicates the capture of analyte. The approach is analogous to,
albeit somewhat different from, that used in recordable compact disks.
Although similar in concept and compatible with CD readers, information is
recorded differently in a recordable compact disk (CD-R) as compared to
the encoding of information via pits in a standard, pressed, CD. In CD-R,
the data layer is separate from the polycarbonate substrate. The
polycarbonate substrate instead has impressed upon it a continuous spiral
groove as a reference alignment guide for the incident laser. An organic
dye is used to form the data layer. Although cyanine was the first
material used for these discs, a metal-stabilized cyanine compound is
generally used instead of "raw" cyanine. An alternative material is
phthalocyanine. One such metallophthalocyanine compound is described in
U.S. Pat. No. 5,580,696.
In CD-R, the organic dye layer is sandwiched between the polycarbonate
substrate and the metalized reflective layer, usually 24 carat gold, but
alternatively silver, of the media. Information is recorded by a recording
laser of appropriate preselected wavelength that selectively melts "pits"
into the dye layer--rather than burning holes in the dye, it simply melts
it slightly, causing it to become non-translucent so that the reading
laser beam is refracted rather than reflected back to the reader's
sensors, as by a physical pit in the standard pressed CD. As in a standard
CD, a lacquer coating protects the information-bearing layers.
a greater number of light-absorbing dyes may be used in this embodiment of
the present invention than may be used in CD-R. Light absorbing dyes are
any compounds that absorb energy from the electromagnetic spectrum,
ideally at wavelength(s) that correspond the to the wavelength(s) of the
light source. As is known in the art, dyes generally consist of conjugated
heterocyclic structures, exemplified by the following classes of dyes: azo
dyes, diazo dyes, triazine dyes, food colorings or biological stains.
Specific dyes include: Coomasie Brilliant Blue R-250 Dye (Biorad Labs,
Richmond, Calif.); Reactive Red 2 (Sigma Chemical Company, St. Louis,
Mo.), bromophenol blue (Sigma); xylene cyanol (Sigma); and phenolphthalein
(Sigma). The Sigma-Aldrich Handbook of Stains, Dyes and Indicators by
Floyd J. Green, published by Aldrich Chemical Company, Inc., (Milwaukee,
Wis.) provides a wealth of data for other dyes. With these data, dyes with
the appropriate light absorption properties can be selected to coincide
with the wavelengths emitted by the light source.
In these embodiments, opaque dye-containing particles, rather than
reflective particles, may be used as a light-responsive signal moiety,
thereby reversing the phase of encoded information. The latex spheres may
vary from 1-100 .mu.m in diameter, preferably 10-90 .mu.m in diameter, and
are most preferably 10-50 .mu.m in diameter. The dye will prevent
reflection of laser light from the metallic layer of the disk substrate.
In yet other embodiments, the signal responsive element may be a
fluorescer, that is, an agent capable of fluorescing, such as fluorescein,
propidium iodide, phycoerythrin, allophycocyanin, Cy-Dyes.RTM., or may be
a chemiluminescer, such as luciferin, high responds to incident light, or
an indicator enzyme that cleaves soluble fluorescent substrates into
insoluble form. Other fluorescent dyes useful in this embodiment include
texas red, rhodamine, green fluorescent protein, and the like. Fluorescent
dyes will prove particularly useful when blue lasers become widely
available.
Direct fluorescence and luminescence measurements can be performed using
detectors and techniques known in the art.
The cleavable spacer embodiments of the present invention permit, inter
alia, fluorescer-quencher and donor fluorescer-acceptor fluorescer pairs.
If these are bound together by the analyte, no fluorescence is observed in
the former case, while acceptor fluorescence is observed in the second
case.
In one possible luminescence approach, an enzyme, such as luciferase, is
bound to a first side member of the spacer or is bound directly to the
assay device substrate in proximity thereto. Luciferin, the enzyme
substrate, is attached to a second side member of the spacer, or is
sequestered, as in a liposome. If there is no binding of biomolecules, the
substrate is removed (alternatively the enzyme). In the case of the
binding, a strong luminescence is observed after the suitable chemicals,
such as ATP and lysing or pore forming agents, have been added.
Dye deposition may also be used, for detection spectrophotometrically. In
these approaches, almost any water insoluble dye can be rendered soluble
by attaching polar groups, such as phosphate or glucose. The solubilizing
groups can be hydrolyzed enzymatically and the corresponding dye
deposited.
The light-reflective, light-scattering, and light-absorptive embodiments of
the current invention preferentially employ a circular assay device as the
substrate for the patterned deposition of cleavable signal elements. In an
especially preferred embodiment, the assay device is compatible with
existing optical disk readers, such as a compact disk (CD) reader or a
digital video disk (DVD) reader, and is therefore preferentially a disk of
about 120 mm in diameter and about 1.2 mm in thickness. By disk is also
intended an annulus.
It will be appreciated, however, that the cleavable reflective signal
elements of the present invention may be deposited in spatially
addressable patterns on substrates that are not circular and essentially
planar, and that such assay devices are necessarily read with detectors
suitably adapted to the substrate's shape.
The maximum number of cleavable signal elements, or biobits, that can be
spatially discriminated on a optical disk is a function of the wavelength
and the numerical aperture of the objective lens. One known way to
increase memory capacity in all sorts of optical memory disks, such as
CD-ROMs, WORM (Write Once Read Many) disks, and magneto-optical disks, is
to decrease the wavelength of the light emitted by the diode laser which
illuminates the data tracks of the optical memory disk. Smaller wavelength
permits discrimination of smaller data spots on the disk, that is, higher
resolution, and thus enhanced data densities. Current CD-ROMs employ a
laser with wavelength of 780 nanometers (nm). Current DVD readers employ a
laser with wavelength between 635 and 650 nm. New diode lasers which emit,
for example, blue light (around 481 nm) would increase the number of
signal elements that could be spatially addressed on a single assay device
disk of the present invention. Another way to achieve blue radiation is by
frequency doubling of infrared laser by non-linear optical material.
Current CD-ROM readers employ both reflection reading and transmission
reading. Both data access methods are compatible with the current
invention. Gold particles are especially suitable for use as a signal
responsive moiety for reflection type CD-ROM readers. Light absorbing dyes
are more suitable for transmission type readers such as the ones discussed
in U.S. Pat. No. 4,037,257.
5.5.3 Other Signal Responsive Moieties
It will be apparent to those skilled in the art that signal responsive
moieties suitable for adaptation to the cleavable spacer of the present
invention are not limited to light-reflecting or light-absorbing metal
particles or dyes. Suitable signal responsive moieties include, but are
not limited to, any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. In some preferred embodiments, suitable signal responsive
moieties include calorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads,
biotin for staining with labeled streptavidin conjugate, magnetic beads
(e.g., Dynabeads.TM.), radiolabels (e.g., .sup.3 H, .sup.125 I, .sup.35 S
.sup.14 C, or .sup.32 p), and enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA).
It will be apparent to those skilled in the art that numerous variations of
signal responsive moieties may be adapted to the cleavable spacers of the
present invention. a number of patents, for example, provide an extensive
teaching of a variety of techniques for producing detectible signals in
biological assays. Such signal responsive moieties are generally suitable
for use in some embodiments of the current inventions. As a non-limiting
illustration, the following is a list of U.S. patents teach the several
signal responsive moieties suitable for some embodiments of the current
invention: U.S. Pat. Nos. 3,646,346, radioactive signal generating means;
U.S. Pat. Nos. 3,654,090, 3,791,932 and U.S. Pat. No. 3,817,838,
enzyme-linked signal generating means; U.S. Pat. No. 3,996,345,
fluorescer-quencher related signal generating means; U.S. Pat. No.
4,062,733, fluorescer or enzyme signal generating means; U.S. Pat. No.
4,104,029, chemiluminescent signal generating means; U.S. Pat. No.
4,160,645, non-enzymatic catalyst generating means; U.S. Pat. No.
4,233,402, enzyme pair signal generating means; U.S. Pat. No. 4,287,300,
enzyme anionic charge label. All above-cited U.S. patents are incorporated
herein by reference for all purposes.
Other signal generating means are also known in the art, for example, U.S.
Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein by reference
for all purposes. a metal chelate complex may be employed to attach signal
generating means to the cleavable spacer molecules or to an antibody
attached as a side member to the spacer molecule. Methods using an organic
chelating agent such a DTPA attached to the antibody was disclosed in U.S.
Pat. No. 4,472,509, incorporated herein by reference for all purposes.
In yet other embodiments, magnetic spheres may be used in place of
reflective spheres and may be oriented by treating the disk with a
magnetic field that is of sufficient strength. Since the empty sites will
not have any magnetic material present, the location of the spacer
molecules remaining can be detected and the information processed to
identify the materials in the test sample. Additionally, reflective or
magnetic material can be added after hybridization of the sample to
provide the signal generating means.
Paramagnetic ions might be used as a signal generating means, for example,
ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt
(II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),
holmium (III) and erbium (III), with gadolinium being particularly
preferred. Ions useful in other contexts, such as X-ray imaging, include
but are not limited to lanthanum (III), gold (III), lead (II), and
especially bismuth (III).
Means of detecting such labels are well known to those of skill in the art.
Thus, for example, radiolabels may be detected using photographic film or
scintillation counters, fluorescent markers may be detected using a
photodetector to detect emitted light. Enzymatic labels are typically
detected by providing the enzyme with a substrate and detecting the
reaction product produced by the action of the enzyme on the substrate,
and calorimetric labels are detected by simply visualizing the colored
label. Colloidal gold label can be detected by measuring scattered light.
A preferred non-reflective signal generating means is biotin, which may be
detected using an avidin or streptavidin compound. The use of such labels
is well known to those of skill in the art and is described, for example,
in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149 and 4,366,241; each incorporated herein by reference for all
purposes.
5.6 Attachment of the Cleavable Spacer Side Members
The side members of the cleavable spacers confer analyte specificity. In a
preferred embodiment, the side members are oligonucleotides.
The oligonucleotides can be added by stepwise synthesis on the cleavable
spacers prior to attachment of the spacers to the derivatized substrate of
the assay device (disk). Alternatively, fully prepared oligonucleotides
may be attached in single step directly to the spacer molecules prior to
the spacer molecule's attachment to the assay device substrate. In such
circumstances, the spacer molecule has protected amino- and/or thiol
groups instead of two protected hydroxyl groups. One protective group is
removed and an oligonucleotide that has, for example, an isocyanate group
at one end is added. a second oligonucleotide is similarly attached as a
second side member to the cleavable spacer molecule.
Alternatively, side member oligonucleotides can be synthesized after the
attachment of the cleavable spacers onto the substrate, either in a single
step using fully prepared oligonucleotides or by stepwise addition. The
latter alternative is expected to be preferred when incorporating a large
number of assays with different analyte specificity on a single assay
device substrate. The general process by which the side members are
attached to cleavable spacers previously immobilized on the substrate,
whether in a single step or by stepwise addition, is herein termed
stamping.
Phosphoramidite chemistry is preferred for preparing the oligonucleotide
side members, although other chemistries can be used. In conventional
solid phase synthesis, oligonucleotides are prepared by using monomeric
phosphoramidites. After conventional synthesis, the oligonucleotides are
then detached from he resinous support and purified by a liquid
chromatograph to remove reactants, including solvents and unreacted
mononucleotides, and to remove shorter oligonucleotides that result from
incomplete synthesis. In certain instances the oligonucleotides cannot be
so purified, and shorter oligonucleotides contaminate the desired
oligonucleotide. This leads to unwanted hybridization. The oligonucleotide
contaminants missing only one nucleotide relative to the desired product
are the most difficult to deal with, because their binding is almost equal
in strength to that of the oligonucleotide having the correct sequence.
In the preparation of oligonucleotides for use as side members in the
cleavable reflective signal elements of the present invention, use of
trimeric or tetrameric phosphoramidites in the synthesis is advantageous
and preferred. Using tetrameric starting materials, for example, 12-mers
can be synthesized in three steps. Unavoidable products of incomplete
synthesis will in this instance be 8-mers and 4-mers, representing failure
of 1 or 2 synthesis steps, respectively. Since the binding of 8-mers is
much weaker than the binding of 12-mers, these contaminants do not cause
any significant interference.
In applying side members to cleavable spacers by the stepwise addition to
spacers immobilized on the surface of the assay device substrate, the
oligonucleotides may advantageously be attached to the cleavable spacers
by chemical printing, which utilizes the formation of the desired
oligonucleotide chemical solution on a printed stamp that is complementary
to the spacer molecule distribution on the solid support. Printing is
rapid and economical. It can also provide very high resolution. a simple
printing method is described, for example, in Science, Vol. 269, pgs.
664-665 (1995).
In this printing method, one of the protecting groups is removed from the
spacer molecule on the assay device substrate. The desired
oligonucleotides are applied to the stamp surface in a manner that will
provide specific oligonucleotides at specific, predetermined locations on
the stamp, and the stamp surface is then applied to the spacer-covered
substrate support surface, thereby depositing the desired oligonucleotides
in the discrete areas in which the spacer molecules reside. Subsequently,
the second protecting group is removed and a different oligonucleotide is
applied to the activated area, again by chemical stamping. Those steps are
illustrated particularly in FIGS. 8A, 8B, 9A, 9B, 13 and 14.
Alternatively, the respective oligonucleotides can be applied by ink-jet
printing, such as by methods described in U.S. Pat. Nos. 4,877,745 and
5,429,807, the disclosures of which are hereby incorporated by reference.
Either of these direct printing methods is rapid. When trimers or tetramers
are used to build oligonucleotides, two printing cycles allows one to
create an array of all possible oligos from 6-mers to 8-mers. To contain
all 8-mers, the assay device must contain 256.times.256 different oligos.
Additional printing cycles increase the length of oligonucleotides
rapidly, although all combinations may not fit onto reasonably sized
surfaces and several assay devices may have to be used to represent all
such combinations.
An alternative printing process useful in the present invention, concave
complementary printing, is shown in FIG. 15. Although only two steps are
shown, very large numbers of oligonucleotides can be printed at the same
time. a mixture of oligonucleotides is synthesized; for example, 12-mers
can be synthesized using a mixture of four phosphoramidites in each step,
and as a last step of the synthesis, a very long spacer is attached to
each oligonucleotide. On the other end a reactive group, such as an
isothiocyanate, is provided. The mixture of oligonucleotides is incubated
with the stamp that will bind complementary oligonucleotides at defined
sites. During the printing process the spacer will attach with the
substrate. The double helices are denatured, for example by heating, and
the stamp and substrate can be separated.
Many other methods for the synthesis of oligonucleotides, and in
particular, for spatially addressable synthesis of oligonucleotides on
solid surfaces, have been developed and are known by those skilled in the
art. Methods that prove particularly useful in the present invention are
further described in U.S. Pat. Nos. 4,542,102; 5,384,261; 5,405,783;
5,412,087; 5,445,934; 5,489, 678; 5,510,270; 5,424,186; 6,624,711; the
disclosures of which are incorporated herein by reference.
Other methods that may prove useful in the present invention generally
include: (1) Stepwise photochemical synthesis, (2) Stepwise jetchemical
synthesis and (3) Fixation of pre-prepared oligonucleotides. Also a glass
capillary array system can be used. In this latter case the synthesis can
be performed parallel in all capillaries as is done in an automated DNA
synthesizer.
Although the oligonucleotide side members have been described herein as DNA
oligonucleotides synthesized using standard deoxyribonucleotide
phosphoramidites, it is known that certain oligonucleotide analogs, such
as pyranosyl-RNA (E. Szathmary, Nature 387:662-663 (1997)) and peptide
nucleic acids, form stronger duplexes with higher fidelity than natural
oligonucleotides. Accordingly, these artificial analogs may be used in the
construction of oligonucleotide side members.
While the oligonucleotide side members are adapted to bind to complementary
oligonucleotides, and are thus useful directly in a nucleic acid probe
assay, it is a further aspect of the invention to conjugate to these
oligonucleotide side members specific binding pair members with utility in
other assays.
In these latter embodiments, the noncovalent attachment of binding pair
members, such as antibodies, to side member oligonucleotides is mediated
through complementarity of side member oligonucleotides and
oligonucleotides that are covalently attached to the binding pair member.
Use of complementary nucleic acid molecules to effectuate noncovalent,
combinatorial assembly of supramolecular structures is described in
further detail in co-owned and copending U.S. patent applications Ser. No.
08/332,514, filed Oct. 31, 1994, 08/424,874, filed Apr. 19, 1995, and
08/627,695, filed Mar. 29, 1996, incorporated herein by reference.
As schematized in FIGS. 3A through 3C, oligonucleotide side members 34a,
34b, 35a, and 35b are coupled noncovalently to modified antibodies 38a,
38b, 38c, and 38d to permit an immunoassay. The noncovalent attachment of
modified antibodies to side members is mediated through complementarity of
side member oligonucleotides and oligonucleotides that are covalently
attached to the antibodies.
Although antibodies are exemplified in FIG. 3, it will be appreciated that
antibody fragments and derivatives such as Fab fragments, single chain
antibodies, chimeric antibodies and the like will also prove useful. In
general, binding pair members useful in this embodiment will generally be
first members of first and second specific binding pairs, exemplified by
antibodies, receptors, etc. that will bind respectively to antigens,
ligands, etc.
5.7 Patterned Deposition Of Cleavable Reflective Signal Elements On The
Assay Device
It will be appreciated from the discussion above that the spatial
distribution of analyte-responsive cleavable reflective signal elements on
the assay device (disk substrate) may be determined at two levels: at the
level of attaching the cleavable spacer itself, and additionally at the
level of attaching the spacer side members. It will be further appreciated
that the spatial distribution of analyte sensitivity may also be
determined by a combination of the two.
One method for controlling the distribution of cleavable spacers in the
first such step is through patterning the substrate with hydrophilic and
hydrophobic domains. At first the hydrophobic surfaces are activated and
the hydrophilic surfaces are deactivated so that a hydrophilic and
functional spot array separated by a hydrophobic unreactive network is
created. If the substrate material is glass, mica, silicon, hydrophilic
plastic or analogous material, the whole surface is first rendered
reactive by treatment with acid or base. The intermediate space between
spots is silanized. This is best performed by using a grid as a stamp. If
on the other hand the substrate is a hydrophobic plastic, it can be
activated by plasma treatment in the presence of ammonia and then
silanized as a hydrophilic substrate. Using resist material in conjunction
with lithographic or mechanical printing to remove the resist at desired
sites, activation can be performed at those sites.
Onto the reactive spots is preferably attached a hydrophilic spacer such as
polyethyleneglycol (PEG). If the substrate contains an amino or a thiol
group, PEG can be preactivated in the other end with a variety of
functional groups, which are known to couple with an amino or thiol group.
These include isocyanate, maleimide, halogenoacetyl and succinimidoester
groups.
A photoresist may also profitably be used to pattern the deposition of
cleavable signal elements. The resist is partially depolymerized by
incident laser light during fabrication and can be dissolved from these
areas. The exposed plastic or metalized plastic is treated chemically, for
example, aminated by ammonia plasma. After the resist is removed, the
spacer, side members, and signaling moiety are connected into the treated
area as needed. The use of photoresists for the patterning of master disks
is well known in the compact disk fabrication arts.
Alternatively, instead of using a resist, a solid mask containing small
holes and other necessary features can be used during ammonia plasma
treatment. Holes have a diameter of about 1 to 3 micrometers. The holes
are located circularly in the mask, forming a spiral track or a pattern
that is a combination of spiral and circular paths. The mask can be metal
or plastic. Several metals, such as aluminum, nickel or gold can be used.
Polycarbonate is a preferred plastic, because it will retain shape well.
Plastics are reactive with the ammonia plasma, however, and a preferred
method for using plastic masks therefore involves depositing a metal layer
on the plastic, by evaporation, sputtering, or other methods known in the
art. Holes may be made in the mask by laser. Those with skill in the art
will appreciate that it is possible to create 1000 1 .mu.m-sized holes in
one second in a thin metal or plastic plate. Alternatively, the holes can
be etched by using conventional methods known in the semiconductor
industry. In the mask approach to patterning the deposition of signal
elements, the mask is pressed against the substrate and the ammonia plasma
applied. The mask may be used repeatedly.
As should appreciated, the spatial distribution of analyte sensitivity may
also be conferred by the patterned application of spacer side arms.
With reference to the printing method above-described, the schematics of
one possible oligonucleotide stamp is shown in FIG. 13. The stamp has
holes which are filled with a certain chemical that will be used to
provide the desired building block of the oligonucleotide being
synthesized. In FIG. 13 each row is filled with the same chemical and
accordingly four different chemicals can be used during one stamping cycle
in the example given in FIG. 13. In commercial systems the number of rows
will be considerably higher, typically 64-256, although lower and higher
numbers of rows can be used in special cases. The linear stamp is
advantageous if all possible oligonucleotides of certain size are to be
fabricated onto the assay device substrate.
In this way all possible hexameric combinations of a given set of
oligonucleotide building blocks can be prepared. For instance, trimer
phosphoramidites can be formed by two reaction cycles by using a 64-row
linear stamp. Each of the 64 different trimer phosphoramidites is fed into
one row of holes. After printing the phosphoramidites, the oxidizer,
deblocker and cap reagent are printed. As these chemicals are the same at
each spot, the stamp can be a flat plate or the whole substrate can be
simply dipped into the reagent solution. The substrate is rotated
90.degree. and the same cycle is repeated. In this way all possible
combinations of trimers have been fabricated. Analogously all combinations
of any set of oligonucleotide amidites can be fabricated.
In FIG. 14 is an example showing the fabrication of all possible
combinations of four different oligonucleotide amidites. After the first
printing cycle all spots in each horizontal row contain the same
oligonucleotide, but each row has a different oligonucleotide. These
oligonucleotide fragments are denoted by numbers 1, 2, 3 and 4 in FIG. 14.
When the stamp is rotated 90.degree. and the printing cycle is repeated
all combinations of four oligonucleotides are formed.
The foregoing orthogonal printing process is particularly advantageous in
the production of signal elements of this invention in the embodiment of
the disk. Orthogonal printing facilitates the distribution of the array of
spacer molecules in a pattern of concentric circles, similar to the
information that is placed onto audio or CD-ROM compact disks in annular
patterns. One preferred variation of an orthogonal printing process
employs superimposition of two sets of spiral stamps with opposite
chirality.
The positioning of the stamp must be accurate within about 1 .mu.m. This
can be achieved mechanically using two to four guiding spike hole pairs or
by an optoelectronically guided microtranslator. a removable reflective
coating may be deposited onto two perpendicular sides of the substrate and
the stamp and their relative positioning measured by an interferometer.
The substrate and stamp can also have a pair of microprisms which must be
perfectly aligned in order for the light pass into the photodetector.
FIGS. 11A through 11G illustrate various useful patterns of spatially
addressable deposition of cleavable reflective signal elements on
circular, planar disk substrates. FIG. 11A particularly identifies an
address line, encodable on the disk substrate, from which the location of
the cleavable spacers may be measured. In FIG. 11A, the cleavable spacer
molecules are deposited in annular tracks. FIG. 11B demonstrates spiral
deposition of cleavable signal elements, and particularly identifies a
central void of the disk annulus particularly adapted to engage rotational
drive means. FIG. 11C demonstrates deposition of cleavable signal elements
in a pattern suitable for assay of multiple samples in parallel, with
concurrent encoding of interpretive software on central tracks. FIG. 11D
schematically represents an embodiment in which the assay device substrate
has further been microfabricated to segregate the individual assay
sectors, thereby permitting rotation of the assay device during sample
addition without sample mixing.
FIG. 11E schematically represents an embodiment in which the assay device
substrate has further been microfabricated to compel unidirectional sample
flow during rotation of the assay device. Techniques for microfabricating
solid surfaces are well known in the art, and are described particularly
in U.S. Pat. Nos. 5,462,839; 5,112,134; 5,164,319; 5,278,048; 5,334,837;
5,345,213, which are incorporated herein by reference.
FIG. 11F demonstrates deposition of cleavable signal elements in a spatial
organization suitable for assaying 20 samples for 50 different analytes
each. FIG. 11G demonstrates the orthogonally intersecting pattern created
by superimposition of spiral patterns with spiral arms of opposite
direction or chirality.
The spatial distribution of cleavable reflective signal elements, or
biobits, on the surface of the assay device may be designed to facilitate
the quantitation of analyte concentration.
Thus, in some embodiments, analyte capture is used for quantification. In
one implementation, the assay device is patterned with a uniform density
of biobits dedicated to each chosen analyte. a test sample is introduced
onto the disk in the center of the disk. By applying rotational force, the
test sample is spread radially to the periphery. In the process of
spreading, analytes are captured by the respective cognate side member of
the cleavable signal element, reducing the concentration of analytes at
the sample front.
With sufficient density of biobits relative to the incident concentration,
all analytes are captured before the sample front reaches the periphery of
the assay device. The concentration of each analyte may then be determined
according to the location of the positive biobit that is farthest from the
sample introduction site.
It will be appreciated that a greater dynamic range of analyte
concentration will be detectable if more biobits are dedicated to the
detected analyte. In the embodiment just described, the uniform density of
biobits would be increased. It will further be appreciated, however, that
the density of biobits need not be constant, and that a linear or
exponentially changing density of biobits may be employed, as measured
from the center of the disk to the periphery, to change the dynamic range
of concentration detection.
In other embodiments and aspects of the present invention, biobits with
different affinities for the chosen analyte may be attached to the assay
device to similar effect, that is, to increase the dynamic range of
concentration detection.
It is further contemplated that other geometries may be used to convey
concentration information. FIG. 16 demonstrates one geometry in which a
single sample is channeled in parallel into four distinct sectors of the
assay device. If either the density of biobits, the affinity of the
biobits, or both density and affinity of biobits in the four sectors
differs, a large dynamic range of concentration may be determined by
detecting the position in each sector of the positive biobit most distal
from the sample application site.
In other embodiments, equilibrium assays are contemplated. Concentration is
thus determined by sampling the entire disk and determining the percentage
of positive biobits per analyte.
In each of these embodiments, generally a number of biobits are dedicated
to detection of positive and negative controls.
In other embodiments, cleavable reflective signal elements (biobits)
specific for multiple different analytes are patterned in a number of
different formats. For example, biobits of distinct specificity are mixed
in each sector of a disk. Alternatively, they may be separated into
different sectors. The ability to pattern specific biobits into predefined
locations and the ability to decipher the identity of biobits by detectors
such as a CD-ROM reader makes flexible designs possible. One of skill in
the art would appreciate that the design of patterns should be tested and
adjusted using test samples containing known analytes of different
concentrations.
5.8 Alternative Assay Device Geometries Without Cleavable Spacers
Although the use of cleavable spacers with analyte-specific first and
second side members is preferred in many cases, alternatives exist that
equally take advantage of optical disk readers for detection. Some of
these alternatives are discussed in various other sections herein.
Alternate geometries that dispense entirely with cleavable spacers are
particularly discussed here.
5.8.1 Detection and Counting of Cells
Viruses are typically nearly spherical particles having diameter less than
0.5 .mu.m. Bacteria are commonly either spherical or rod shaped; their
largest dimension is usually less than 2 .mu.m excluding flagella and
other similar external fibers. These pathogens are somewhat smaller than,
or about the same size as, the gold spheres used in the cleavable signal
elements of the present invention. Their interaction simultaneously with
two side members of the cleavable signal element above-described may,
therefore, be sterically inhibited.
To detect such pathogens using the cleavable spacer embodiments presented
hereinabove, the pathogens in the sample may be lysed, and the proteins
and nucleic acid fragments identified as above-described. By detecting
several components of the pathogens, the assay can be made highly
reliable. However, the lysis and subsequent sample processing take several
steps which require instrumentation and take time. Direct detection of
cells would be advantageous.
Thus, an alternative geometry dispenses altogether with the cleavable
spacers. One analyte-specific side member is attached directly to the
substrate surface of the assay device in spatially addressable fashion.
The second side member, specific for a second site of the chosen analyte,
is attached directly to the signal responsive moiety. In preferred
embodiments, that moiety is a gold sphere. In this alternative geometry,
recognition of analyte creates a direct sandwich of the formula:
substrate-first side member-analyte-second side member-signal responsive
moiety. This geometry might be said to be a limiting case in which "m" in
the formula for the cleavable spacer is zero.
For detecting E. coli, for instance, recognition structures, such as
antibodies, may be used that are specific for flagellin. There are about
40,000 molecules of flagellin per flagella, and 0-100 flagella per cell.
Flagellin is strikingly diverse among different bacterial species. Other
proteins presenting attractive targets for detecting E. coli include
fimbriae (common pili), F-pilus, OmpA, OmpC, OmpF.
This assay geometry is also useful for detecting, counting, and
characterizing eukaryotic cells, that is, for assays in which eukaryotic
cells are the analyte to be detected.
Cell counting has been traditionally been done by visual counting of
stained cells under a microscope. Automated flow cytometry has, for many
purposes, now supplanted or augmented manual inspection. See, e.g., M. G.
Ormerod (ed.), Cytometry: a Practical Approach, 2d ed., Oxford University
Press (1997); J. P. Robinson (ed.) and Z. Darzynkiewicz, Handbook of Flow
Cytometry Methods, John Wiley & Sons(1993); a. L. Givan, Cytometry: First
Principles John Wiley & Sons (1992), all of which are hereby incorporated
by reference. In addition to the number of cells, automated flow
cytometers further report the average size distribution of the cells.
Although they have not previously been so recognized or described, optical
disk readers are, in essence, scanning confocal laser microscopes. As
such, they can be used, with proper software, to study the detailed
structure of biological and other specimens. Cell counting and cell shape
measurement are two examples of these applications. FIG. 33 depicts one
geometry, based upon this principle, useful for detecting eukaryotic
cells.
The detection of eukaryotic cells in the present invention is best
performed by attaching, directly to the device substrate surface, a first
structure capable of recognizing and binding to the desired cells, such as
an antibody. A second structure capable of recognizing and binding to the
desired cells, such as a second antibody, is attached directly to the
surface of a signal responsive moiety, such as a metal microsphere.
The first and second antibodies (or other recognition structures) may be
identical, may be nonidentical but recognize the same protein, or may
recognize different structures entirely. Use of distinct antibodies will
increase specificity. It is also possible to use a mixture of antibodies,
either for the first recognition structure, the second, or both, in order
to broaden the detection to several cell types.
As is well recognized, cell surface proteins present particularly good
targets for cellular recognition in assays. Extracellular matrix and
adhesion proteins may also be used, either as targets or themselves as
recognition molecules.
TABLE 5
Cell surface structures
Matrix proteins
MAG (myelin)
MUC18 (melanoma)
Selectins (carbohydrate
binding proteins)
Restrictin (neural cells)
Serglycin (mast cells and
other myeloid cells)
SPARC/Osteonectin (bone)
Syndecan (epithelial
cells)
Tenascin (developing
cells, tumor cells, neural
and muscle cells)
Thrombospondin
(inflammation)
von Willebrand Factor
(platelet aggregation)
Selective cell-cell binding protein pairs
Cell 1 protein Cell 2 protein
GP Ib-IX (platelet) vWF (platelet)
Integrin .alpha.1.beta.1 Collagen, Laminin
ICAM-1 and ICAM-2 LFA-1 (leukocytes)
(endothelium, monocytes,
lymphocytes)
L1 (neurons, Schwann L1
cells)
LFA-5 or CD58 CD2 (T lymphocytes)
(monocytes, B lymphocytes)
MBP (hepatocyte) mannose
NCAM (several cell types) NCAM
PECAM-1 or CD31 PECAM-1
(platelets, white and
endothelial cells)
PH-20 Protein (sperm) zona pellucidal protein
E-Selectin or ELAM-1 NeuAc.alpha.2, 3Gal.beta.1, 4[Fuca1, 3]
(endothelial cells) GlcNAc.beta.1, 3Gal.beta.-Carbohydr-
Prot
TAG-1 (axons) Integrins
VCAM-1 (endothelial cells) Integrin VLA4 (lymphocytic
and monocytic cells)
To the above nonexhaustive list may be added, as particularly useful,
antibodies to CD antigens that have been defined on the surface of immune
system cells. Of particular interest in this regard is CD4, for purposes
of following T helper counts in individuals with AIDS.
The sample can be any biological fluid, such as blood, saliva, semen, etc.
Alternatively, the cells may be cultured, or from a gently homogenized
tissue sample.
Prior to assay, certain cell types may be enriched or depleted, as by
separation using magnetic beads (Miltenyi Biotec, Auburn, Calif.). In this
case, signal responsive moieties, e.g., plastic beads, will already be
attached to the cells of interest prior to addition to the assay device,
and no other microspheres or other signal responsive moieties are needed
on the disk at that specific assay site.
Furthermore, magnetic beads can be used to accelerate the binding of the
cells onto the assay device surface. a pulsating and rotating magnetic
field will allow the cell to contact, with high frequency, various assay
sites at high frequency. Contact with the appropriate recognition
structure will thereafter constrain movement. The frequency of pulsing can
be 0.1-1,000,000 Hz.
Ultrasound is another way to accelerate the binding. Ultrasound will
provide the energy for the high frequency movement of cells in the sample
across the assay device substrate, but does not concentrate the sample at
the interface. It is advantageous to use a static or pulsating magnetic
field in conjunction with application of ultrasound.
By labeling the surface of cells relatively uniformly, their individual
sizes and shapes can be measured by the optical disk drive functioning as
a scanning confocal microscope. Many staining methods can be used. Cells
can be coated by small latex or metal particles, or stained with
immunogold silver stain, detection of which does not depend on the
wavelength of the incident laser light (M. A. Hayat (ed.), Immunogold
Silver Staining, CRC Press (1995)). Membrane-specific dyes allow the
measurement of cell size, and, through intensity changes associated with
the gradient of the membrane surface, permit reconstruction of the
approximate topography of the cell.
But staining need not be limited to decoration of the surface by
microspheres or other signal responsive moieties. For example, cells may
be stained internally, so that they absorb enough laser light to prevent
reflection from a reflective layer of the assay device. In yet another
class of stains, the degree of staining correlates with some enzymatic
activity, permitting study of the specific metabolic activity of the
cells. An example is the nitroblue tetrazolium reduction test for
neutrophil activity.
The confocal nature of the CD- or DVD-Drive also allows the study of thin
tissue specimens. If only the side of the sample that is in contact with
the assay device surface is stained, it will be preferentially detected,
because the incident laser light is focussed into about micrometer sized
spot in that plane. The part of the sample that is further removed from
the surface will give only a weak diffuse background, because that part of
the sample is not stained, and additionally because the light cone probes
a relatively large area and all effects are averaged out.
This particular geometry, in which one analyte-specific moiety is attached
directly to substrate and another is attached directly to the signal
responsive element, may also prove useful in detecting nucleic acid
hybridization, as shown in FIG. 17.
In this alternative geometry, if the signal responsive moiety is
reflective, the information encoding is similar to that in the geometries
presented earlier--the presence of analyte is signaled by reflection.
Alternatively, if the signal responsive moiety is opaque, e.g. through
incorporation of dye, the encoding is reversed: the presence of analyte is
signaled by absence of reflection from the metallic layer of the device
substrate.
Magnetic plastic spheres may provide particular advantages in this
alternative geometry. Because they contain magnetic particles inside, they
are less transparent than latex spheres. Furthermore, magnetism can be
used to remove weakly bound spheres that are otherwise difficult to
remove, as, e.g., latex spheres, because their density is close to that of
water and centrifugal force would prove ineffectual.
A further variant of this alternative geometry takes advantage of
agglutination in a reflection assay, as shown in FIG. 18. In this
alternative, the signal responsive moieties are preferably microspheres.
These microspheres are relatively small (30-600 nm), so that one alone
does not block the light efficiently.
5.8.2 Detection of Aldehydes and Ketones
Chemical assays may also be adapted to detection using optical disk
readers, without the use of cleavable spacers.
Aldehydes and ketones can be detected by immobilizing phenylhydrazine onto
the detection surface of the assay device, preferably intermediated by a
spacer molecule. If the assay device substrate is coated with gold, the
spacer may be polyethylene glycol that has a thiol group at the end distal
to that with the phenylhydrazine group.
The sample that contains an aldehyde or ketone is added. Hydrazone
formation inactivates phenylhydrazine moieties to the extent that is
proportional to the carbonyl concentration. Plastic spheres containing
aldehyde groups (Bangs Laboratories, Inc., Indiana) are added. These
plastic spheres will be bound covalently by the remaining phenylhydrazine
moieties. The number of bound plastic spheres, as read by an optical disk
reader, is inversely proportional to the concentration of an aldehyde or
ketone.
5.9 Classification of Assay Geometries
As has been discussed and demonstrated hereinabove, virtually any
analyte-specific assay may be adapted for use with the assay devices of
the present invention. The sole requirement is that the assay's
analyte-specific recognition be adapted to signal elements suitable for
detection by an optical disk reader. Many of these assay methods are
known, but their adaptation for detection using an optical disk-based
reader is new.
Preferred embodiments of the assays of the present invention use the
cleavable reflective signal elements of this invention. Others, however,
dispense with the cleavable spacer side members, with specificity
conferred by the cleavage site itself, while still others dispense
entirely with cleavable spacers. Given the variety of assay geometries
that may usefully be employed, a summary of those which prove particularly
useful is presented here. The summary is illustrative, not exhaustive, and
is not to be construed as limiting.
Assay methods, as adapted for use in the assay devices of the present
invention, are schematized in FIGS. 34 and 35. FIG. 34 depicts assays
without cleavable spacers; FIG. 35 depicts the corresponding assays with
cleavable spacer. In these figures "R" and "S" represent the recognition
molecules, whether disposed on cleavable spacer side members or not, and
"X" and "Y" represent the analytes to be detected, or detectable moieties
thereon. The signal-responsive moiety, suitable for detection in an
optical disk reader, is shown as a sphere.
As has been discussed hereinabove, "R" and "X" represent cognate members of
a specific binding pair, such as antibody-antigen, receptor-ligand,
enzyme-substrate, enzyme-inhibitor, complementary oligonucleotides, or the
like. Similarly, "S" and "Y" represent cognate members of a specific
binding pair. For the chemical assays described above, the "specific
binding pair" may alternatively represent chemical function groups with
reactive specificity for one another.
FIG. 34A depicts a traditional "sandwich" assay. If "R" and "S" are
antibodies, and "X" and "Y" are epitopes displayed by the analyte to be
detected, this represents a sandwich immunoassay If "R" and "S" are
oligonucleotides, and "X" and "Y" are complementary sequences on a nucleic
acid to be detected, this represents a nucleic acid hybridization assay.
In either case, the principle is clear: presence of the appropriate
analyte in the sample serves to tether the signal responsive moiety to the
assay device substrate, generating a detectable signal at that location.
The geometry also serves the converse purpose. Thus, if "R" and "S" are
identical epitopes of an antigen, this geometry permits detection of an
antibody that binds thereto.
FIG. 34B depicts a replacement assay. Recognition molecule "R" is attached
to the signal responsive moiety. The analyte to be detected, or an
analogue thereof, "X", is immobilized on the assay device substrate
surface. Analyte present in the sample, shown as free "X", will displace
the binding by the surface-immobilized "X", liberating the signal
responsive moiety. The signal is lost at that location, the inverse of the
signal direction in the first geometry, but equally informative.
FIG. 34C represents a competitive assay. It is analogous to replacement
assay, but in this case the sample is mixed first with the recognition
molecule-signal responsive moiety conjugate and it is this mixture that is
added onto the substrate.
FIGS. 35A-C depict the incorporation of the cleavable spacer into the
assays of FIGS. 34A-C. The spacer can be a single molecule, but it may
also contain particles or a part of a bulk material, such as substrate
plastic, rubber, glass, metal, or the like.
As detailed above, cleavable spacers offer several advantages in these
latter geometries. First, all components are immobilized onto the assay
site during manufacturing. Second, as a consequence of immobilization,
less reagents are needed. Third, the kinetics are improved, because all
components are maintained in close proximity to one another.
Several modifications of the schematized methods are readily apparent. For
example, with reference to FIGS. 34B and 34C, the recognition molecule can
be immobilized, while the analyte or its analog is conjugated with the
signal responsive moiety. As would be recognized by those skilled in the
assay arts, it is also possible to form various combinations of these
assays. For example, even if the antigen is so small that the traditional
"sandwich" assay is not feasible, a dimeric antigen, where "X" and "Y" are
identical antigens, can be artificially prepared and be used in
conjunction with a competitive assay (FIGS. 34C and 35C). The dimeric
antigen is added together with the sample, and the univalent sample
antigen prevents competitively the bridging by the dimeric antigen.
5.10 Continuous Monitoring Devices Incorporating An Optical Waveguide
It will be appreciated that each of the above-described assay device
geometries is particularly suited for discontinuous, also termed static or
batch, assay. That is, the obligatory cleavage step precludes repeated or
continuous assay using the same cleavable signal elements. While physical
segregation of cleavable spacers on the assay device, e.g. as exemplified
in FIG. 11D, will permit multiple uses of the assay device itself, it
remains true even in this geometry that each of the cleavable signal
elements may be used only once to signal the presence or absence of
analyte.
Another embodiment of the invention thus combines the cleavable signal
elements above-described with an optical waveguide, thereby permitting
repeated, or even continuous, monitoring for analyte. In another aspect,
the continuous monitoring embodiment may be converted, after detection of
analyte, to spatially-addressable static detection, as above-described.
The continuous monitoring assay devices profit from the ability to adapt
the assay device substrate to serve as an optical waveguide. Incident
light is directed into the device substrate via a radially disposed mirror
integrated into the assay device itself; upon application of incident
light, an evanescent wave propagates through the device substrate through
internal reflectance. The presently preferred plastic compositions of the
assay device substrate are particularly well suited for adaptation to
serve as optical waveguides, although glass may also be used.
The internal reflectance of the evanescent wave is not total, however;
light necessarily escapes the substrate. Escaping light interacts with the
light-scattering or light-reflective signal moiety of attached signal
elements; the light so scattered or so reflected may be measured.
The degree of interaction of the evanescent wave with a light-scattering or
light-reflective signal moiety of an attached signal element will depend
exponentially on the distance between the signal moiety (e.g., a gold
microsphere) and the internally-reflective substrate; this distance, in
turn, depends upon the differential presence or absence of the chosen
analyte. With deposition of a plurality of signal elements, the intensity
of the light scattered or reflected from the waveguide is strongly
correlated with the concentration of the analyte.
In general, light will travel radially through the waveguide. To detect
signaling events, the internally reflected light can be directed to exit
the waveguide at a defined point, where the remaining luminescence may be
assessed. Alternatively, since the light-scattering or reflective signal
element moieties will also cause significant back scattering of the
escaping light, the change in intensity of back scattered light may be
measured. The intensity change in the back scattered light is much easier
to detect than that of a forward light beam. Thus it might be advantageous
to measure the back scattered light.
Optimization of the light-transmitting properties of the waveguide itself
may include the deposition of cladding, or of partially reflective
surfaces, on one or more surfaces, internal or external, of the waveguide;
however, as described above, some leakage of light from the waveguide is
essential for analyte detection. Such optimization is within the skill in
the optical arts.
Although a mirror is preferred for directing incident light into the
optical waveguide when visible or near infrared (NIR) radiation is used,
prisms or diffraction gratings will also find use, especially for NIR or
longer wavelength light. FIG. 26 demonstrates one embodiment in which
uncollimated, but focused light, is first collimated into (nearly)
parallel rays by a lens. The collimated beam is then directed by a prism
to a diffraction grating integral to the assay device, then into the
waveguide. The lens and prism may be in a modified detector, with the
diffraction grating alone integrated into the substrate itself in lieu of
a mirror.
The source of light for illuminating the waveguide may, in embodiments
suitable for detection in CD-ROM or DVD readers, be the detector's
in-built laser itself. Certain modifications of commercial laser-based
detectors must be made, however, to ensure proper alignment.
The continuous monitoring principle may be better understood through
reference to the figures. FIG. 19 shows a top view of an assay device of
the present invention, as adapted for continuous monitoring. a radially
disposed mirror directs incident light into the plane of the assay device
substrate which is adapted to function as an optical waveguide. Also shown
in FIG. 19 are circumferentially disposed sample application inlets for
each of 20 spatially-segregated assay sectors. It will be appreciated that
the assay device may also be constructed so that sample is applied more
medially, nearer the mirror, so that rotation of the assay device drives
sample toward the periphery through centrifugal force.
FIG. 20 shows further detail of the continuous monitoring assay device of
FIG. 19, with FIG. 20A showing a top view of a single assay sector and
FIG. 20B showing a side view. Particularly demonstrated are the spatially
addressable assay sites, each containing a plurality of cleavable signal
elements, the mirror, sample inlet port and a port for outflow, for
outflow either of sample fluid or of sample gas (should sample be applied
in the gaseous phase), and for outflow of air and other gases entrained in
a liquid sample stream.
The side view shown in FIG. 20B further demonstrates a first assay device
substrate 20 to which are attached cleavable signal elements, as in the
static assay geometries described hereinabove. In the present example,
substrate 20 is adapted for use as an optical waveguide. FIG. 20B also
shows a second assay device substrate 53, substantially parallel to and
separated from the first assay device substrate 20, and a gap
therebetween, also termed a sample cavity, through which sample flows from
sample inlet to outlet.
In preferred embodiments, the sample cavity is hydrophilic so that the
wetting by liquid sample is perfect and no air bubbles are retained, and
the total volume of the cavity is about 1-100 .mu.l, preferably 10-50
.mu.l, most preferably about 5 .mu.l. Furthermore, it is preferred that
the outlet be hydrophobic.
It will be appreciated that the total depth of the assay device may be
adjusted--through adjustment of the width of substrate 20, adjustment of
the width of substrate 53, and adjustment of the width of the sample
cavity, as required by the requirements of the detection device. Thus, as
set forth in Table 1 above, commercially available CD and DVD disks have a
depth of 1.2 mm. Although a depth of 1.2 mm is most preferred for such
disks, such detection devices will typically accommodate disks as wide as
2.4 mm. Thus, the continuous monitoring assay devices of the present
invention will have a depth of 1.0-2.4 mm, preferably 1.2-2.0 mm, most
preferably 1.2 mm.
In these embodiments, the assay device will preferentially be made of two
disks of optically clear polycarbonate, each having a diameter of 120 mm,
i.e., the same diameter as conventional CDS. During manufacture, the two
disks will be assembled to form a hollow interior, and the resulting
cavity may additionally be divided into sectors through which the liquid
samples will flow. It will be appreciated, however, that other substrates,
as described above, may also be used depending on their suitability for
adaptation to function as optical waveguides. It will further be
appreciated that the static assay geometries which do not use a substrate
adapted for use as an optical waveguide may nonetheless also utilize a
hollow interior geometry, and similar sample application techniques.
Plastic polycarbonate disks suitable for the optical waveguide embodiments
may be purchased from Disk Manufacturing, Inc., Wilmington, Del. ("DMI").
The top disk will have a circular 45.degree. tilted gold mirror evaporated
near the center. The address information may simply be a zone of
evaporated gold near the center. The mirror and address information may be
deposited simultaneously.
FIG. 21 shows side views of an assay site with two signal elements during
continuous monitoring for dimeric analytes.
FIG. 21A shows a first and a second cleavable reflective signal element
attached to derivatized assay device substrate surface 21 of assay
substrate 20. Assay substrate 20 is adapted for use as an optical
waveguide. a first analyte-specific side member 34a is attached directly
to the derivatized surface 21 of assay device substrate 20, and a second
analyte-specific side member 34b is attached directly to the signal
responsive moiety, a metal microsphere 40, of a first signal element. In
this exemplification, the cleavable spacer does not itself contain side
members. Also shown are a third side member 35a and fourth side member
35b, neither of which is specific for the chosen analyte; the second
signal element thus cannot recognize the chosen analyte.
FIG. 21B demonstrates analyte-specific recognition by the first and second
side members, 34a and 34b, tethering the first signal-responsive moiety to
the substrate 20. This tethering is optionally assisted by application of
centrifugal force, as shown. Also shown, side members 35a and 35b, which
cannot recognize the chosen analyte, do not tether the second signal
element to the substrate. Upon cessation of rotation of the assay device,
only the first signal element is brought into proximity to the optical
waveguide substrate, as shown in FIG. 21C.
In this proximal position, each bound gold sphere will give a reflective
signal to waveguide light leakage; this, in turn, will alter the light
intensity within the waveguide to a detectable degree. This change in
light intensity may be registered by the detector, and will indicate the
recognition of analyte by one of the signal elements.
FIG. 21D-21F shows a similar effect without application of centrifugal
force. And in contrast to the dimeric analyte detected in FIGS. 21A-21C,
the analyte itself contains a plurality of sites for attachment to the
side members.
It is anticipated that the detector for assessing changes in waveguide
transmittance in the continuous assay embodiments of this invention will
have a more limited ability to discriminate the spatial location of
signals than will the detector used for detection of reflection of the
perpendicularly directed incident light. Thus, FIG. 22 demonstrates the
combination of the spatially addressable, cleavable signal elements of the
earlier-described static assay devices, with the continuous monitoring,
optical waveguide geometry described here.
Once analyte is detected through change in the amount of light within the
waveguide, or alternatively, through detecting a change in the amount of
light escaping from the waveguide, the assay device may be exposed to a
cleavage agent, as described for the static, or batch, devices. For
siloxane-containing spacers, a solution of sodium fluoride, with
concentration of 1 mM to 1 M, preferably 50 mM to 500 mM, most preferably
100 mM (0.1 M) will be used.
FIG. 22A demonstrates application of sodium fluoride as cleavage agent.
FIGS. 22B and 22C demonstrate the differential signal provided after
cleavage. As with the static, non-waveguide geometries, once cleavage has
been performed, the cleaved signal elements (biobits) may not be used
again.
It should be recognized that the hollow geometry is particularly suited for
creation of physically segregated assay sectors, as, e.g., through
interposition of interior walls. In this latter case, introduction of
cleavage agent to one sector does not preclude subsequent continuous
monitoring, and later cleavage, of other sectors on the same assay device.
The spatial discrimination of the waveguide detector will be sufficient,
however, to identify whether signal emanates from any of the individually
segregated assay sectors. The waveguide will indicate the sector where the
detection occurred, and the one-to-one correspondence between sample and
sector will identify the positive analyte-containing sample.
Subsequently, cleavage of cleavable spacers in that sector may be used to
identify the nature and/or concentration of the analyte in the sample.
5.10.1 High Volume Screening of Drug Candidates
The continuous waveguide geometry is particularly well suited for high
volume, rapid screening of drug candidates. The process provides both
highly reliable and accurate results at a relatively low cost, and is
particularly suitable for screening chemical libraries, prepared by either
parallel synthesis or the split-and-mix method.
Although both parallel synthesis and split-and-mix chemical libraries can
be screened by the continuous monitoring assay device (BCD), each will
require a different design configuration within the BCD envelope. For
parallel screening applications, the assay device (BCD) will contain
upwards of 100 sectors, preferably more than 200 sectors, most preferably
200-400 sectors, with 400 sectors being presently the most preferred. For
split-and-mix screening, the assay device will be sectored for each
sublibrary; for example, screening of peptides will require 20 sectors in
the BCD, corresponding to the 20 natural amino acids.
About 0.5 billion total Biobits will be fixated onto the waveguide disk
during initial manufacture, and the total will be divided into radially
oriented linear areas called assay sites. Each assay site will contain
about 50,000 identical Biobits. Accordingly, one BCD will have 10,000
assay sites, which limits the number of assays per BCD to 10,000.The BCD
will be further divided into identical sectors, and each sector will be
used to study one sample.
It is to be noted that a sample may contain one compound (parallel
synthesis), or one million compounds (split-and-mix synthesis). The number
of assay sites in any one sector will set the upper limit for the number
of target biomolecules. The practical upper limit for the number of
sectors per BCD is approximately 400. Thus, in parallel screening, 400
compounds can be screened against 25 target biomolecules (400
sectors.times.25 target biomolecule=10,000 assay sites). In the
split-and-mix protocol the number of samples will almost always be less
than 25, and each sector can contain 400 target molecules. Because in this
case each sample can contain up to one million compounds, 25 million
compounds will be able to be screened simultaneously against 400 target
biomolecules. For the sake of simplicity, FIG. 2 depicts a sector that has
only 40 assay sites.
In high volume drug screening, analyte-specific side members will
preferentially be disposed as shown in FIG. 21, rather than being disposed
on either side of the spacer's cleavage site, as shown in FIG. 1, although
the geometry shown in FIG. 1 remains feasible. As with the static assay
elements and geometries, the side members may be single or double stranded
DNA fragments, which are useful in the screening of gene-regulating
agents; antibodies, antibody derivatives, or antibody fragments, to screen
autoimmune disease or allergy drugs; enzymes, to screen for enzyme
inhibitors; receptors, for screening for artificial ligands; and ligands,
for screening for cognate receptors.
In many cases of drug screening, as well as in standard immunoassays, the
analyte chosen for detection is a small organic molecule which can
interact with only one cognate binding partner at time. These so-called
univalent analytes are unable in the present invention to form the
tethering loop required either (1) to secure the signal moiety in
proximity to the optical waveguide, or (2) to secure the signal moiety to
the substrate after addition of cleaving agent.
The problem of univalent small analytes has previously been addressed in
development of standard immunoassays. Most of the existing strategies for
solving this problem in standard immunoassays are readily adaptable to the
novel cleavable signal element and waveguide assay device of the present
invention. Therefore, only two particular strategies will be described
here: (1) use of a replacement assay, and (2) use of dimeric or polymeric
analyte candidates.
In the replacement assay, the tethering loop is premade using a surrogate
ligand with modest affinity for the first and second side members. The
surrogate ligand can be of biological origin, but preferably is a known
artificial ligand, so that its binding affinity can be adjusted if
necessary. The surrogate ligand will be suitable for binding
simultaneously to both first and second side members. Each side member
contains a receptor specific for the surrogate ligand and specific also
for the chosen analyte. If the sample contains a higher affinity,
univalent analyte for the same receptor, the sample analyte will replace
the stationary surrogate ligand; since the sample analyte is univalent,
the tethering loop is broken. If sufficient receptors are so blocked, the
distance between the gold sphere and the waveguide will increase, thus
changing the intensity of the light transmitted by the optical waveguide.
Upon optional subsequent cleavage, such blocked receptors will be lost. In
this approach, the drug candidates are in a soluble form and unlabeled.
Alternatively, the binding of dimeric or polymeric drug candidates can be
measured. Dimeric molecules are able to bind two similar recognition
molecules and will form a loop between a gold sphere and the waveguide.
Two binding events will serve as a redundant check for good binding. Thus,
nonspecific binding and a false signal due to impurities is largely
eliminated. Although not ideal, the dimers more closely mimic actual drug
molecules than do fluorescently labeled drug candidates in other,
existing, approaches, since a fluorescent label may interfere with the
binding process. The other half of the dimer is unlikely to do so any more
than another similar molecule in close proximity.
In order to eliminate the effect of the spacer, several variants of the
same drug candidates, connecting the spacer in different positions, should
be synthesized. Actually, it is conceivable that some dimers might
themselves serve as drugs, because they might induce dimerization of the
receptors, which is an essential part of the natural function of single
.alpha.-helix receptors.
When detection is done by the replacement method, there is virtually no
restriction on the method used for synthesis of the chemical libraries.
Chemicals are used as such and no labels are needed. However, when a
binding assay is performed by the BCD, two or more similar molecules must
be bound together.
Synthesis performed on a solid support automatically produces particles
that have identical molecules connected onto their surfaces by a spacer.
In parallel synthesis different types of particles are separated, and in
split-and-mix synthesis several different types of particles are mixed.
Importantly, in both cases a certain particle contains only one type of
molecule on its surface (excluding impurities). Thus, these particles can
be used directly in the binding assay on the BCD.
Often it is preferable that drug candidates not be bound onto large solid
particles, but instead be soluble in the binding assay. Dimeric molecules
can be conveniently prepared using a Y-shaped spacer. The spacer is singly
connected with the solid support and synthesis is performed in both ends
of the branches. The spacer is again cleavable, so that after completion
of the synthesis it is cleaved near the intersection and the dimeric drug
candidate is released for testing.
Four hundred assay sectors fit into one BCD. One chemical compound is
tested in each assay sector. Accordingly, four hundred chemicals can be
tested simultaneously in one BCD. As discussed earlier, in this case each
assay sector can contain 25 assay sites. Each assay site is dedicated for
a certain recognition molecule. Thus, four hundred compounds may be tested
simultaneously against twenty-five recognition molecules; therefore, the
total number of tests is 10, 000.
Split-And-Mix
Each drug candidate should have at least a 100 nM concentration in the
first test, i.e., 3.times.10.sup.11 molecules in 200 .mu.l, which is a
typical test volume in the split-and-mix assays. One million compounds
would have a combined concentration of 10 mM. Average molecular weight of
400 D gives a total mass of 40 mg per milliliter. This is close to the
upper limit before interference may be expected. Solubility of compounds
might be limiting when the highest possible concentrations are used. The
solvent is commonly water, although alcohol or some other biocompatible
solvent may used in conjunction with water.
The following example is actually a hybrid of parallel and split-and-mix
screening. The interaction of 25 biomolecules and all hexapeptides is
measured. It is supposed that the BCD contains 10,000 assay sites. These
are divided into 400 identical sectors of 25 assay sites each,
corresponding to 25 different biomolecules. Thus, 400 different chemical
libraries could be tested simultaneously against all 25 biomolecules.
There are 64 million different hexapeptides containing 20 of the most
common amino acids. All hexapeptides are conveniently divided into 20
sublibraries so that each sublibrary has a certain known amino acid in a
given position. For example, the last amino acid is alanine in one
sublibrary, while other positions contain all combinations. In another
sublibrary, the last amino acid is arginine, etc. This principle can be
further expanded to produce 400 sublibraries as is explained in the
following.
All hexapeptides can be synthesized in 400 groups so that, first, all
possible tetrapeptides are synthesized in one column. Without detaching
the tetrapeptides, the solid support is divided into 20 equal parts and a
different amino acid is coupled with tetrapeptides in each of these baths.
Pentapeptides are obtained in 20 sublibraries. Each of these sublibraries
is further divided into 20 equal parts and again a different amino acid is
coupled with pentapeptides in these baths giving a total of 400
sublibraries. In each of these cases, the last two amino acids are known
while the first four vary freely (FIG. 25, where AA is an amino acid).
Each of the 400 sublibraries is injected into a dedicated sector in the
BCD. The most interesting hexapeptides will be identified and one is
selected for the next phase (denoted by a star in FIG. 25). At a later
time, all can be studied in a similar manner. The last two amino acids of
the lead candidate will be known. Next, the process is repeated so that
the central two amino acids define 400 sublibraries. The last two amino
acids are fixed by the result obtained in the first round. New testing
will indicate the two central amino acids that give the best result. a
third similar cycle will reveal all six amino acids in the most active
hexapeptide.
Any library of chemicals can be studied in a similar manner. The mixtures
could be made by combining smaller libraries into larger ones and storing
samples of the intermediate ones. Alternative synthesis strategies can be
used to create mixtures of millions of compounds. This is analogous to the
hexapeptide example given above. In general, the starting materials and
reactions can be any compatible combination.
The Biobit is able to detect any biomolecules for which recognition
molecules are available. Oligonucleotides can be best recognized by
complementary oligonucleotides. For example, to recognize a 22-mer
oligonucleotide in the sample two 11-mer oligonucleotides can be used for
the recognition. The other is complementary to 3'-end and the other to
5'-end of the sample oligonucleotide.
This is called (a,b)-recognition in general and in this special case it
would be (11,11)-recognition.
Receptors, antibodies, enzymes, etc., can be used as recognition molecules.
The molecules that interact with them, such as agonists, antagonists,
antigens, inhibitors, etc., are herein collectively called ligands. The
ligand may be naturally occurring compound, or it may be an existing drug.
The purpose is to find a new compound that will bind so strongly with the
biomolecule that the ligand will be replaced. In this case, the gold
sphere will be lost when the spacer is cleaved.
In order to perform drug mass screening on the BCD, biomolecules must be
attached onto some specific areas. This is accomplished by first
conjugating a biomolecule with an oligonucleotide that is complementary
with a stationary oligonucleotide on a given area. The recognition
molecule-oligonucleotide conjugate will hybridize with the complementary
oligonucleotide and the biomolecule is automatically located in the chosen
area. The second recognition molecule is similarly attached onto each
assay site. If a replacement assay is performed then the ligand of each
biomolecule is similarly located on the same area.
Importantly, this method of attaching biomolecules onto the BCD is based on
a self-assembly and can be performed by any ink-jet or automatic pipetting
station. Thus, the operator will be able to use proprietary and other
biomolecules in the assays while avoiding secret disclosure. The BCD can
be provided as a blank platform where the operator will be able to attach
all interesting biomolecules, or certain standard assays can be included
in the production phase, while the operator will be able to add his own
assays into the dedicated area as necessary.
5.10.2 Battlefield Bioanalyzer
The continuous waveguide geometry of the assay device of the present
invention is also well suited for use under rigorous field conditions, and
is particularly useful for use in portable instruments for continuous
monitoring and analysis of environmental conditions. The solid state and
essentially digital nature of the assay device finds particular utility
under conditions of severe environmental stress, such as a battlefield.
Thus, the continuous waveguide embodiments of the present invention are
well suited for a battlefield analyzer, also termed herein a battlefield
bioanalyzer. Such a device is useful for continuous monitoring of the
battlefield atmospheric environment, and for rapid identification of a
large spectrum of pathogens and toxins (Agents) which may be present,
especially in conjunction with a sample collector that filters ambient air
and solubilizes the resulting sample.
The BCD sample cavity will be sectored to provide space for detection of
Agents.
During continuous monitoring, aqueous samples are pumped in a pulsating
manner into the stationary BCD sample cavity through a detachable
capillary plugged into the hollow interior via a central edge of the BCD.
Each sample circulates for about 5 minutes, then exits through a second
capillary near the inlet port, in a continuous manner for as long as
monitoring is deemed necessary. Both capillaries will be coupled to the
BCD during continuous monitoring, but decoupled when sample identification
is needed.
The first sector of the BCD is the primary area for detecting an incoming
Agent. It contains all possible Biobits for various Agents, i.e., it
contains a plurality of signal elements with collective specificity for
every one of the predicted spectrum of Agents for which monitoring is
desired. Thus, continuous monitoring is possible without rotating the BCD.
If a threshold is exceeded in this first sector, indicating the presence of
one or more Agents, the sample identification process is automatically
triggered and performed within the same BCD. Other sectors will contain
some subgroups of the Biobits spatially segregated so that the specific
class of pathogen or toxin can be further identified.
It is to be noted that in the above manner, the waveguide will also be able
to indicate a positive detection event in any sector of the BCD when the
BCD is rotated.
After the computer has initiated the specific identification process,
sodium fluoride (50 mM-100 mM) is pumped through the BCD inlet and outlet
capillaries with the same pump as used for the monitoring samples. This
solution will essentially cut the cleavable spacers holding the non-bound
gold spheres to the waveguide substrate. The gold spheres are either
flushed out of the BCD cavity or they will fall onto the bottom disk. In
both cases they will give a zero signal. The cleavage will last only a few
seconds. The CD-ROM laser will then "assay" the sample by reading
perpendicularly through the waveguide disk and determining the exact
number and location of all remaining gold spheres bound to the waveguide
substrate. In this identification process, the CD-ROM computer will attach
a value of one to all remaining bound spheres, while the absence of a
sphere will have a zero value.
As the computer software will have been programmed to recognize the
particular BCD sector in which each specific recognition biomolecule will
have been placed, and which Agent will bind to each biomolecule, the
computer will quickly identify the specific Agent present. Each Agent can
be identified in various ways. For example, surface and core proteins of a
virus can be identified, and some gene fragments can be identified.
Individual viruses can easily be identified in ten different ways. This
capability will increase reliability.
Final identification and quantification of the sample will be performed by
perpendicular site-specific reading of the BCD.
The fastest way to identify biological warfare agents is to detect and
identify whole pathogens, i.e., viruses, fungi and bacteria as such, using
surface proteins as the chosen analyte for detection in immunoassays.
Direct detection of pathogens through immunoassay is a particularly favored
assay for use in the battlefield bioanalyzer.
The instrument for the reading of the BCD is the computer with a CD-ROM.
The sample will be collected and concentrated by a separate unit that will
feed the sample into the CD-ROM through a tubing that must by retrofitted
into a commercially available CD-ROM.
5.11 Sample Delivery Devices
General principles of sample delivery have been described hereinabove
(section 5.1.7). Devices that facilitate such delivery are described in
this section. Other variants will readily suggest themselves to those
skilled in the assay arts. The following embodiments are thus
illustrative, not exhaustive.
5.11.1 General Structural Features
Briefly, the sample delivery device and method of this invention utilize a
multiwell plate, so dimensioned as conveniently to align in registration
with the assay sites of the assay device. Where the assay device is
fashioned as a disk, for example for reading in an optical disk reader,
the multiwell plate is circular.
Because these multiwell plates are, in most applications, not used in the
actual analysis, their manufacture is typically not constrained as to the
optical quality of the material. In such cases the material can be
plastic, metal or a combination of these, preferably but not limited to
polyethylene, polypropylene, polyvinylchloride, polybutadiene,
polytetrafluoroethylene or aluminum or some other metal coated with these
plastics. However, if the sample application well plate is integral to the
assay device, or is to be left approximated to the assay device during
reading, the choice of the material is more stringent. If optical reading
is performed through the well plate, it must be transparent and preferably
non-fluorescent. Examples are polymethylacrylate, polycarbonate,
polyvinylchloride, and cellulose acetate.
The multiwell plate can be single self-supporting structure or it can
consist of several layers, most notably a rigid supporting structure and a
thin, malleable, disposable film.
The sample aliquot can be brought into the contact with the chip or disk by
rotating around the diagonal or normal of the plate. The rotation around
the diagonal is 180.degree. and the gravity will bring the aliquot onto
the surface of the assay substrate. After the 180.degree. rotation, a
wagging motion can be maintained in order to increase the interaction
between the aliquot and the assay site. When the rotation is around the
surface normal utilized, the centrifugal force will bring the aliquot onto
the assay site. In this case it is preferential to load sample, reagents
and washing solutions into serially connected wells. a waste collection
well is the last in the chain of wells.
5.11.2 Single Self-supporting Well Plate
FIG. 36 depicts a simple circular multiwell plate having 112 wells.
Diameter varies between 5 mm-500 mm, and thickness between 100 .mu.m-100
mm. In a typical circular embodiment, each well has a diameter of 1 mm-50
mm and depth of 1 .mu.m-50 mm. The plate has a thickness of 0.2-20 mm. In
the present example, the plate is 1.5 mm thick and one well has diameter
of about 5 mm and depth of 5 mm. The well is oval-shaped and hydrophilic,
so that the liquid can easily flow when a the plate is rotated. The volume
of the well is 125 .mu.l, sufficient to hold a typical sample of 5-75
.mu.l.
Each well delivers one sample onto one assay site of the assay device. As
discussed hereinabove, each assay site may contain signal elements
specific for a number of different analytes. Thus, these assay sites on
the assay device are herein alternatively denominated panels, to denote,
as in the clinical laboratory arts, that the set of analytes detected at
that site is informative as to a potential diagnosis or condition.
The wells are arranged along 16 equally-spaced diagonals (FIGS. 36 and
37a). There are six or eight wells on each diagonal An eight tip pipetter
can dispense samples simultaneously into each diagonal set of wells (FIG.
37B). The circular well plate can be rotated an angle of 90.degree./8
(=11.degree. 15') between pipetting steps. Altogether, 16 pipetting steps
are needed to fill all 112 wells.
The density of the wells can be increased by organizing the wells spirally.
Then all nearest neighbor distances can be identical or nearly identical.
In such a case, existing commercial pipetting stations must be modified
accordingly.
When an assay device--also termed a bio-CD, biocompatible CD, or BCD--is
apposed to the well plate, the air must get out; conversely, when the
assay device is removed, the air return. In order to facilitate such air
flow, the well plate may have a plurality of air holes (FIG. 38).
Preferably, there is at least one air hole between each pair of wells
(FIG. 38). Air holes are optionally present in the perimeter, where air
access will occur nonetheless.
5.11.3. Capillary Well Plate
It may often be the case that the volume of each sample is so small that
the sample forms only a film in a well. In such cases, gravity flow may be
constrained.
FIG. 39 depicts a well plate that can be used for very small volumes. The
well is the only hydrophilic part of the structure. Around the sample well
is a shallow hydrophobic indentation. This can accommodate any excess
sample when the well plate and BCD are compressed together. The bottom of
the well communicates to another side of the plate via an air capillary.
The sample cannot penetrate into this capillary, because it is very narrow
and hydrophobic, yet this capillary provides replacement air, if there is
so little sample that it cannot otherwise contact the surface.
5.11.4. Vacuum Well Plate
Although the cost of the sample well plates will be low, it might
nonetheless be preferable to reduce the amount of disposables. This can be
achieved by using a permanent well plate structure in which only the
surface film is disposable, as shown in FIG. 41. The disposable film alone
contacts the sample, permitting reuse of the well plate manifold.
The film can have a thickness between 10 .mu.m-1 mm, and may be made of any
elastic material. It may be secured over the surface using a supporting
ring.
The wells of the reusable manifold are connected to a compartment that can
be evacuated (FIG. 41). After the film is on the surface the valve that
connects the well plate with a vacuum line is opened. While the air is
removed from the wells the elastic film will tightly cover the wells (FIG.
41B and 41C). The valve can be closed and the vacuum line disconnected
(FIG. 41D). Samples can now be pipetted (FIG. 41 E). After the samples
have been incubated with the BCD (FIG. 41 F-H) and the BCD has been
removed, the film can be removed. The film will form a bag that can be
sealed and disposed (FIG. 41L). The well plate base itself is never in
contact with any liquid and can be used repeatedly.
The same well plate base can also be used without a vacuum line (FIG. 42
A-E). After the film has been assembled onto the surface, the wells can be
formed by a mechanical stamp. While the stamp is in the lower position,
the valve is closed. Although there is no vacuum, the film must line the
wells, because no replacement air can get underneath the film. The well
plate can now be used as described earlier.
5.11.5. Centrifugal Well Plate
Instead of gravity, centrifugal force can be used to drive the liquid into
contact with the BCD, if necessary, with force much greater than gravity.
Axial rotation allows minimal instrument size. The drive of the optical
disk detector may itself be used to accomplish this purpose.
In embodiments that contemplate centrifugal application of sample, it is
possible to load sample, reagent and washing solutions simultaneously onto
a centrifugal well plate (FIG. 43A-43E). During rotation these solutions
pass the assay site or panel in the correct order. At the conclusion of
the spin, the assay site may be covered by a buffer or by air, depending
of the volumes of various liquids and the receiving reservoir. In either
case the result can be read immediately, if the whole operation is
performed inside an optical disk drive, reducing the assay to a single
step.
5.11.6 Carousels and Jukeboxes
The multiwell sample application plate is placed in a rotation instrument
manually or by a robot.
If the wells are protruding from the bottom, the support may have holes
which can accommodate these protrusions. After the samples are in the
wells, the BCD is placed on top of the well plate either manually or by a
robot. Proper orientation and registration of the BCD is critical. The BCD
can have mechanical and/or optical markings that make the correct
registration possible during all steps. Mechanical slots or holes are
preferred, because the system can be designed so that if these features
are not aligned properly, the BCD is not leveled correctly, pipetting is
physically impossible, and the system can alarm the operator.
Alternatively, optical or electrooptical registration may be used,
according to techniques known in the art.
The structure supporting the well plate can have features complementary to
the slot(s) in the BCD. After the BCD is oriented properly it can be
clamped together with the well plate from the edges of the perimeter
and/or central hole. On top of the BCD there can be another supporting
plate.
The structure supporting the well plate can contain active components, such
as magnets. These magnets can coincide with assay panels on the assay
device or with certain assay sites. In some assays, as described
hereinabove, magnetic spheres are mixed with the sample, in which certain
analyte(s) bind with these magnetic spheres. Subsequently, these magnetic
spheres can be attracted onto the surface of the BCD by magnetic field.
The binding kinetics will be greatly increased. The removal of extra
spheres after the incubation can be greatly facilitated by an opposite
magnetic field on the other side of the BCD.
5.11.7. Use of Sampling Well Plates in Clinical Laboratories
The assay devices and sample application well plates of this invention can
be used in clinical medical laboratories and in other laboratories where
multiple samples are analyzed.
In large clinical laboratories, samples are conventionally moved by means
of conveyor belts. The system resembles railway network. Thus, samples
that are intended for certain assays are diverted from the track onto a
side track. Sample is placed first into a multiwell plate, and from there,
in the present invention, applied to a BCD assay device that has an
appropriate analyte-detection panel. All tests of that panel will be
automatically performed, because that is easier and cheaper than excluding
some assays at that point. But all tests need not be reported. In this
sense the BCD is like a random access analyzer, which allows any
combination of assays from a certain panel.
Pipetting of samples into multiwell plates is the rate-limiting step.
Accordingly, several pipetting stations can be located along one sidetrack
(FIG. 44). The multiwell disks should be maintained in constant humidity
and temperature, preferably high humidity and low temperature, during
pipetting. For example, the multiwell plate can be maintained in a
temperature controlled box that has a slit or series of holes in the cover
for the pipetting tips. The tips can always enter into the same place and
the multiwell plate is horizontally rotated around the central axis. When
all wells are full, the multiwell plate is transferred into another
thermostated chamber that has relatively high temperature, typically
physiological temperature, and high humidity (FIG. 44). The BCD is placed
on the top of the multiwell plate and the samples are incubated on the top
of the BCD. The BCD can be washed in the same chamber, dried by blowing
warm air and read by CD- or DVD-drive.
The procedure is otherwise similar in small clinical laboratories and in
hospitals, but these typically use test tube racks instead of conveyor
belts. However, even small laboratories have pipetting robots and the
process is basically the same as described above.
In field use, the samples must be often pipetted manually. Especially in
this case it is preferable that the sample is put in always using the same
fixed hole, with the disk rotating after each addition so that a new well
is aligned below this hole.
5.11.8. Adding Reagents and Washing Solutions
The assay devices of the present invention, as intended for use in large
clinical laboratories, contain multiple assay sites, each with signal
elements specific for a plurality of analytes, termed a panel. Panels are
generally configured so that the protocols are identical for each test in
that panel, i.e., temperature, reaction time, reagents and washing
solutions are the same. Thus, at one time only one reagent or washing
solution is added onto the BCD. Accordingly, the same solution is added
into all wells of the multiwell plate. a dispenser can be dedicated for
each reagent and washing solution. This allows the continuous use of the
same tips and tubing without any disposable parts.
The invention may be better understood by reference to the following
examples, which are offered by way of illustration and not by way of
limitation.
6. EXAMPLES
6.1 Example 1. Synthesis of a Spacer with Cleavable Siloxane Site
A representative cleavable spacer, shown schematically in FIG. 5, is
synthesized as follows.
In brief, the synthesis is begun by constructing the central portion of the
spacer molecule first. Both ends of the poly(ethyleneglycol) are then
silanized, e.g. with chlorodimethylsilane to afford a compound of the
formula of Compound I.
The silane groups then are derivatized with an alkenoic acid, straight or
branched chain (e.g., CH.dbd.CH(CH.sub.2).sub.n COOH, n=1-11, although the
number of carbon atoms is immaterial, such as vinyl acetic acid, acrylic
acid and the like) having a terminal double bond, such as vinyl acetic
acid to form a compound having the structural formula of Compound II, and
reacted further to provide a protected hydroxyl group on each side of the
silane to provide for later attachment of oligonucleotides as illustrated
by the compound having the structural formula of Compound III. Various
common reactants can be used for this purpose, and N-acryloyl serine and
TMT-serine methyl ester, when allowed to react in the presence of a
catalyst such as chloroplatinic acid, are exemplifications of preferred
reactants.
The resulting ester is partially hydrolyzed by the addition of an alkali
metal hydroxide, such as sodium hydroxide, in an alcoholic solvent, and
the adjacent protected hydroxyl group is preferentially hydrolyzed to
yield a compound represented by the structural formula of Compound IV.
Amino terminated poly(ethyleneglycol) is derivatized at one end with a thio
ester, such as 3-(2-pyridyldithio)propionic acid N-hydroxy succinimide
ester, and coupled with Compound IV to yield a compound represented by the
structural formula of Compound VI. The terminal ester group is hydrolyzed
to yield the acid, which is further reacted with methoxyacetic acid, to
afford the compound represented by the structural formula of Compound
VIII. That compound is treated with aminated poly(ethyleneglycol) to form
the completed spacer molecule substantially as illustrated in FIG. 5.
In detail, the synthesis is performed as follows:
Preparation 1: Compound I
To a mixture of poly(ethyleneglycol) (10 g, 10 mmol, av. MW 1,000 Aldrich
Chemical Company) and triethylamine (TEA) (2.1 g, 21 mmol) in 100 ml of
dichlormethane (DCM), is added dropwise 2.0 g of chlorodimethylsilane in
20 ml of DCM with cooling in an ice bath. After 10 minutes, the reaction
mixture is filtered and the filtrate is applied into a 200 g silica
column. The column is eluted with DCM/MeOH 19:1, and the eluant affords
poly(ethyleneglycol), di(dimethylsilyl) ether, the compound represented by
the structural formula of Compound I.
##STR1##
Preparation 2: Compound II
Compound I (10 g, 9 mmol) and vinylacetic acid (1.72 g, 20 mmol) is
dissolved into 60 ml of ethyl acetate (EtOAc). A catalytic amount (40 mg)
of chloroplatinic acid is added, and the mixture is heated to boiling and
boiled for 1 hour. After cooling, the solution is applied directly into a
200 g. silica column. The column is eluted with EtOAc and EtOAc/MeOH 9:1,
and the eluant affords poly(ethyleneglycol), di
(2-carboxyethyldimethylsilyl) ether, the compound represented by the
structural formula of Compound II.
##STR2##
Preparation 3: Compound III
Compound II (9.5 g, 8 mmol) and trimethoxytrityl-serine methyl ester (7.0
g, 16 mmol) are dissolved into 100 ml of DCM. Dicyclohexylcarbodiimide
(DCC) (3.25 g, 16 mmol) in 30 ml of DCM is added dropwise at room
temperature. After 1 hour the reaction mixture is filtered. The filtrate
is applied directly into 300 g silica column. The column is eluted with
DCM/TEA 99:1 and then with DCM/MeOH/TEA 94:5:1. The eluant affords the
compound represented by the structural formula of Compound III.
##STR3##
Preparation 4: Compound IV
Compound III (10 g, 5 mmol) is dissolved into 100 ml of EtOH and partially
hydrolyzed by adding 10 ml 0.5 M NaOH in EtOH. The mixture is slightly
acidified by adding 300 mg (5 mmol) acetic acid. The TMT-group proximal to
the carboxylate group is preferentially hydrolyzed. After 30 min the
mixture is made slightly basic by adding 0.5 ml tetraethylamine (TEA). The
EtOH solution is fractionated by HPLC using a reverse phase column eluted
with EtOH/Water/TEA 90:9:1. The eluant affords the compound represented by
the structural formula of Compound IV.
##STR4##
Preparation 5: Compound V
O,O'-Bis(aminopropyl)polyethyleneglycol (9.5 g, 5 mmol, av. MW 1900),
triethylamine (0.5 g, 5 mmol) and 3-(2-pyridyldithio) propionic acid
N-hydroxysuccinimide ester (0.77 g, 2.5 mmol) are dissolved into 150 ml of
DCM. The mixture is stirred 1 hour at room temperature, concentrated into
half volume and fractionated in 200 g silica column. The column is eluted
with DCM/MeOH 95:5, to afford the compound represented by the structural
formula of Compound V.
##STR5##
Preparation 6: Compound VI
Compound IV (3.5 g, 2 mmol) and Compound V (4.4 g, 2 mmol) are dissolved
into 100 ml of DCM and 450 mg (2.2 mmol) DCC in 5 ml of DCM is added.
After 1 hour the mixture is filtered, and fractionated in 150 g silica
column. The column is eluted with DCM/MeOH/TEA 94/5/1, to afford the
compound represented by the structural formula of Compound VI.
##STR6##
Preparation 7: Compound VII
Compound VI (6.0 g, 1.5 mmol) is dissolved into 50 ml of EtOH and 3 ml of
0.5 M NaOH in EtOH is added. After 30 min the product is purified by
reverse phase HPLC using EtOH/water/TEA EtOH/Water/TEA 90:9:1 as an
eluent, to afford the compound represented by the structural formula of
Compound VII.
##STR7##
Preparation 8: Compound VIII
Compound VII (4.0 g, 1 mmol) is dissolved into 80 ml of DCM. The mixture of
320 mg (2 mmol) of methoxyacetic acid anhydride and 202 mg (2 mmol) of
triethylamine in 5 ml of DCM is added. the mixture is evaporated by rotary
evaporator into dryness. The residue is purified by reverse phase HPLC
using EtOH/water/TEA EtOH/Water/TEA 90:9:1 as an eluent, to afford the
compound represented by the structural formula of Compound VIII.
##STR8##
Preparation 9: Compound IX
Compound VIII (4.0 g, 1 mmol) and 0,0'-bis(aminopropyl)poly-ethyleneglycol
(4.8 g, 2.5 mmol, av. MW 1900) are dissolved into 100 ml of DCM, 230 mg
(1,1 mmol) DCC in 5 ml of DCM is added. After 1 hour the mixture is
filtered and the mixture is fractionated in 100 g silica column using
DCM/MeOH/TEA 94/5/1 as an eluent, to afford the compound represented by
the structural formula of Compound IX, substantially as schematically
represented in FIG. 5.
##STR9##
6.2 Example 2. Synthesis of a Cleavable Magnesium Dicarboxylate Spacer
Recognizing Human IgG
Onto a gold-coated polycarbonate disk is added by ink-jet printer 2 .mu.l
of 10 .mu.M biotindisulfide water solution in 64 circular spots having a
diameter of 5 mm. Onto these same spots is added by ink-jet printer 2
.mu.l of a mixture of 1 .mu.M streptavidin and 1 .mu.M albumin.
Goat anti-human IgG (Bioprocessing, Inc., Scarborough, Me; Covalent
Immunology, Monroe, N.H.) is reduced by thioethanolamine to produce
univalent halves, each of which consists of one heavy chain and one light
chain (HL). Thioethanolamine is removed by dialysis and
maleimido-polyethyleneglycol-biotin (MAL-PEG-BIO; MW 3, 400, Shearwater
Polymers, Inc., Alabama) is added. A small amount of thioethanolamine is
added to render maleimido groups unreactive. The mixture is dialyzed
against 10 mM phosphate buffer (pH 7) in a dialysis tube (molecular weight
cut-off 30,000).
To this antibody derivative (Ab-PEG-BIO) is added a ten fold excess of
BIO-PEG-carboxylic acid and a one hundred fold excess of BIO-PEG-OMe in 1
.mu.M MgCl.sub.2. Two (2) .mu.l of this mixture is added, by ink jet
printer, onto the spots previously printed on the assay disk. The disk is
washed.
At this point, slightly fewer than 1% of streptavidin sites earlier-spotted
on the disk display the goat anti-human antibody half (HL) at the end of a
PEG spacer, somewhat fewer than 9% display carboxylic acid groups at the
end of a PEG spacer, and about 90% display hydroxymethyl groups, which are
inert in the present case.
Into a suspension of 10 mg streptavidin-coated latex beads (1micrometer in
diameter) is added 0.1 mg of Ab-PEG-BIO, prepared as above-described, 0.1
mg of BIO-PEG-carboxylic acid and 1 mg of BIO-PEG-OMe in pH 7 phosphate
buffer. The mixture is filtered through a 0.2 gm filter. As with the disk
surface, the beads display analyte-specific groups (PEG-Ab), carboxylic
acid groups, and carboxymethyl groups that are functionally inert in the
assay.
The beads are suspended in distilled water and the suspension added
uniformly onto the surface of the disk. The disk is shaken gently about
one hour to permit adherence of beads through ionic bond formation between
carboxylic acid groups displayed on the beads and carboxylic acid groups
presented from the surface of the assay device. Extra beads are removed by
gentle washing. The wash solution may contain a polyalcohol, such as
glycerol, mannitol, starch or the like to stabilize proteins during the
storage.
A sample containing human IgG is pipetted (10 .mu.l) onto each assay spot.
The assay device is incubated in a humidified incubator. Following
incubation, the assay disk is washed with an excess of 25 mM phosphate
buffer (pH 7) containing 100 mM sodium chloride.
Human IgG in the sample binds both to PEG-Ab that is directly adherent to
the assay disk surface and to PEG-Ab displayed by beads tethered adjacent
thereto by magnesium dicarboxylate groups.
The magnesium dicarboxylate groups are cleaved by addition of 10 .mu.l 50
mM EDTA, which chelates magnesium. Latex spheres that have not bound human
IgG are lost. Latex spheres that have bound human IgG that is additionally
bound to surface adherent Ab, are retained. The unbound spheres are washed
away with water. The disk is dried and read in an optical disk drive. The
concentration of human IgG is proportional to the signal generated by the
latex spheres.
6.3 Example 3. Detection of HIV-1 in a Nucleic Acid Assay
HIV-1 proviral DNA from clinical samples is amplified as follows,
essentially as described in U.S. Pat. No. 5,599,662, incorporated herein
by reference.
Peripheral blood monocytes are isolated by standard Ficoll-Hypaque density
gradient methods. Following isolation of the cells, the DNA is extracted
as described in Butcher and Spadoro, Clin. Immunol. Newsletter 12:73-76
(1992), incorporated herein by reference.
Polymerase chain reaction is performed in a 100 .mu.l reaction volume, of
which 50 .mu.l is contributed by the sample. The reaction contains the
following reagents at the following initial concentrations:
10 mM Tris-HCl (pH 8.4)
50 mM KCl
200 .mu.M each DATP, dCTP, dGTP, and dUTP
25 pmoles of primer 1, of sequence shown below
25 pmoles of primer 2, of sequence shown below
3.0 mM MgCl.sub.2
10% glycerol
2.0 units of Taq DNA polymerase (Perkin-Elmer)
2.0 units UNG (Perkin-Elmer)
Primer 1: 5'-TGA GAC ACC AGG AAT TAG ATA TCA GTA CAA TGT-3' (SEQ ID NO: 10)
Primer 2: 5'-CTA AAT CAG ATC CTA CAT ATA AGT CAT CCA GT-3' (SEQ ID NO: 11)
Amplification is carried out in a TC9600 DNA thermal cycler (Perkin Elmer,
Norwal, Conn.) using the following temperature profile: (1)
pre-incubation--50.degree. C. for 2 minutes; (2) initial cycle--denature
at 94.degree. C. for 30 seconds, anneal at 50.degree. C. for 30 seconds,
extend at 72.degree. C. for 30 seconds; (3) cycles 2 to 4--denature at
94.degree. C. for 30 seconds, anneal for 30 seconds, extend at 72.degree.
C. for 30 seconds, with the annealing temperature increasing in 2.degree.
C. increments (to 58.degree. C.) as compared to cycle 1; (4) cycles 5 to
39--denature at 90.degree. C. for 30 seconds, anneal at 60.degree. C. for
30 seconds, extend at 72.degree. C. for 30 seconds.
Following the temperature cycling, the reaction mixture is heated to
90.degree. C. for 2 minutes and diluted to 1 ml. Alternatively, the sample
is stored at -20 .degree. C., and after thawing, heated to 90.degree. C.
for 2 minutes then diluted to 1 ml.
Cleavable spacers with siloxane moiety are synthesized and attached in a
uniform density to a derivatized 120 mm polycarbonate disk substrate
essentially as set forth in sections 5.2 and 5.3 and Example 1
hereinabove. The following side members are then stamped on the cleavable
spacers:
first side member: 5'-TAG ATA TCA GTA CAA-3' (SEQ. ID NO. 12)
second side member: 3'-TAT TCA GTA GGT ACA-5' (SEQ. ID NO. 13)
A suspension of gold microspheres, 1-3 .mu.m in diameter, is added dropwise
to the disk, which is gently rotated to distribute the gold particles.
Gold particles are added until the effluent contains the same density of
particles as the initial suspension, thus ensuring saturation of the
cleavable spacers.
Sample is applied at room temperature dropwise near the center of the assay
device which is rotated at a continuous speed Rotation is halted after the
sample front reaches the periphery, and the disk is incubated stationary
at room temperature for 3-5 minutes.
One ml of sample buffer is added dropwise as a wash while the disk is
rotated. One ml of 100 mM sodium fluoride is added and distributed by disk
rotation. The disk is incubated stationary for 1-2 minutes, then 5 ml of
sample buffer is added dropwise during vigorous rotation of the assay
disk.
The disk is dried, then read directly in a CD-ROM reader programmed to
assay each predetermined site upon which cleavable spacers were deposited.
6.4 Example 4. Increased Specificity of a Nucleic Acid Hybridization Assay
In a direct nucleic acid hybridization assay, the side elements of the
cleavable signal element are oligonucleotides designed to hybridize with
distinct sites on a chosen, predetermined, nucleic acid to be detected in
the sample. For many applications of this methodology, cross-reactivity
with sample oligonucleotides having even a single mismatched nucleotide
should be minimized. In particular, nucleic acid hybridization assays
adapted to use the cleavable reflective signal element of the present
invention for detection of point mutations, as, e.g., for detection of
point mutations in the BRCA1 and BRCA2 genes that predispose to breast and
ovarian cancers, must be able to discriminate as between nucleic acid
samples containing a single mismatched nucleotide.
The longer the oligonucleotide side elements of the cleavable signal
element--and thus the longer the sequence that is complementary as between
the side elements and the nucleic acid sample--the greater the possibility
of erroneously recognizing a mismatched sample, since the strength of
hybridization, even given the presence of a mismatch, will be reasonably
high.
Thus, one way to reduce erroneous recognition of mismatched nucleic acid
sequences is to reduce the length of the side element oligonucleotides.
Specificity is increased by shortening side-arms to 8-mers or even to
6-mers. These will still hybridize at room temperature, depending on
stringency of wash, conditions of which are well known in the art. The
mismatched oligonucleotides would use five or fewer nucleotides for
pairing and will form highly unstable binding at room temperature.
This solution, however, presents its own problem: the relatively short
overall length, 12-16 nucleotides, used for recognition leads to a
concomitantly reduced overall strength of the hybridization required to
restrain the signal responsive moiety of the cleaved signal elements. Use
of ligase, as depicted in FIGS. 2E-2F, partly solves this problem.
Ligation will not only provide a stronger bond, but will further act to
ensure selectivity, since DNA ligase will not join oligonucleotides if
there is a mismatch near the end of the oligonucleotides. Because the
oligonucleotides are short, no mismatched base pairs are accepted. Ligase
serves as a very strict double-check for the match of the oligos.
An alternative, and complementary, solution, uses the triple recognition
principle illustrated in FIG. 2D-2E constructively to shorten the test
sample sequence available for hybridization to the cleavable signal
element side elements. A soluble specificity-enhancing oligonucleotide,
for example an 8-mer, which is complementary to the central part of the
sample oligonucleotide, is added to the sample solution prior to
contacting the assay device with the fluid sample. This 8-mer hybridizes
well under the testing conditions. The side elements of the cleavable
signal elements recognize six nucleotides in the immediate vicinity of the
preformed duplex.
Ligation will ensure selectivity and will also provide a strong bond.
Ligase will not join oligonucleotides if there is a mismatch near the end
of the oligonucleotides. Because the oligonucleotides are short, no
mismatched base pairs are accepted. Ligase serves as a very strict
double-check for the match of the oligos.
Currently DNA ligase T4 is preferred. It couples the 3'-hydroxy and the
5'-phosphate termini of hybridized oligonucleotides, if there is no gap or
mismatching oligonucleotides nearby. It requires ATP and Mg.sup.++ for the
full activity. DNAs that lack the 5'-phosphate can be rendered a suitable
substrate for ligation by phosphorylation with T4 polynucleotide or
similar kinase.
It will be apparent that the soluble specificity-enhancing oligonucleotide,
shown here as an 8-mer, that is added to the test sample may be designed
to position the potential mismatch near the sample ends, where mismatch
will be most disfavored for binding to the side elements.
Moreover, because addition of ligase ensures a covalent loop, stringency of
wash may be increased by addition of chaotropic agents and/or by heating
to remove any unselective oligonucleotides.
The "blocked" sample oligonucleotide suitable for and capable of binding
correctly to the side elements may be mimicked, however, by a sample
nucleic acid that possesses the requisite terminal hexanucleotide
sequences directly connected to one another without the intervening 8-mer
sequence.
As shown in FIG. 2D, further addition to the sample of a 10-mer with
sequence equally drawn from the first side element oligonucleotide
sequence and second side element oligonucleotide sequence will prevent
such binding upon contacting the assay device of the present invention.
The combination 8+10+8 of the specificity-enhancing soluble
oligonucleotides is presently preferred, but other combinations, such as
7+9+7 and 8+8+8 may be used.
A further method to increase specificity includes use of so-called padlock
probes, in which circularized oligonucleotides are catenated, permitting
extensive washing to remove weakly bound probes. Padlock probes can
achieve a 50:1 discrimination between complementary and singly mismatched
oligonucleotides (Nilsson et al., Science 265:2085 (1994)), while with
conventional probes this ratio is typically between 2:1 and 10:1.
Oligonucleotide side members having the following sequences are prepared by
automated synthesis so that each of them contains a terminal thio (or
aliphatic amino) group, depending on the attachment site with the
cleavable spacer molecule (5' end or 3' end).
Ia: 5'-CGGGTGTGG (SEQ. ID. Ib: CGGCCGCGG-3' (SEQ.
NO. 1) ID. NO. 5)
IIa: 5'-CGGGTGTGA (SEQ. ID. IIb: CGGCCGCGG-3' (SEQ.
NO. 2) ID. NO. 5)
IIIa: 5'-CGGGTGTGC (SEQ. ID. IIIb: CGGCCGCGG-3' (SEQ.
NO. 3) ID. NO. 5)
IVa: 5'-CGGGTGTGT (SEQ. ID. IVb: CGGCCGCGG-3' (SEQ.
NO. 4) ID. NO. 5)
The cleavable spacer molecules are synthesized with two aliphatic amino
groups, in place of the protected hydroxy groups above-described, and one
group is protected by monomethoxytrityl (MMT, acid labile) and the other
group is protected by fluorenyloxycarbonyl (FMOC, base labile). After the
removal of the FMOC-group, the amino function is allowed to react under
aqueous conditions with 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic
acid N-hydroxysuccinimide ester (SMMC). Thiol derivatized Ia is added to
the spacer molecule and allowed to couple to the spacer molecule.
Subsequently, MMT is removed by treatment with acetic acid, and after
washing with buffer, pH 8, SMCC is added, and oligonucleotide IIb is
allowed to couple with the spacer molecule. The spacer molecules prepared
above are attached to a polycarbonate substrate.
A test sample containing 5'-GCCCACACCGCCGGCGCC-3' (SEQ. ID NO. 6) is
prepared and allowed to contact the cleavable signal element at a
temperature that approximates the T.sub.m of the side members Ia and Ib.
The temperature of the sample solution is heated to about 20 degrees
Centigrade above the T.sub.m. Subsequently, the signal element is treated
with 0.1M sodium fluoride solution and washed. Spacer molecules remaining
attached to the surface signal the presence of, and tethering by,
5'-GCCCACACCGCCGGCGCC-3' (SEQ. ID NO. 6)
The foregoing process is applied to the analysis of 5'GCCCACACTGCCGGCGCC-3'
(SEQ. ID NO. 7) 5'GCCCACACGGCCGGCGCC-3' (SEQ. ID NO. 8) and
5'-GCCCACAGCCGGCGCC-3' (SEQ. ID NO. 9), using, respectively, spacer
molecules incorporating side members IIa and IIb, IIIa and IIIb, and IVa
and IVb.
6.5 Example 5. Noncleavable Spacer Assay for Detection of Spermidine
Spermidine (N-(3-aminopropyl)-1,4-butanediamine) has one secondary and two
primary aliphatic amino groups. Recognition of spermidine can be
accomplished by any functional groups that can be coupled with amino
groups with sufficiently high specificity. Because the presence of thiol
groups introduced by other molecules in a sample can interfere with the
amino group assay, however, the presence of thiol groups must be assayed
simultaneously with amino groups.
Noncleavable aliphatic spacers terminating in carboxylic groups are
synthesized and disposed on the solid surface substrate of an assay device
as described hereinabove. Plastic microspheres are coated by standard
techniques to display maleimido groups.
Two aliquots of each of three samples are separately incubated with the
maleimido-coated plastic spheres, one aliquot per sample at pH 6, the
other aliquot at pH 8. Amino groups present on components of the sample
react at pH 8 with the maleimido group. In the presence of spermidine,
reaction proceeds with modification of spheres to display amino groups.
Thiol-containing components react only at pH 6 with the maleimido groups.
Into all aliquots (two per sample) is then added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The aliquots are
then applied to separate assay sites of the assay device. The device is
washed, and then read in an optical disk reader.
In the presence of spermidine, plastic microspheres display an amino group
available for bonding to the carboxylic group of the spacers. In the
presence of EDAC, a peptide bond tethers the plastic sphere to the assay
device substrate. Thiols form unstable thioester bonds that hydrolyze
relatively fast.
For sample 1, binding is observed only for the aliquot incubated at pH 8,
confirming the presence of diamine, diagnostic of spermidine, in the
sample.
For sample 2, no binding is reported at pH 8, indicating the absence of
spermidine.
For sample 3, a positive result is reported for both pH 8 and pH 6,
indicating the presence of aminothiol in the sample, rendering the pH 8
test inconclusive for presence of spermidine. A separate test is thus
performed, as follows. To differentiate diamines and aminothiols, the test
with carboxylated plastic beads is performed as described above. Only
diamine will form a stable bridge between two carboxylic groups. Finally,
to detect any dithiol in the sample, both the plastic spheres and the
asswsay site should be functionalized with maleimido groups and the test
is performed at pH 6.
In the other embodiment the cleavable spacer can be used to bind the
plastic sphere onto the BCD surface. The recognition protocol is analogous
to one described above, except that the spacers must be cleaved in the end
of the assay.
The present invention is not to be limited in scope by the exemplified
embodiments and examples, which are intended as illustrations of
individual aspects of the invention. Indeed, various modifications thereto
and equivalents and variations thereof in addition to those shown and
described herein will become apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such modifications are
intended to be and are included within the scope of the appended claims.
All publications, patents, patent applications, and provisional patent
applications cited herein are incorporated by reference in their entirety.
Top