U.S. patent application number 12/188340 was filed with the patent office on 2009-09-24 for multiplexed molecular analysis systems.
Invention is credited to William J. BALCH.
Application Number | 20090239759 12/188340 |
Document ID | / |
Family ID | 21877589 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090239759 |
Kind Code |
A1 |
BALCH; William J. |
September 24, 2009 |
MULTIPLEXED MOLECULAR ANALYSIS SYSTEMS
Abstract
A method and apparatus for analyzing molecular structures within
a sample substance using an array having a plurality of test sites
upon which the sample substance is applied. The invention is also
directed to a method and apparatus for constructing molecular
arrays having a plurality of test sites. The invention allows for
definitive high throughput analysis of multiple analytes in complex
mixtures of sample substances. A combinatorial analysis process is
described that results in the creation of an array of integrated
chemical devices. These devices operate in parallel, each unit
providing specific sets of data that, when taken as a whole, give a
complete answer for a defined experiment. This approach is uniquely
capable of rapidly providing a high density of information from
limited amounts of sample in a cost-effective manner.
Inventors: |
BALCH; William J.; (The
Woodlands, TX) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD., SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
21877589 |
Appl. No.: |
12/188340 |
Filed: |
August 8, 2008 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10316077 |
Dec 11, 2002 |
7413852 |
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12188340 |
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09625086 |
Jul 25, 2000 |
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10316077 |
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09217154 |
Dec 21, 1998 |
6331441 |
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09625086 |
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09002170 |
Dec 31, 1997 |
6083763 |
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09217154 |
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60034627 |
Dec 31, 1996 |
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Current U.S.
Class: |
506/9 ; 506/39;
536/24.3 |
Current CPC
Class: |
G01N 35/028 20130101;
B01J 2219/00378 20130101; Y10T 436/115831 20150115; Y10S 435/808
20130101; B01J 2219/00626 20130101; Y10S 436/809 20130101; B01L
3/0262 20130101; G01N 35/0099 20130101; B01J 2219/00621 20130101;
C40B 60/14 20130101; B01J 2219/00596 20130101; Y10T 436/11
20150115; B01J 2219/00659 20130101; B01J 2219/00605 20130101; G01N
33/54366 20130101; G01N 2035/1039 20130101; B01J 2219/00637
20130101; B01L 3/0268 20130101; G01N 2035/1041 20130101; B01L
3/5085 20130101; G01N 35/1074 20130101; B01J 2219/00511 20130101;
B01J 2219/00612 20130101; G01N 35/1065 20130101; G01N 2035/1034
20130101; Y10S 436/80 20130101; B01J 19/0046 20130101; B01J
2219/00707 20130101; Y10S 436/804 20130101; B01J 2219/00722
20130101; B01J 2219/0061 20130101; B01J 2219/00617 20130101; B01J
2219/00317 20130101; B01L 2300/0893 20130101; C40B 40/06 20130101;
G01N 2035/00237 20130101; Y10S 436/805 20130101; B01J 2219/00369
20130101; B01J 2219/00585 20130101; B01J 2219/00315 20130101 |
Class at
Publication: |
506/9 ; 506/39;
536/24.3 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 60/12 20060101 C40B060/12; C07H 21/04 20060101
C07H021/04 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made at least in part with funds from the
National Aeronautics and Space Administration, Grant Number NAGW
4530.
Claims
1. A device for preparing a reaction substrate for conducting
multiplexed microassays to determine binding between a target
analyte and a capture probe, including: a plurality of capillary
tubes, each tube having a proximal end and a distal end; an
attachment site for holding said capillary tubes at a point spaced
from the distal ends of such capillary tubes; an array template for
slidably holding each capillary tube near its distal end, and for
allowing the distal end of each capillary tube to move with respect
to the attachment site; at least one manifold for positioning the
proximal end of each capillary tube within a corresponding supply
chamber, wherein each supply chamber is capable of supplying a
liquid reagent to at least one corresponding capillary tube; and a
positioning device for precisely positioning the array template and
said capillary tubes with respect to said reaction substrate and
depositing liquid reagents from said capillary tubes onto said
reaction substrate as biosites.
2. The device of claim 1, wherein said plurality of capillary tubes
comprises about 2 to about 10,000 tubes, having a center to center
spacing of about 80 .mu.m to about 5 mm.
3. The device of claim 1, wherein each supply chamber supplies only
one capillary tube.
4. The device of claim 1, wherein a supply chamber supplies a
plurality of capillary tubes.
5. The device of claim 1, wherein said capillary tubes comprise
stainless steel, plastic, rubber, glass, or fused silica coated
with polyimide.
6. The device of claim 1, wherein said device includes a plurality
of array templates.
7. The device of claim 1, further comprising a positioning device
for precisely positioning said reaction substrate.
8. The device of claim 1, wherein said capillaries have an inside
diameter of about 10 to about 200 .mu.m, and an outside diameter of
about 80 to about 500 .mu.m.
9. The device of claim 1, wherein said array template comprises an
array of sleeves, each sleeve having an inside diameter sufficient
to permit a capillary to slide through, and a length sufficient to
allow a precise pattern to be maintained while depositing fluids
onto said reaction substrate.
10. The device of claim 1, wherein said array template comprises a
plurality of holes formed in a rigid material.
11. The device of claim 1, wherein said array template comprises a
rigidly formed or held mesh.
12. The device of claim 1, further comprising a housing for
containing said supply chambers.
13. The device of claim 12, wherein said housing is capable of
maintaining an inert atmosphere.
14. The device of claim 12, wherein said housing is capable of
maintaining an elevated or reduced temperature.
15. The device of claim 12, wherein said housing may be pressurized
to a predetermined pressure.
16. The device of claim 15, wherein said pressure is modulated to
control the flow of liquid reagents through said capillary
tubes.
17. The device of claim 1, wherein said supply chambers are
positioned higher than the distal end of said capillary tubes to
provide a pressure head.
18. The device of claim 1, wherein deposit of capture probe is
controlled by electrophoresis.
19. The device of claim 1, wherein supply of liquid reagent is
controlled by electro-osmosis.
20. The device of claim 1, wherein said device includes multiple
sets of supply chambers, each for supplying liquid reagent to a
subset of said capillary tubes.
21. The device of claim 1, wherein said capillary tubes are free to
flex between said attachment site and said array template.
22. The device of claim 1, wherein the reaction substrate is
pre-etched to define a pattern of reactive areas matching the
geometry of the deposited biosites.
23. The device of claim 1, wherein said biosites are deposited
substantially simultaneously.
24. The device of claim 1, wherein each biosite is fluidically
isolated from each other biosite.
25. A reaction substrate for conducting multiplexed microassays to
determine binding of a target molecule and a capture probe/target
probe complex, said reaction substrate including an array of
biosites, each biosite comprising a single type of capture probe
bound to said substrate, each capture probe capable of binding to a
corresponding target probe having a capture probe specific domain
which specifically binds with a corresponding capture probe, and a
target analyte specific domain which specifically binds with a
target analyte.
26. The reaction substrate of claim 25, wherein said reaction
substrate is about 50 .mu.m to about 300 .mu.m in thickness.
27. The reaction substrate of claim 25, wherein each biosite on the
reaction substrate comprises a capture probe different from the
capture probe in every other biosite on said reaction
substrate.
28. The reaction substrate of claim 25, wherein said array of
biosites comprises from about 2 to about 10,000 biosites.
29. The reaction substrate of claim 25, further including a target
probe having a capture probe specific domain which specifically
binds with a corresponding capture probe, and a target analyte
specific domain which specifically binds with a target analyte.
30. The reaction substrate of claim 29, wherein each capture probe
is a first oligonucleotide, each capture probe specific domain is a
second oligonucleotide, and each target analyte specific domain is
a third oligonucleotide.
31. The reaction substrate of claim 29, wherein each capture probe
is a hapten, each capture probe specific domain is a hapten binding
polypeptide, and each target analyte specific domain is an
oligonucleotide.
32. The reaction substrate of claim 31, wherein each hapten binding
polypeptide is selected from the group consisting of an antibody, a
Fab, an F(ab'), an Fv, and SCA and a CDR.
33. The reaction substrate of claim 29, wherein each capture probe
is a first oligonucleotide, each capture probe specific domain is a
second oligonucleotide, and each target analyte specific domain is
a hapten binding polypeptide.
34. The reaction substrate of claim 29, wherein each capture probe
is a hapten binding polypeptide, each capture probe specific domain
is a hapten, and each target analyte specific domain is an
oligonucleotide.
35. The reaction substrate of claim 34, wherein each hapten binding
polypeptide is selected from the group consisting of an antibody,
an FV, and an Fab.
36. The reaction substrate of claim 29, wherein each capture probe
is an avidin, each capture probe specific domain is a biotin, and
each target analyte specific domain is an oligonucleotide.
37. The reaction substrate of claim 30, 31, 35, or 36, wherein each
target analyte specific domain is an oligonucleotide nucleic acid
amplification primer.
38. The reaction substrate of claim 25, wherein at least one
reaction substrate is contained in at least one reaction
chamber.
39. The reaction substrate of claim 38, wherein a plurality of
reaction substrates are contained within each reaction chamber.
40. The reaction substrate of claim 40, further including a
reaction vessel, wherein a plurality of reaction chambers are
contained within said reaction vessel.
41. The reaction substrate of claim 39, wherein said reaction
vessel comprises about 2 to about 10,000 reaction chambers.
42. The reaction substrate of claim 25, wherein said reaction
substrate is optically clear.
43. The reaction substrate of claim 25, wherein each capture probe
has a percentage base composition in the range of about 30-40% G,
30-40% C, 10-20% A, and 10-20% T.
44. The set of claim 25, wherein each capture probe has a length
ranging from 2 to 30 bases.
45. The set of claim 44, wherein each capture probe has a length
ranging from 5 to 25 bases.
46. The set of claim 45, wherein each capture probe has a length
ranging from 10 to 20 bases.
47. The set of claim 25, wherein each capture probe has a length of
about 16 bases.
48. The set of claim 25, wherein each capture probe has a length
that differs by no more than one base from the average length of
all capture probes.
49. The set of claim 25, wherein each capture probe possesses an
overall gross base composition which is substantially similar to
the overall gross base composition of all other capture probes.
50. The set of claim 25, wherein each capture probe has a sequence
homology that differs from the sequence homology of each other
capture probe by at least 20%.
51. The set of claim 50, wherein each capture probe has a sequence
homology that differs from the sequence homology of each other
capture probe by at least 40%.
52. The set of claim 51, wherein each capture probe has a sequence
homology that differs from the sequence homology of each other
capture probe by at least 50%.
53. The set of claim 52, wherein each capture probe has a sequence
homology that differs from the sequence homology of each other
capture probe by at least 60%.
54. The set of claim 25, wherein the sequence homology of any two
capture probes is less than 80%.
55. A reaction substrate for use in conducting multiplexed
microassays to determine binding between a target molecule and a
capture probe, said reaction substrate having an array of parallel
printed biosites, wherein each biosite comprises a single type of
capture probe bound to said reaction substrate.
56. A method for preparing a reaction substrate for use as an assay
device, comprising the step of parallel printing an array of
biosites on said reaction substrate, wherein each biosite comprises
a single type of capture probe bound to said reaction
substrate.
57. The method of claim 56, further including the step of binding a
plurality of target probes to said array of biosites, wherein each
target probe binds to a specific capture probe within said
army.
58. The method of claim 56, wherein each biosite comprises a spot
of about 25 to about 200 .mu.m in diameter.
59. The method of claim 56, wherein said reaction substrate is
optically clear and has a thickness of about 50 to about 300
.mu.m.
60. A method for identifying target analytes in a plurality of
separate samples, each target analyze capable of binding a
corresponding capture probe, including the steps of parallel
printing an array of biosites on a reaction substrate, each biosite
comprising a single type of capture probe bound to said substrate;
contacting each biosite with a sample including at least one target
analyze; and determining the presence or absence of binding of a
target analyte at each biosite in said array.
61. The method of claim 60, wherein each target analyte is
detectably labeled.
62. The method of claim 61, wherein each target analyte is labeled
with a fluorescent label.
63. The method of claim 61, wherein each target analyte is labeled
with an electroluminescent label.
64. The method of claim 61, wherein each target analyte is labeled
with a radioisotope label.
65. The method of claim 60, wherein the presence or absence of
binding at each biosite is determined using an optical sensing
array.
66. The method of claim 65, wherein the optical sensing array is
disposed in close proximity to the reaction substrate.
67. The method of claim 65, wherein the optical sensing array is
lensless.
68. An apparatus for preparing a reaction substrate for use as an
assay device, including: an attachment site for holding a plurality
of flexible capillary tubes at a point spaced from the distal ends
of such capillary tubes; a structure for positioning the proximal
end of each capillary tube within a corresponding supply chamber,
each supply chamber capable of supplying a liquid reagent to at
least one corresponding capillary tube; a print head for holding
said plurality of capillary tubes near their distal ends and for
precisely positioning the distal ends of said capillary tubes with
respect to said reaction substrate to deposit liquid reagents onto
said reaction substrate as biosites.
69. The apparatus of claim 68, where the print head includes an
ink-jet deposition device.
70. A method for detecting labeled sample molecules, comprising the
steps of: providing a reaction substrate having a plurality of
biosites, each biosite being attached to the reaction substrate,
the reaction substrate having an embedded conductive maternal
connected to an electrical source; contacting each biosite with a
sample of labeled molecules; initiating an electrochemical event
within the labeled molecules which releases electromagnetic energy;
detecting the released electromagnetic energy.
71. The method of claim 70, wherein the sample molecules are
fluorescently labeled.
72. The method of claim 70, wherein the sample molecules are
electroluminescently labeled.
73. The method of claim 70, wherein the released electromagnetic
energy is detected using, an optical sensing array.
74. The method of claim 73, wherein the optical sensing array is
disposed in close proximity to the reaction substrate.
75. The method of claim 73, wherein the optical sensing army is
lensless.
76. A method for detecting particle emissions from labeled sample
molecules, comprising the steps of: providing a reaction substrate
having a plurality of biosites, each biosite comprising a single
type of capture probe bound to said substrate; providing a lensless
array of particle detectors disposed in close proximity to said
plurality of biosites; contacting each biosite with a sample of
labeled molecules; converting, in said particle detectors, particle
emissions emanating directly from said sample into corresponding
electrical signals; forming an image from said electric signals
representing the quantitative presence or absence of the labeled
molecules on each biosite from said sample.
77. The method of claim 76, wherein the sample molecules are
fluorescently labeled.
78. The method of claim 76, wherein the sample molecules are
chemiluminescently labeled.
79. The method of claim 76, wherein the sample molecules are
radioisotope labeled.
80. A set of nucleic acid capture probes, each configured to be
bound to a reaction substrate, each capture probe having a length
sufficient to provide dissimilarity among capture probes, each
capture probe having a percentage base composition in the range of
about 30-40% G, 30-40% C, 10-20% A, and 10-20% T, wherein the set
is selected such that the nucleic acid sequence of each capture
probe in the set is substantially dissimilar from the nucleic acid
sequence of all other capture probes in the set.
81. The set of claim 80, wherein the length ranges from 2 to 30
bases.
82. The set of claim 81, wherein the length ranges from 5 to 25
bases.
83. The set of claim 82, wherein the length ranges from 10 to 20
bases.
84. The set of claim 80, wherein the length is about 16 bases.
85. The set of claim 80, wherein the lengths of said capture probes
differ by no more than one base from the average length of the
capture probes.
86. The set of claim 80, wherein each capture probe possesses an
overall gross base composition which is substantially similar to
the overall gross base composition of all other capture probes of
the set.
87. The set of claim 80, wherein each capture probe of a given
binding specificity has a nucleic acid sequence that differs from
the nucleic acid sequence of each other capture probe of different
specificity by at least 20%.
88. The set of claim 87, wherein each capture probe of a given
binding specificity has a nucleic acid sequence that differs from
the nucleic acid sequence of each other capture probe of different
binding specificity by at least 40%.
89. The set of claim 88, wherein each capture probe of a given
binding specificity has a nucleic acid sequence that differs from
the nucleic acid sequence of each other capture probe of different
binding specificity by at least 50%.
90. The set of claim 89, wherein each capture probe of a given
binding specificity has a nucleic acid sequence that differs from
the nucleic acid sequence of each other capture probe of different
binding specificity by at least 60%.
91. The set of claim 90, wherein each capture probe of a given
binding specificity has a nucleic acid sequence that differs from
the nucleic acid sequence of each other capture probe of different
binding specificity by at least 80%.
92. The set of claim 80, wherein the set includes at least 16
capture probes.
Description
[0001] This application is based on U.S. provisional application
60/034,627, filed 31 Dec. 1996, incorporated herein by
reference.
TECHNICAL FIELD
[0003] This invention relates to a multiplexed molecular analysis
apparatus and method for the detection and quantification of one or
more molecular structures in a sample.
BACKGROUND
[0004] It is very desirable to rapidly detect and quantify one or
more molecular structures in a sample. The molecular structures
typically comprise ligands, such as antibodies and anti-antibodies.
Ligands are molecules which are recognized by a particular
receptor. Ligands may include, without limitation, agonists and
antagonists for cell membrane receptors, toxins; venoms,
oligosaccharides, proteins, bacteria and monoclonal antibodies. For
example, DNA or RNA sequence analysis is very useful in genetic and
infectious disease diagnosis, toxicology testing, genetic research;
agriculture and pharmaceutical development. Likewise, cell and
antibody detection is important in numerous disease
diagnostics.
[0005] In particular, nucleic acid-based analyses often require
sequence identification and/or analysis such as in vitro diagnostic
assays and methods development, high throughput screening of
natural products for biological activity, and rapid screening of
perishable items such as donated blood, tissues, or food products
for a wide army of pathogens. In. all of these cases there are
fundamental constraints to the analysis, e.g., limited sample,
time, or often both.
[0006] In these fields of use, a balance must be achieved between
accuracy, speed, and sensitivity in the contest of the constraints
mentioned earlier. Most existing methodologies are generally not
multiplexed. That is, optimization of analysis conditions and
interpretation of results are performed in simplified single
determination assays. However, this can be problematic if a
definitive diagnosis is required since nucleic acid hybridization
techniques require prior knowledge of the pathogen to be screened.
If symptoms are ambiguous, or indicative of any number of different
disease organisms, an assay that would screen for numerous possible
causative agents would be highly desirable. Moreover, if symptoms
are complex, possibly caused by multiple pathogens, an assay that
functioned as a "decision tree" which indicated with increasing
specificity the organism involved, would be of high diagnostic
value.
[0007] Multiplexing requires additional controls to maintain
accuracy. False positive or negative results due to contamination,
degradation of sample, presence of inhibitors or cross reactants,
and inter/intra strand interactions should be considered when
designing the analysis conditions.
Conventional Technologies and Limitations
Sanger Sequencing
[0008] Of all the existing techniques, one of the most definitive
is the traditional Sanger sequencing technique. This technique is
invaluable for identifying previously unknown or unsuspected
pathogens. It is also valuable in determining mutations that confer
drug resistance to specific strains of disease organisms. These
analyses are generally research oriented. The end result of this
research, e.g., sequence determination of a specific pathogen, can
be used to design probes for identification applications in a
clinical setting.
[0009] However, there are constraints to employing this technique
in a clinical lab. The primary constraints are cost and throughput
due to the inherent labor intensive procedures, requiring multiple
steps to be performed by skilled personnel. For example, typical
analysis usually requires more than a day for completion. Of more
concern is the potential for ambiguity when multiple strains of a
pathogen are present in one sample. Virulence of the pathogen is
often determined by the strain. An example is HPV, also known as
human papilloma virus. Seventy strains of HPV are commonly known to
exist. Two strains, in particular, are strongly associated with an
increased risk of cervical cancer, hence the aggressiveness of
treatment of screening for malignancy is determined by the presence
of an HPV strain. Multiple strains cause indeterminate results when
using sequencing methodologies. The ideal assay would be
multiplexed with the selectivity to identify all strains
involved.
Blotting Techniques
[0010] Blotting techniques, such as those used in Southern and
Northern analyses, have been used extensively as the primary method
of detection for clinically relevant nucleic acids. The samples are
prepared quickly to protect them from endogenous nucleases and then
subjected to a restriction enzyme digest or polymerase chain
reaction (PCR) analysis to obtain nucleic acid fragments suitable
for the assay. Separation by size is carried out using gel
electrophoresis. The denatured fragments are then made available
for hybridization to labeled probes by blotting onto a membrane
that binds the target nucleic acid. To identify multiple fragments,
probes are applied sequentially with appropriate washing and
hybridization steps. This can lead to a loss of signal and an
increase in background due to non-specific binding. While blotting
techniques are sensitive and inexpensive, they are labor intensive
and dependent on the skill of the technician. They also do not
allow for a high degree of multiplexing due to the problems
associated with sequential applications of different probes.
Microplate Assays
[0011] Microplate assays have been developed to exploit binding
assays, e.g., an ELISA assay, receptor binding and nucleic acid
probe hybridization techniques. Typically, with one microplate,
e.g., micro-well titer plate, only one reading per well can be
taken, e.g., by light emission analysis. These assays function in
either one of two ways: (1) hybridization in solution; or (2)
hybridization to a surface bound molecule. In the latter case, only
a single element is immobilized per well. This, of course, limits
the amount of information that can be determined per unit of
sample. Practical considerations, such as sample size, labor costs,
analysis time place limits on the use of microplates in multiplex
analyses. With only a single analysis, reaction, or determination
per well, a multiple pathogen screen with the appropriate controls
would consume a significant portion of a typical 96 well format
microplate. In the case where strain determination is to be made,
multiple plates must be used: Distributing a patient sample over
such a large number of wells becomes highly impractical due to
limitations on available sample material. Thus, available patient
sample volumes inherently limit the analysis and dilution of the
sample to increase volume seriously affects sensitivity.
Polymerase Chain Reaction
[0012] Although, the polymerase chain reaction (PCR) can be used to
amplify the target sequence and improve the sensitivity of the
assay, there are practical limitations to the number of sequences
that can be amplified in a sample. For example, most multiplexed
PCR reactions for clinical use do not amplify more than a few
target sequences per reaction. The resulting amplicons must still
be analyzed either by Sanger sequencing, gel electrophoresis, or
hybridization techniques such as Southern blotting or microplate
assays. The sample components, by PCR's selective amplification,
will be less likely to have aberrant results due to cross
reactants. This will not be totally eliminated and controls should
be employed. In addition, PCR enhances the likelihood of false
positive results from contamination, thus requiring environmental
controls. PCR controls must also include an amplification positive
control to ensure against false negatives. Inhibitors to the PCR
process such as hemoglobin are common in clinical samples. As a
result, the PCR process for multiplexed analysis is subject to most
of the problems outlined previously. A high density of information
needs to be acquired to ensure a correct diagnostic determination.
Overall, PCR is not practical for quantitative assays, or for broad
screening of a large number of pathogens.
Probe-Based Hybridization Assays
[0013] Recently, probe hybridization assays have been performed in
array formats on solid surfaces, also called "chip formats." A
large number of hybridization reactions using very small amounts of
sample can be conducted using these chip formats thereby
facilitating information rich analyses utilizing reasonable sample
volumes.
[0014] Various strategies have been implemented to enhance the
accuracy of these probe-based hybridization assays. One strategy
deals with the problems of maintaining selectivity with assays that
have many nucleic acid probes with varying GC content. Stringency
conditions used to eliminate single base mismatched cross reactants
to GC rich probes will strip AT rich probes of their perfect match.
Strategies to combat this problem range from using electrical
fields at individually addressable probe sites for stringency
control to providing separate micro-volume reaction chambers so
that separate wash conditions can be maintained. This latter
example would be analogous to a miniaturized microplate. Other
systems use enzymes as "proof readers" to allow for discrimination
against mismatches while using less stringent conditions.
[0015] Although the above discussion addresses the problem of
mismatches, nucleic acid hybridization is subject to other errors
as well. False negatives pose a significant problem and are often
caused by the following conditions: [0016] 1) Unavailability of the
binding domain often caused by intra-strand folding in the target
or probe molecule, protein binding, cross reactant DNA/RNA
competitive binding, or degradation of target molecule. [0017] 2)
Non-amplification of target molecule due to the presence of small
molecule inhibitors, degradation of sample, and/or high ionic
strength. [0018] 3) Problems with labeling systems are often
problematic in sandwich assays. Sandwich assays, consisting of
labeled probes complementary to secondary sites on the bound target
molecule, are commonly used in hybridization experiments. These
sites are subject to the above mentioned binding domain problems.
Enzymatic chemiluminescent systems are subject to inhibitors of the
enzyme or substrate and endogenous peroxidases can cause false
positives by oxidizing the chemiluminescent substrate.
SUMMARY
[0019] The instant invention provides for both a multiplexed
environment to rapidly determine optimal assay parameters, as well
as a fast, cost-effective, and accurate system for the quantitative
analysis of target analytes, thereby circumventing the limitations
of single determination assays. The optimization of a multiplexed
assay can be carried out by experimental interrogation to determine
the appropriate solution conditions for hybridization and
stringency washes. The development of these optimal chemical
environments will be highly dependent on the characteristics of the
army of bound capture probe molecules, their complementary target
molecules, and the nature of the sample matrix.
[0020] Multiplexed molecular analyses are often required to provide
an answer for specific problems. For example, determining which
infectious agent out of a panel of possible organisms is causing a
specific set of disease symptoms requires many analyses. Capture
probe arrays offer the opportunity to provide these multivariate
answers. However, the use of single probe array platforms does not
always provide enough information to solve these kinds of problems.
Recent innovative adaptations of proximal charge-coupled device
(CCD) technology has made it feasible to quantitatively detect and
image molecular probe arrays incorporated into the bottom of
microplate wells. This creates a high throughput platform of
exceptional utility, capable of addressing several applications,
with very complex analysis parameters.
Uses
[0021] The multiplexed molecular analysis system of the instant
invention is useful for analyzing and quantifying several molecular
targets within a sample substance using an array having, a
plurality of biosites upon which the sample substance is applied.
For example, this invention can be used with microarrays in a
microplate for multiplexed diagnostics, drug discovery and
screening, analysis, gene expression analysis, cell sorting, and
microorganic monitoring (see examples below for each use).
Proximal CCD Imaging with Multiplexer Arrays
[0022] One application of the microplate based arrays of this
invention is in parallel processing of a large number of samples.
Large clinical labs process thousands of samples a day. A
microplate configured with a four by four (4.times.4) matrix of
biosites in each of the 96 wells would be able to perform a total
of 1536 nearly simultaneous tests from 96 different patient samples
utilizing the proximal CCD imager as illustrated in FIG. 1. FIG. 1
is a diagram showing a multiplexed molecular analysis detection
imaging system. Moreover, a microplate configured with 15.times.15
arrays of probe elements in each of 96-wells enables a total of
21,600 nearly simultaneous hybridization analyses, which becomes
significant for analyzing gene expression from specific cells.
[0023] Throughput is also important when screening natural products
for biological activity. A matrix of biosites that model binding
sites of interest may be placed in the bottom of each well and
interrogated with an unknown product. Thousands of molecules may be
screened per day against these biosite arrays.
Creation of Hierarchical Arrays
[0024] Another use of the microplate based arrays is for the
creation of hierarchical arrays for complex analyses. In this
format, multiple arrays operate in parallel to provide an answer to
a complex assay. The example of the diagnostic assay provided in
the Background section illustrates some of the parameters which
should be considered in order to provide an accurate result. For
any specific analysis, a set of probe elements most be chosen. The
selected probe elements should be able to selectively associate
with defined targets without significant cross association to other
macromolecules expected from either the patient or other organisms
commonly associated with a specific sample type. Controls most be
designed to prevent false positive or negative results from the
sources outlined in the Background section. Once this is done, a
combinatorial process can be used to identify the optimal
association and selectivity conditions for the defined analysis.
For nucleic acid applications, these conditions are highly
dependent on the capture probe length and composition, target base
composition, and sample matrix. The number of arrays to be used
depends on a number of different factors, e.g., the controls to be
implemented and the differences in base composition of the capture
probes. Ultimately, a set of integrated chemical devices emerge
that can rapidly, efficiently, and accurately provide an answer for
the molecular analysis of interest.
[0025] Another use of the hierarchical arrays and the reaction
vessel based arrays would be for screening samples for a broad
range of possible targets. In one case, a diagnostic test is
perforated to search for the cause of a defined set of symptoms. In
most cases this narrows the range of possible organisms to a small
number. Conversely, to screen donated blood or tissue for a broader
range of disease organisms, a decision tree approach could be
employed. Here an initial array or set of arrays could be chosen to
screen for broader classes of pathogens using probes for highly
conserved nucleic acid regions. Results from this would indicate
which additional array sets within the microplate to sample next,
moving to greater and greater specificity. If enough sample is
available, as might be the case with donated blood or tissue, all
of the decision tree elements could be interrogated simultaneously.
If sample quantity is limiting, the approach could be directed in a
serial fashion.
[0026] Assay development for any multiplex analysis is time
consuming. The microplate based arrays as described herein can be
used to speed the process for capture probe/target binding or
hybridization. A defined array can be deposited into each well of a
microplate and then the association reactions are carried out using
"gradients" of conditions that vary in two dimensions. For example,
consider a 96 well microplate containing nucleic acids arranged in
8 rows by 12 columns. In one step of the optimization; the effects
of pH on various substrate compositions might be examined to see
how this affects hybridization specificity. Twelve different pH's,
one for each column, and S different surface chemistries, one for
each row could be used under otherwise identical
hybridization-conditions to measure the effects on hybridization
for each capture probe/target element in the array. This type of
analysis will become essential as array technology becomes widely
used and is amenable to any receptor/ligand binding type
experiment.
[0027] The hierarchical array format, consisting of defined sets of
arrays with individually optimized chemical environments
functioning in parallel to provide an answer to a complex analysis,
can be implemented in other ways. Instead of a batch process, where
a series of analysis sets are present in each microplate, a
hierarchical array analysis set can be fashioned into a flow cell
arrangement. This would be specific to a particular analysis and
consist of the appropriate array sets and the necessary fluidics to
take a single sample and deliver the appropriate aliquot to each
array in the set. The fluidics will deliver the appropriate
association and wash fluids to perform the reactions, as defined
for each array in the set.
ADVANTAGES
[0028] The multiplexed molecular analysis system of the instant
invention has many advantages over the conventional systems. Some
of these advantages are discussed below.
High Throughput
[0029] Multiple DNA/RN.A probe arrays can be fabricated in the
bottom of 96 well microtiter plates which offer the potential of
performing 1,536 (96.times.16) to 21,600 (96.times.225)
hybridization tests per microtiter plate. Each well will contemn
probe army of N elements dispensed onto plastic or glass and bonded
to the microtiter plate. Moreover, by coupling the microtiter trays
to a direct (lensless) CCD proximal/imager, all 1,536 to 21,600
hybridization tests can be quantitatively accessed within seconds
at room temperature. Such proximal CCD detection approach enables
unprecedented speed and resolution due to the inherently high
collection efficiency and parallel imaging operation. The upper
limit to the hybridization tests per microtiter plate exceeds
100,000 based on a 100..mu.m center-to-center spacing of
biosites.
Low Cost
[0030] Since the capture probe volumes dispensed on the reaction
substrate can be limited to about 50 picoliters (pL), only 10
nanoliters (nL) of capture probe reagent is required to produce
over 1,500 distinct binding tests. The dispensing of the probe
arrays on plastic rolls or on thin glass sheets can be efficiently
performed in an assembly-line fashion with a modular ink-jet or
capillary deposition system.
Automated Operation
[0031] The multiplexed assay can be designed in a standard 96 well
microtiter plate format for room temperature operation to
accommodate conventional robotic systems utilized for sample
delivery and preparation. Also, the proximal CCD-based imager with
a graphical user interface will enable the automation of the
parallel acquisition of the numerous hybridization test results.
The CCD imaging system software provides automated filtering,
thresholding, labeling, statistical analysis and quantitative
graphical display of each probe/target binding area within
seconds.
Versatility
[0032] The proximal CCD detector/imager utilized in a particular
embodiment of the multiplexed molecular analysis system
accommodates numerous molecule labeling strategies including
fluorescence, chemiluminescence and radioisotopes. Consequently, a
single instrument can be employed for a variety of reporter groups
used separately or together in a multiplexed manner for maximal
information extraction.
High Resolution
[0033] The accompanying proximal CCD detector/imager offers high
spatial and digital resolution. In the preferred embodiment, CCD
pixel sizes of approximately 25.times.25 .mu.m.sup.2 will support
the imaging of hundreds to thousands of individual biosites on a
reaction substrate. Together with 16 bit digital imaging, a highly
quantitative image of the high density of biosites is achieved.
Fast Time-to-Market
[0034] Since the approach outlined is based on previously
demonstrated proximal CCD detection and imaging coupled with
microarrays dispensed in conventional sized microtiter plates, the
overall molecular analysis system is expected to provide a fast
time-to-market solution to complex multicomponent molecular-based
analyses.
[0035] Overall, the invention disclosed provides a method and
apparatus for both a multiplexed environment to rapidly determine
the optimal assay parameters as well as a fast, cost-effective, and
accurate system for the quantitative analysis of molecules, thereby
circumventing the limitations of single determination assays.
[0036] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a diagram showing a multiplexed molecular analysis
detection/imaging system.
[0038] FIG. 2 is a printed computer image obtained with the
proximal CCD imager showing deposited DNA probe biosites with
ink-jet printing.
[0039] FIG. 3 is a diagram showing the biosite deposition system
using staggered ink jet dispensing, modules.
[0040] FIG. 4 is a diagram showing the biosite deposition system
using multiple capillaries. FIG. 4a is a diagram showing biosite
deposition with array templates. FIG. 4b is a diagram showing
biosite deposition into nanoliter wells.
[0041] FIG. 5a is a diagram illustrating the Universal Array
concept.
[0042] FIG. 5b is a diagram showing direct binding for a target
probe associated with the Universal Array.
[0043] FIG. 5c is a printed computer image showing a
multi-microtiter well proximal CCD image of a 4.times.4 Universal
Array.
[0044] FIG. 5d is a printed computer image showing a single
microtiter well proximal CCD image of a 4.times.4 Universal
Array.
[0045] FIG. 6 is a diagram illustrating an ECL implementation in a
reaction vessel with proximal CCD imaging.
[0046] FIG. 7 is a diagram showing fabrication of ECL reaction
vessels.
[0047] FIG. 8 is a chemical drawing showing lanthanide
chelators.
[0048] FIG. 9 is a diagram showing the electronics schematic of a
multiplexed molecular analysis system.
[0049] FIG. 10a is a diagram showing a CCD sensor array for the
proximal CCD imager.
[0050] FIG. 10b is a diagram showing the tiling of CCD sensors to
form a large format proximal CCD imager.
[0051] FIG. 10c is a diagram showing an alternative tiling scheme
of multiple CCD sensors used to form a large format proximal CCD
imager.
[0052] FIG. 11 is a printed computer image showing microarrays
within a microplate reaction vessel. One single reaction chamber is
shown as an insert.
[0053] FIG. 12 is a diagram showing glass and polypropylene surface
coupling chemistries.
[0054] FIG. 13 is a diagram showing genotyping by universal point
mutation scanning.
[0055] FIG. 14 is a diagram showing microtiter-based throughput
genotyping.
[0056] FIG. 15 is a diagram showing homogeneous in situ microarray
detection of multiplexed PCR amplicons.
[0057] FIG. 16 is a diagram showing homogeneous in situ microarray
detection of multiplexed gap-ligase chain reaction products.
[0058] FIG. 17 is a diagram showing small molecule universal array
(drug screening/discovery).
[0059] FIG. 18 is a diagram illustrating spatial addresses of small
molecules covalently immobilized on amino-derivitized thin-bottom,
glass microtiter wells.
[0060] FIG. 19A is a printed computer image showing specific
imaging of biotin-addressable biosites detected using
streptavidin:HRP conjugate (4.times.4 single well microarray).
[0061] FIG. 19B is a printed computer image showing specific
imaging of digoxigenin-addressable biosites detected using
anti-digoxigenin:HRP conjugate (4.times.4 single well
microarray).
[0062] FIG. 19C is a printed computer image showing specific
imaging of fluorescein-addressable biosites detected using
anti-fluorescein:HRP conjugate (4.times.4 single well
microarray).
[0063] FIG. 19D is a printed computer image showing simultaneous
imaging of fluorescein, biotin, and digoxigenin biosites detected
using anti-fluorescein, anti-digoxigenin and steptavidin:HRP
conjugates (4.times.4. single well microarray).
DETAILED DESCRIPTION
Definitions
[0064] For the purpose of this invention, different words and
phrases are defined as follows:
[0065] By "target molecules or target analyte" is meant the
molecules of interest in a substance which are to be interrogated
by binding to the capture probes immobilized in an array.
[0066] By "mRNA target molecule or mRNA target-analyte" is meant a
substance containing identical mRNA components or a mixture of
disparate mRNAs.
[0067] By "capture probe, probe molecules or probes" is meant the
molecules which are deposited as biosites onto the reaction
substrate for interrogating the target molecules. Probes are meant
to include nucleic acids, DNA, RNA, receptors, ligands, antibodies,
anti-antibodies, antigens, proteins, and also small chemical
compounds such as drugs, haptens, or peptides.
[0068] The term "hapten, binding polypeptide" includes intact
antibody molecules as well as fragments thereof, such as Fab,
F(ab).sub.2, Fv, single chain antibody (SCA), and single
complementary-determining region (CDR). For purposes of the
invention, "hapten" and "epitope" are considered
interchangeable.
[0069] The term "array" refers to a two-dimensional spatial
grouping or an arrangement.
[0070] By "hierarchical array" is meant an array arranged in an
hierarchy or arranged in a graded or ranked series. Examples of
different "hierarchical arrays" comprising the multiplexed assay of
the invention include, but are not limited to, an array of a 96
well microtiter plate, wherein there are N probe sites or biosites
per well, wherein there are 10.sup.7 to 10.sup.10 molecules for
each probe site or biosite, wherein an array of M depositors are
used to deposit probes in each probe site onto the film substrate
that forms the bottom of the well in a 96 microtiter well reaction
chamber. The depositors can deposit the probes via many different
mechanisms, e.g., ink jet deposition, capillary, and
photolithography.
[0071] The term "probe arrays" refers to the array of N different
biosites deposited on a reaction substrate which serve to
interrogate mixtures of target molecules or multiple sites on a
single target molecule administered to the surface of the army.
[0072] The term "oligonucleotide probe arrays" refers to probe
arrays wherein the probes are constructed of nucleic acids.
[0073] By "charge coupled device;" also referred to as CCD, is
meant a well-known electronic device which outputs an electrical
signal proportional to the incident energy upon the CCD surface in
a spatially addressable manner.
[0074] The term "CCD proximal detection" refers to the use of CCD
technology for detection and imaging in which the CCD is proximal
to the sample to be analyzed, thereby avoiding the need for
conventional lenses.
[0075] By "ligands" is meant molecules which are recognized by a
particular receptor. "Ligands" may include, without limitation,
agonists and antagonists for cell membrane receptors, toxins,
venoms, oligosaccharides, proteins, bacteria and monoclonal
antibodies.
[0076] By "multiplexed" is meant many or a multiple number.
[0077] By "multiplexed diagnostic assay" is meant a method for
performing in parallel a large set or number of diagnostic assays.
Thus a set of parallel reactions can be handled with the same
effort as a single sample in previously described methods. Hence, a
greater number of assays can be handled within a fixed period of
time. The parallel set of reactions or multiplexed assay must be
deciphered at the end of the process. This is done by labeling or
tagging the biosite, as defined herein.
[0078] The term "reaction vessel" refers to an array of reaction
chambers as defined below. An example of a reaction vessel is a 96
well microtiter plate.
[0079] By "reaction chamber" is meant the environment in which the
hybridization or other binding association takes place.
Commercially available reaction vessels contain at least one
reaction chamber, but can contain 8, 24, 96 or 384 reaction
chambers. For this invention, "reaction chamber(s)," "well(s),"
"reaction site(s)," "reaction substrate(s)," "array hybridization
site(s)," "hybridization chamber(s)," and "hybridization well(s),"
are used interchangeably. An example of a reaction chamber is one
of the 96 microtiter wells in a 96 well microtiter plate.
[0080] By "biosite" is meant the biological molecules or capture
probes that are deposited on the top surface of the reaction
substrate, or base material. Under appropriate conditions, an
association or hybridization can occur between the capture probe
and a target molecule. The component strands of the biological
molecule form the biosite since there is the potential of a
reaction occurring between each component strand of the biological
molecule and the target molecule. For example, each reaction
chamber can contain at least one biosite. The maximum number of
biosites per reaction chamber will depend on the size of the
reaction vessel and on the practical optical resolution of the
accompanying detector/imager. For example, an array of 16
(4.times.4 array) biosites may be deposited on the hybridization
substrate or base material that eventually forms the bottom of the
entire reaction vessel. Each biosite comprises a circle of
approximately 25-200 microns (.mu.m) in diameter. Thus, for a 16
biosite array, each of the 16.times.200 .mu.m diameter area
contains a uniform field of probes attached to the hybridization
substrate (base material) in a concentration which is highly
dependent on the probe size and the well size. Each 25-200 .mu.m
diameter area can contain millions of probe molecules. Also, each
of the 16 different biosites (probe sites) can contain one type of
probe. Thus, 16 different probe types can be assayed in an array
containing 16 biosites (4.times.4 array) per reaction chamber. As
another example, four separate 10.times.10 arrays (400 biosites)
can be generated to fit into one well of a 96 well microtiter plate
with sufficient spacing between each of the 400 biosites. For this
10.times.10 format, 400 hybridization experiments are possible
within a single reaction chamber corresponding to 38,400
(96.times.400) assays/hybridization that can be performed nearly
simultaneously.
[0081] By "reaction substrate" is meant the substrate that the
biosites or probe sites are deposited on by using the depositors.
Examples of "reaction substrates" include, without limitation,
nylon membrane, polypropylene, polystyrene, vinyl, other plastics
and glass.
[0082] By "modular deposition array" is meant an array of
depositor. The number of depositors depends primarily on the
dimensions of the reaction substrate. For example, there can be
four depositors fitted nest to each other, staggered regarding the
front to back position of each depositor. Each depositor can be
directly coupled to a housing reservoir. The housing reservoir
holds a solution, e.g., a solution containing a desired probe at an
appropriate concentration. The number of injection mechanisms again
depends on the design of the depositor, e.g., ranging from one to
several injection mechanisms per depositor.
[0083] By "array formats on solid surfaces" is meant chip formats
or microarrays.
[0084] By "throughput" is meant the number of analyses completed in
a given unit of time.
[0085] By "decision tree approach" is meant a sequential routing
approach in which at each step an assessment is made which directs
the subsequent step.
[0086] By "hybridization detection" is meant to include, without
limitation, a means of two or more components to interact through
hybridization, an association, linking, coupling, chemical bonding,
covalent association, lock and key association, and reaction. For
the purpose of this invention, these terms are used
interchangeably.
[0087] By "methods of detecting (or detection) the
association/hybridization" is meant to include, without limitation,
fluorescent labeling, radioisotope labeling, chemiluminescence
labeling, bioluminescence labeling, calorimetric labeling. Labeling
can be achieved by one of the many different methods known to those
with skill in this art.
[0088] The term "luminescence" refers to, without limitation,
electrical (electro), chemical, fluorescence, phosphorescence,
bioluminescence, and the like. However, for this invention,
electrochemiluminescence or electrical chemiluminescence (ECL)
labeling is included as another method of detection which does not
require a wash step to remove excess target molecules from the
solution, and is highly sensitive. For the electrochemiluminescence
or electrical chemiluminescence method of detection, once
hybridization/association has occurred and a voltage has been
applied, only the labeled target molecules associated with the
biosite will emit light and be detected. The residual excess label
in the solution not associated with the biosite will therefore not
emit light.
[0089] This application is related to the following pending United
States patent, applications, incorporated herein by reference: U.S.
Ser. No. 07/794,036 entitled "Method and Apparatus for Molecular
Detection" filed Nov. 11, 1991, U.S. Ser. No. 08/353,957 entitled
"Multiple Molecule Detection Apparatus" issued Jul. 2, 1996, U.S.
Ser. No. 08/457,096 entitled "Multiple Molecule Detection Method"
filed, Jun. 1, 1995, U.S. Ser. No. 07/872,582 entitled "Optical and
Electrical Methods and Apparatus for Molecule Detection" filed Apr.
23, 1992, U.S. Ser. No. 08/511,649 entitled "Optical and Electrical
Methods and Apparatus for Molecule Detection" filed Aug. 7, 1995,
and U.S. Ser. No. 08/201,651 entitled "Method and Apparatus for
Detection and Imaging Particles" filed Feb. 25, 1994.
Overview
[0090] The multiplexed molecular analysis system of the invention
can be divided into four aspects:
[0091] A. Preparing the sample for subsequent association to a
probe array within the reaction chamber. This includes all
front-end processes such as purification, isolation, denaturation
and labeling required to extract the target molecules from the
sample.
[0092] B. Binding target molecules to the biosites within
specialized reaction chambers in sufficient concentrations for
association to occur. Following association, non-specific binding
of target molecules is often minimized by washing out the reaction
chambers.
[0093] C. Detecting and/or imaging the association (hybridization)
of the target molecules with the biosites within each reaction
chamber by proximal detection/imaging.
[0094] D. Processing the images to determine information about the
target molecules such as the presence and amount of specific
molecular constituents within a given sample that leads to the
analysis output.
[0095] The advantage of the instant invention lies in the
particular implementation of the above four procedures/steps, in
particular in the method and apparatus for:
[0096] STEP 1. Biosite Deposition. Biosite deposition relates to
constructing microarrays.
[0097] STEP 2. Self Assembling Arrays--Universal Arrays. Creating
and constructing self assembling probe arrays or universal arrays
enables on-line configuration of the biosites wherein an unvarying
probe array (capture probes) is activated by binding to a cognate
set of adapters (target probes) to yield a modified probe array
which is specifically configured for analysis of a target or target
mixture. For this invention, "cognate" is defined for nucleic acids
as a sequence which is complementary by the means of Watson-Crick
pairing to another sequence.
[0098] STEP 3. Molecular Labeling Strategies. Molecular labeling
strategies relates to versatile labeling of the target molecules
(fluorescence, chemiluminescence, etc.) consistent with proximal
large area detection/imaging.
[0099] STEP 4. Detection System. A detection system relates to
parallel detection and/or imaging in the reaction vessel containing
the reaction chambers using a proximal large area
detector/imager.
Step 1--Biosite Deposition
[0100] Biosite deposition relates to constructing microarrays.
There are many different methods that may be used for depositing
biosites into/onto the reaction chamber: Three of these approaches
are taught below.
1. Ink Jet Deposition
[0101] Ink jet printing can be employed for printing the biological
fluids to form the biosites. This approach provides very low
droplet volumes (=100 pL with 75 .mu.m diameter spot size) which
minimizes reagents used and therefore cost. Moreover, the printing
process can be accelerated to thousands of droplets per second,
thereby enabling a high throughput production capability for the
reaction vessels.
[0102] One Method useful for this invention utilizes
electromechanically driven ink jets which produce a volumetric
change in the fluid by applying a voltage across a piezoelectric
material (See Hansell, U.S. Pat. No. 2,512,743, 1950). The
volumetric changes causes a subsequent pressure transient in the
fluid which can be directed to form a drop on demand (D. Bogg et
at., IBM Jour Res Develop (1984) 29:214-321.
[0103] Individual ink jet devices can be integrated in a modular
fashion to enable the printing of multiple fluids. For example,
MicroFab Inc. has developed an ink jet based DNA probe array
printer constructed of eight modular dispensing units, each with an
integral 500 mL reservoir independently addressed by respective
drive and control electronics.
[0104] FIG. 2 depicts a printed computer image showing DNA probe
biosites deposited with ink jet printing. FIG. 2 illustrates actual
biosite deposition whereby an array of 100 DNA probe biosites per 1
cm.sup.2 was ink jet deposited onto a glass substrate. The array
consists of alternating columns of match and mismatch I2mer probes
which were subsequently hybridized to a,I2mer single-stranded DNA
target. The mismatch columns correspond to an A-A mismatch in the
probe/target complex. The actual image was captured within 1 second
by the proximal CCD detector/imager described below.
[0105] L banks of modular ink-jet devices containing M depositors
per module can be assembled in a staggered fashion to print
L.times.M different biosites on the bottom surface of the reaction
chambers as illustrated in FIG. 3. FIG. 3 is a diagram showing the
biosite deposition system using staggered ink-jet dispensing
modules. Here the reaction vessel is moved by a precision
motor-controlled stage underneath the ink-jet devices for rapid
printing. By constructing additional banks of modular ink-jet
devices and/or miniaturizing the individual depositors, an
arbitrarily large number of distinct biosites can be printed in the
reaction vessels. Alternatively, the printing can be performed on
thin substrates such as glass or plastics which are subsequently
bonded to form the bottom of the reaction vessels.
2. Capillary Deposition
[0106] Another approach to biosite deposition involves the use of
capillary tubing to dispense small amounts of the biosite solution
onto the reaction substrate as illustrated in FIGS. 4,4a, and 4b.
FIG. 4 depicts a biosite deposition system using multiple
capillaries. FIG. 4a is a diagram showing biosite deposition with
array templates. FIG. 4b is a diagram showing biosite deposition
into nanoliter wells. As shown, a storage vessel which contains the
appropriate solutions is pressurized momentarily to prime tubes
held in appropriate position by a manifold to initiate the
capillary dispensing action. With very small inner diameter
capillaries (<50 .mu.m), continuous pressure may be applied.
Pressure pulses of varying duration can be utilized to deliver
larger volumes of solution. Upon contact with the reaction
substrate, the capillary tubes simultaneously deliver small volumes
of the biosite solutions at precise locations controlled by spatial
arrangement of the bundled capillaries.
[0107] In this invention, the storage vessel allows for sampling
either from a standard format microtiter plate or a customized
plate designed to hold small volumes of liquid, allowing the
capillary to efficiently dispense picoliter volumes of liquid to
many thousands of biosites with minimal loss to evaporation or
possibility of cross contamination.
[0108] The lid of the storage vessel holder can be attached to a
Z-axis motion control device to allow for automated changes of the
biosite solutions contained in the microplates which may be
delivered by a robotic arm. This is useful for printing sets of
arrays containing large numbers of solutions, such as small
molecule libraries used in drug discovery.
[0109] Also in this invention, the capillary tubing may be made of
fused silica coated with an outer layer of polyimide. These tubes
are available commercially in any length with various widths and
internal diameters. The preferred dimensions are 80 to 500 .mu.m
outer diameter (OD) and 10 to 200 .mu.m inner diameter (ID). The
capillary bundles may be affixed to a robot arm and held in a
precise pattern by threading the capillaries through array
templates. An array template is a structure designed to maintain
the capillaries in the desired configuration and spacing, and may
consist of, without limitation, a metal grid or mesh, a
rigidly-held fabric mesh, a bundle of "sleeve" tubes having an
inner diameter sufficient to admit the fluid delivery capillaries,
or a solid block having holes or channels, e.g., a perforated
aluminum block.
[0110] The embodiment depicted employs 190 .mu.m OD capillaries,
which are threaded through an attachment site at the top of the
printing fixture. The tubes extend down from the attachment site
through an area that allows for the capillaries to flex during
printing. Below the flex region the capillaries are threaded
through an array template or a set of fused silica sleeves held in
a grid pattern by the aluminum holder assemblies. The capillary
sleeves/array template constitute an important innovation. The
array templates/capillary sleeves also allow the capillary tubing
to travel smoothly and independently with respect to each other in
the vertical axis during printing.
[0111] The printing system can print high density probe arrays
covering the bottom surface of microplate wells. o accomplish this,
the printing system must be able to maintain a precise printing
pattern and accommodate irregular surfaces. Rigid tubes could be
used to maintain a precise pattern, however, they cannot readily
accommodate irregular surfaces. Flexible tubes will print on uneven
surfaces but will not maintain a precise printing pattern. The
rigid sleeves, which extend below the aluminum holder assembly
approximately 2 cm. support the flexible 190 .mu.m OD fused silica
capillary tubing and provide the structural rigidity necessary to
maintain a precise grid pattern over this distance. The sleeves
also allow the 190 .mu.m tubing to travel smoothly in the Z-axis
during printing. This ability coupled with the flexibility of the
small OD capillary tubing allows for successful printing on
surfaces that are not completely flat or absolutely perpendicular
to the printing fixture. Since the robot arm extends 0.1 mm to 0.3
mm beyond the point where the capillary bundle contacts the
surface, the capillaries flex in the deflection zone illustrated in
FIG. 4 resulting in total surface contact among all capillaries in
the bundle. When the printing fixture withdraws from the substrate,
the capillaries straighten, returning to their original positions.
The highly parallel nature of the capillary-bundle printing
technique allows for microarrays containing from two to over 10,000
chemically unique biosites to be created with a single "stamp." The
printer can print these arrays at a rate of approximately one per
second. This represents a greater than 10-fold increase in speed
over existing technologies such as photolithographic in situ
synthesis or robotic deposition using conventional load and
dispense technology.
[0112] In photolithographic microarray synthesis, a series of masks
are sequentially applied to build the nucleic acid probes a base at
a time. An array of oligonucleotide probes each 12 bases long would
require 48 masks (12 nucleotide positions.times.4 bases). This
process takes approximately 16 hours to complete a wafer containing
43 microarrays.
[0113] Current robotic microarray printing or gridding systems are
universally based on various load and dispense techniques. These
techniques can be split into two categories. Active loading systems
such as syringe needles or capillaries draw up enough solution to
dispense multiple biosites or array elements before returning to
reload or collect a new probe solution. Pin style printing or
gridding systems can only print one biosite per pin at a time. The
pins are dipped into the probe solutions momentarily and the amount
of solution adhering to the pin is sufficient to print a single
biosite. Both categories have limitations that are resolved by the
capillary bundle printing system described herein.
[0114] Production capacity is a primary constraint in microarray
manufacturing limiting the use of microarrays in high volume
applications such as drug discovery due to the cost and limited
availability. For photolithographic in situ synthesis, the
constraint is the number of individual masks that must be applied
to create an array of probes with the necessary length to be
effective. To increase capacity, the production systems must be
duplicated. Current capacities for this approach (approximately
80,000 arrays for 1997) do not meet the needs for the drug
discovery market, where a single company may screen over 100,000
samples per year.
[0115] Robotic printing systems currently manufacture microarrays
in a largely serial fashion. The geometry of the fluid reservoir is
often responsible for the limited degree of parallel biosite
deposition. This can be explained by illustrating the process
needed to produce a microarray. A "micro" array has a small overall
dimension, typically smaller than 2 cm by 2 cm. The actual size is
determined by the number of array elements and their spacing, with
an emphasis on reducing the overall size as much as possible to
reduce reagent costs and sample requirements. If a parallel
printing approach is implemented using multiple pins or depositors,
the geometries of these depositors must allow them to interface
with the probe solution reservoirs and still be able to fit within
the confines of the area to be occupied by the microarray. If a 1
cm.sup.2 100 element microarray (10.times.10) is to be constructed
using a standard 384 well microplate with wells spaced 4.5 mm on
center as the probe solution reservoir, only 4 depositors can be
used to print simultaneously within the microarray. A total of 25
cycles of loading and printing would be required to complete the
array. In comparison, this array would be manufactured with a
single print step for a capillary bundle printer with 100
capillaries. This is 50 times faster than robotic depositors using
a load and dispense technique. If the same array is condensed into
a 0.5 cm.sup.2 area, then only one depositor can be used, resulting
in a 200 fold differential in manufacturing time compared with the
capillary bundle printer.
[0116] An important feature of the capillary bundle printer is the
manner in which it interfaces to the printing solution storage
vessel. The capillary bundles have a printing (distal) end and a
storage vessel end. The printing solution is held in a sealed
container that positions every capillary in the printing bundle via
a manifold so that each capillary dips into a specific well (supply
chamber) of a microtiter plate, one capillary per well. Current
multi-well microtiter plates are available with 96, 384, or 1536
wells, and can contain up to 96, 384, or 1536 individual probe
solutions, respectively. For microarrays containing more probe
elements, multiple printing solution reservoirs or storage vessels
can be interfaced to a single print head, as illustrated in FIG.
4a. This design concept eliminates the geometry problems associated
with load and dispense systems. The flexible fused silica
capillaries can be gathered together with the array templates or
sleeves to create a print head with capillaries spaced as close as
200 .mu.m center to center.
[0117] The enclosed printing solution storage vessel is purged with
an inert gas during the priming step of the printing process, which
also serves to maintain an inert environment for the probe
solutions. Contamination of the probe solutions is minimized
because of the single direction of flow through the capillaries.
The printing end does not dip into the reservoir after every print
cycle as in the load and dispense techniques. This is important
with contact printing where the depositors touch the surface of the
chip or slide that will contain the microarray. These surfaces are
chemically treated to interact or bind to the probe solutions.
Residual reactive chemicals, or even dust and dirt could be
introduced into the probe solution supply chambers with load and
dispense systems.
[0118] Often, the solution to be printed is available in limited
quantity or is very expensive. This is often the case in
pharmaceutical drug discover applications where small molecule
libraries, containing hundreds of thousands of unique chemical
structures that have been synthesized or collected and purified
from natural sources, are used in high throughput screens of as
many potential disease targets as possible. These libraries must be
used as efficiently as possible. The amount of fluid that is
required for each printing system varies depending on the design.
Most require a minimum of 100 microliters (uL) and are able to
print less than 1,000 slides, with a significant amount of solution
lost to washing between print cycles. The capillary array printer
requires only 3 .mu.L with less than 1 .mu.L used for the initial
priming: This volume of printing solution is sufficient to print
between 20,000 to 30,000 microarrays with each capillary dispensing
50 to 100 .mu.L per array. Load and dispense systems deliver
anywhere from 800 .mu.L to several .mu.L per array.
[0119] The highly parallel approach allows probe solution
deposition in a microarray geometry (less than 2 cm.times.2 cm)
independent of the geometry of the probe solution storage vessel.
This permits production of an entire microarray containing from 2
to >10,000 unique capture probes (biosites) in a single stamp of
the print head.
[0120] The flexible fused silica tubing (or other suitable material
such as glass, Teflon or other relatively inert plastic or rubber,
or thin, flexible metal, such as stainless steel) originating at
the printing storage vessel, pass through a series of arraying
templates or sleeves that are held at specific locations in the
print head. An attachment site holds the capillaries in a fixed
position that does not generally allow horizontal or vertical
movement. The capillaries extend down from this anchor point
through an open area ("flexation zone") and into a set of array
templates or sleeves. These lower array templates or sleeves serve
to. hold the printing capillaries in a geometry that matches the
microarray to be printed. The array templates limit the lateral
movement of the printing capillaries to preserve the correct
printing pattern, while allowing unrestricted vertical movement of
each printing capillary independently of each other. This feature
allows the print head to print on slightly irregular or uneven
surfaces. The print head moves downward to contact the substrate
that is to receive the probe solutions, after the initial contact,
the downward movement continues (the distance depends on the
surface, from 100 .mu.m to a few .mu.m) to ensure that all of the
printing capillaries contact the surface. The flexation zone
positioned between the attachment site (that is holding the
capillaries fixed) and the array templates or sleeves allows each
capillary, to bend so as to accommodate the "overdrive" of the
print head. When the print head moves up away from the substrate,
the printing capillaries straighten out again.
[0121] The capillary bundle originates in an enclosure containing
discreet fluid supply chambers, such as the wells in a microtiter
plate. Each capillary is inserted into a specific well, which
usually contains a unique probe solution with respect to the other
wells. The storage vessel can be momentarily pressurized to begin
the fluid flow in all of the capillaries simultaneously to prime
the printer. After priming, continuous flow of the probe solutions
through the capillaries is thereafter facilitated by adjusting the
head height .DELTA.H (the vertical distance from the upper fluid
reservoir and the printing tips, as shown in FIG. 4), or by
electro-osmotic or electrophoretic force (where the tubes, storage
vessels, and reaction chambers are appropriately modified to
maintain and modulate an electro-osmotic and/or electrophoretic
potential). The chamber can maintain an inert environment by
pressurizing the chamber with an inert gas, such as nitrogen or
argon.
[0122] Fluid volumes deposited at each biosite can be modified by
adjusting the head height, by applying pressure to the printing
solution storage vessel, by changing the length or inner dimension
of the printing capillaries, or by adjusting the surface tension of
the probe solution or the substrate that is being printed.
[0123] The prime and continuous print with multiple capillaries
prevents contamination of the probe solution that can occur with
load and dispense systems, which must contact the surface and them
return to the probe solution to draw more fluid. The continuous
printing of the capillary bundle printer is extremely efficient and
proves to be an enabling technique for applications that require
the use of small volumes of probe solution. The small outer and
inner diameters of the printing capillaries allow for printing as
many as 10,000 spots per .mu.L from a total volume of less than 5
.mu.L.
[0124] In an alternative embodiment, the capillary tubes may be
essentially rigid tubes (e.g., stainless steel) mounted in flexible
or movable fashion at the attachment site, and slidably held by an
array template. In this embodiment, the plurality of capillary
tubes can be pressed against a reaction substrate and "even up" at
their distal ends by moving lengthwise through the array template,
thus accommodating uneven deposition surfaces.
3. Photolithography/Capillary Deposition
[0125] To increase the spatial resolution and precision of the
capillary deposition approach, a combined photolithographic
chemical masking and capillary approach is taught herein. The first
photolithographic step selectively activates the precise biosite
areas on the reaction substrate. Once selective activation has been
achieved, the resulting capillary deposition results in uniform
biosite distribution.
[0126] Many different substrates can be used for this invention,
e.g., glass or plastic substrates. With glass substrates, the
procedure begins by coating the surface with an aminosilane to
aminate the surface. This amine is then reacted with a UV sensitive
protecting group, such as the succimidyl ester of
.alpha.(4,5-dimethoxy-2-nitrobenzyl) referred to as "caged"
succimidate. Discrete spots of free amine are revealed on the caged
succimidate surface by local irradiation with a UV excitation
source (UV laser or mercury arc). This reveals free acid groups
which can then react with amine modified oligonucleotide probes.
Such a process provides for local biosite modification, surrounded
by substrate areas with a relatively high surface tension,
unreacted sites.
[0127] When using plastic substrates, the procedure begins by
coating aminated plastic with an amine blocking group such as a
trityl which is poorly water soluble and hence produces a coating
with high surface tension. Next, an excitation source (eximer or IR
laser for example) is used to selectively remove trityl by
light-induced heating. The biosite areas are then activated with
bifunctional NHS ester or an equivalent. The net result is similar
for glass wherein the locally activated biosite areas will have low
aqueous surface tension which are surrounded by relatively high
surface tension, thereby constraining the capillary dispensing to
the biosite area.
Step 2--Self Assembling Arrays--Universal Arrays
[0128] Creating and constructing self assembling probe arrays or
universal arrays enables on-line configuration of the biosites
wherein an unvarying probe array (capture probes) is activated by
binding to a cognate set of adapters (target probes) to yield a
modified probe array which is specifically configured for analysis
of a target or target mixture.
[0129] The Universal Array format overcomes significant obstacles
that currently prevent probe array technology from being
implemented in a commercially broad manner. Fundamentally, probe
arrays that allow for highly parallel analysis of binding events
require specialized equipment to manufacture and sophisticated
instrumentation to interpret the binding patterns. Unfortunately,
the current manufacturing processes for making biosite arrays, such
as ink-jet, robotic deposition, or photolithographic in-situ
synthesis are relatively inflexible. These techniques are designed
to make a large number of specific arrays to cover the cost of
setup and operation. Hence, small volume custom arrays would be
prohibitively expensive.
[0130] In contrast, the Universal Arrays system as taught herein
solves this problem by taking advantage of efficient high volume
manufacturing techniques for the capture probe arrays only. In this
fashion, each customer can use a pre-manufactured, high density
biosite capture array that is readily "tailored or customized" by
the end-user for their specific target analyte screening. For this
invention, "target analyte" is defined as the solution-state solute
to be analyzed via binding to the probe array. In short,
customization of the array can be performed in the customer's
laboratory. The end-user synthesizes or produces bifunctional
target probes containing two separate binding domains, one binding
domain cognate to a specific member in the may (capture domain) and
another binding domain specific for the target analytes of interest
(target domain). In an actual assay, end-users add their customized
bifunctional probes to a solution phase mixture of analytes and
incubate in a reaction chamber containing a pre-manufactured
universal capture army. Alternatively, the capture army is
incubated first with the bifunctional probes followed by an
addition of the analyte mixture (see FIG. 5). FIG. 5a is a diagram
showing a Universal Array. The analytes self assemble onto the
array in a sandwich mode by selective binding of their bifunctional
probes to both the complementary portions of the target and the
capture array. The resulting addressable, self assembled arrays is
easily analyzed with the complimentary proximal detector/imager.
Teachings for constructing the surface bound capture probes and
target probes are outlined below.
1. Capture Probes
[0131] The surface bound universal capture probes are arranged in
an array of biosites attached to a solid support. Each biosite
consists of a multitude of specific molecules distinct in function
or composition from those found in every other biosite in the
array. These capture probes are designed to have a specific
composition or sequence to provide rapid and efficient binding to
the capture domain of the target probes. The specific composition
is also chosen to minimize cross association between capture probes
and their specific target probes.
[0132] Specifically for a nucleic acid capture probe the surface
bound capture array should be designed for optimum length, base
composition, sequence, chemistry, and dissimilarity between
probes.
[0133] The length of the nucleic acid capture probe should be in
the range of 2-30 bases and preferably in the range of 5-25 bases.
More preferably, the length ranges from about 10-20 bases and most
preferably is at or about 16 bases in length to allow for
sufficient dissimilarity among capture probes. Length is also
adjusted in this range to increase target probe binding affinity so
that capture probe arrays can be activated by addition of target
probe mixtures as dilute as 10.sup.9M. This allows target probes to
be synthesized in small scale and inexpensively. Also, length is
adjusted to this range to reduce the rate of target probe
dissociation from capture probe arrays. This allows the activated
capture probe arrays to be washed thoroughly to remove unbound
target probes, without dissociation of specifically bound target
probes from the surface. With capture probes in such a size range,
the complex formed by and between the target probe and capture
probe interaction is stable throughout subsequent air drying, and
can be stored indefinitely with refrigeration.
[0134] A preferred percentage base composition for capture probe
array sets is in the range of at or around 30-40% G, 30-40% C,
10-20% A, 10-20% T. Relatively G-C rich capture probes are
desirable such that the thermodynamic stability of the resulting
capture/target probe pairing will be high, thus allowing for
surface activation at low added target probe concentrations (e.g.,
in the range of 10.sup.9M). Nearest neighbor frequency in the
capture probe set should minimize G-G or C-C nearest neighbor3.
This criterion minimizes the possibility of side reactions,
mediated via G-quartet formation during capture probe attachment to
the surface, or during the capture probe-target probe binding
step.
[0135] For capture probe sets it is desirable to obtain a set
structure such that each member of the capture probe set is
maximally dissimilar from all others. To obtain such maximally
dissimilar sets, the following algorithm can be employed. [0136] 1)
The set size is defined. In a preferred embodiment, 16, 24, 36, 48,
49, 64. 81, 96 and 100 constitute useful sizes. [0137] 2) The
overall sequence structure of the capture probe set is defined. The
length and base composition as described above are used to define
such parameters in general, the number of G bases and C bases are
held equal as are the number of A bases and T bases. This equality
optimizes the configurational diversity of the final sets. Thus,
such sets will be described by the equation
G.sub.nC.sub.nA.sub.mT.sub.m. [0138] 3) For a set structure defined
by m and n, a random number generator is employed to produce a set
of random sequence isomers. [0139] 4) One member of the random
sequence set is selected to be used as element #1 of the set.
[0140] 5) The maximum similarity allowable among set members is
defined. Similarity is defined in terms of local pair-wise base
comparison. For example, when two oligomer strands of identical
length n are aligned such that 5' and 3' ends are in register, the
lack of mismatches refers to the situation where at all positions
1-n, bases in the two strands are identical. Complete mismatching
refers to the situation wherein, at all positions 1-n, bases in the
two strands are different. For example, a useful maximum similarity
might be 10 or more mismatches within a set of 16, 16mer capture
probes. [0141] 6) A second member of the random sequence set is
selected and its similarity to element #1 is determined. If element
#2 possesses less than the maximum allowable similarity to element
#1, it will be kept in the set. If element #2 possesses greater
than the maximum allowable similarity, it is discarded and a new
sequence is chosen for comparison. This process is repeated until a
second element has been determined. [0142] 7) In a sequential
manner, additional members of the random sequence set are chosen
which satisfy the dissimilarity constraints with respect to all
previously selected elements.
[0143] Standard deoxyribonucleic acid base homologues, or
homologues with modified purine or pyrimine bases, or modified
backbone chemistries such as phosphoramidate, methyl phosphonate,
or PNA may be employed in synthesis of capture probes.
[0144] The capture probe should be linked to a solid support. This
can be done by coupling the probe by its 3' or 5' terminus.
Attachment can be obtained via synthesis of the capture probe as a
3' 5' biotinylated derivative, or as a 3'/5' amine modified
derivative, a 3'/5' carboxylated derivative, a 3'/5' thiol
derivative, or as a chemical equivalent. Such end-modified capture
probes are chemically linked to an underlying microtiter substrate,
via interaction with a streptaviden film (for biotin), coupling to
surface carboxylic acids or epoxide groups or alkyl halides or
isothiocyanates (for amines) to epoxides or alkyl halides (for
thiols) or to surface amines (for carboxylic acids). Other
attachment chemistries readily known to those skilled in the art
can be substituted without altering general performance
characteristics of the capture probe arrays. Capture probe arrays
can be fabricated by such chemistries using either robotic or micro
ink jet technology.
[0145] In order to minimize cross hybridization during the target
probe activation step capture probe sets are constructed such that
every member of the capture probe set has a length which is
identical or differs by no more than 1 base from the average length
of the set, and possesses an overall gross base composition which
is identical or substantially similar to all other members of the
set. These two criteria interact to allow the free energy of all
target probe/capture probe pairings to be identical. The above
described algorithm generates such sets of probes.
[0146] It is important that the sequence of each member of the
capture probe set differ from every other member of the capture
probe set by at least 20%, preferably 40%, more preferably 50% and
most preferably 60%. This extent of sequence homology (less than
80% between any two members of the set) prohibits target probes
from binding to members of the probe set other than that to which
it has been designed.
[0147] There are numerous capture probe sets that satisfy the
general design criteria as outlined above. Presented below is a
specific example of a 16 element capture probe set generated by the
above described algorithm which adequately satisfies the above
criteria.
[0148] For this example, capture probe length is held at 16 bases
and base composition is fixed at G.sub.5C.sub.5T.sub.3A.sub.3 among
all 16 members of the set. There are no more than 3 G-G or C-C
pairings per capture probe element. This particular capture probe
set is designed to be linked to microtiter support via an amine
linkage at its 3' terminus. However, a 5' amine linkage, or other
chemistries could have been used as well.
[0149] The top-most array element (#1) has been chosen as a
standard. Detailed inspection of this set shows that every member
of the set differs from every other member of the set by at least
10 base mismatches, thus satisfying the criterion of no more than
50% homology between capture probe set elements.
TABLE-US-00001 SEQUENCE # CAPTURE PROBES, 16 MERS SEQ ID NO:1
5'-TGATTCAGACCGGCCG-3'a SEQ ID NO:2 5'-CCCGGGGCGTCTTAAC-3'a SEQ ID
NO:3 5'-GGACGCCATATGCGCT-3'a SEQ ID NO:4 5'-TGAGGGCTCCGCCATA-3'a
SEQ ID NO:5 5'-AACCCGTGACGTGTGC-3'a SEQ ID NO:6
5'-AGCATCGCCGGTCCTG-3'a SEQ ID NO:7 5'-CCTGCAAGGCTGACGT-3'a SEQ ID
NO:8 5'-CAGTTGTCGACCCCGG-3'a SEQ ID NO:9 5'-CGGCGCGTCCAATTCG-3'a
SEQ ID NO:10 5'-ATCGATCTGAGGGCCC-3'a SEQ ID NO:11
5'-GTACATGCGGCCTGCA-3'a SEQ ID NO:12 5'-TAGCCGCTCGCTAGAG-3'a SEQ ID
NO:13 5'-CCTAGTGATGACCGGC-3'a SEQ ID NO:14 5'-GTCTGAGGGCAACCTC-3'a
SEQ ID NO:15 5'-CTAGCTGGCTACGCAG 3'a SEQ ID NO:16
5'-GCCATCCGCTTGGAGC-3'a a = amine linkage to solid support, such as
a 3' propanolamine, coupled to a carboxylate modified surface via
amide linkage or epoxide modified surfaces.
TABLE-US-00002 ELEMENTAL TARGET PROBES SEQUENCE # (coenate to
capture probes) SEQ ID NO:17 3'-TTACTAAGTCTGGCCGGC-5' SEQ ID NO:18
3'-TTGGGCCCCGCAGAATTG-5' SEQ ID NO:19 3'-TTCCTGCGGTATACGCGA-5' SEQ
ID NO:20 -TTACTCCCGAGGCGGTAT-5' SEQ ID NO:21
3'-TTTTGGGCACTGCACACG-5' SEQ ID NO:22 3'-TTTCGTAGCGGCCAGGAC-5' SEQ
ID NO:23 3-TTGGACGTTCCGACTGCA-5' SEQ ID NO:24 3'-TTGTCAACAGCTGGGGC
C-5' SEQ ID NO:25 3-TTGCCGCGCAGGTTAAGC-5' SEQ ID NO:26
3-TTTAGCTAGACTCCCGGG-5' SEQ ID NO:27 3'-TTCATGTACGCCGGACGT-5' SEQ
ID NO:28 3-TTATCGGCGAGCGATCTC-5' SEQ ID NO:29
3-TTGGATCACTACTGGCCG-5' SEQ ID NO:30 3'-TTCAGACTCCCGTTGGAG-5' SEQ
ID NO:31 3-TTGATCGACCGATGCGTC-5' SEQ ID NO:32
3'-TTCGGTAGGCGAACCTCG-5'
2. Target Probes
[0150] A target probe set is designed and constructed to bind to
the capture probe set in a specific manner, i.e., each target probe
element binds to only one element of the capture probe set. Thus, a
mixture of target probes can be administered to a capture probe
array formed on the bottom of a microtiter well, or equivalent
surface. For the nucleic acid embodiment of the Universal Army,
subsequent to binding, the target probe set will partition itself
among capture probe set members via Watson-Crick base pairing,
thereby delivering a unique binding domain (cognate to analyte) to
each site in the probe array.
[0151] There are two general methods that can be employed by the
end-user to synthesize customized nucleic acid-based bifunctional
target probes. The simplest and most direct method is to synthesize
a single oligonucleotide that contains the two domains (capture and
analyte) separated by a linker region using a standard automated
DNA synthesizer. As a class, the bifunctional target probes for a
nucleic acid embodiment possess a structural domain cognate to the
capture probe which is the Watson-Crick complement to one element
of the capture probe set. Its length and base sequence is thus
defined by that of the capture probe via standard rules of
antiparallel Watson-Crick duplex formation. In addition, the target
probe also contains one of the following structural domains:
[0152] a. Cognate to a Small Segment of a Solution State Nucleic
Acid Target Analyte
[0153] This is the component of the target probe which is
complementary via WatsonCrick pairing to the solution state target
nucleic acid to be analyzed. In general, its sequence has no
correlation to that of the domain which is cognate to the capture
probe. However, several general design criteria should be met.
[0154] First, for ease of target probe synthesis, the unique domain
in the range of about 5-30 bases in length, and preferably in the
range of about 10-25 bases in length. With shorter target probe
domains, analyte binding affinity is insufficient, and longer
target probe domains present synthesis difficulties.
[0155] Second, when the unique sequence is equal in length or
longer than the capture probe set, the unique element should
possess a sequence which is no more than 80% homologous to the
Watson-Crick complement of any capture probe element. This
criterion eliminates inappropriate association of the unique target
probe segment with members of the capture probe set.
[0156] b. Cognate to a Priming Site Used for Biochemical
Amplification Such as PCR and LCR
[0157] This domain essentially creates nucleic acid amplification
primers with tails complementary to capture probe sites in a
Universal Army. After amplification, the resulting amplicon sets
can be directly hybridized to the capture probe array and analyzed
as described below.
[0158] c. Chemically Modified for Direct Linkage
[0159] Another method of synthesizing bifunctional DNA target
probes consists of individually and separately synthesizing analyte
and capture sequence oligos that are chemically altered to
incorporate a reactive functionality which will allow subsequent
chemical linkage of the two domains into a single bifunctional
molecule. In general, the 5' or 3' terminus of each oligo is
chemically altered to facilitate condensation of the two sequences
in a head to tail or tail to tail manner. A number of methods are
known to those skilled in the art of nucleic acid synthesis that
generate a variety of suitable functionalities for condensation of
the two oligos. Preferred functionalities include carboxyl groups,
phosphate groups, amino groups, thiol groups, and hydroxyl groups.
Further, chemical activation of these functionalities with homo- or
heterobifunctional activating reagents allows for condensation of
the activated oligo with the second functionalized oligo sequence.
Some examples of the various functionalities and activating
reagents that lead to condensation are listed below:
TABLE-US-00003 Terminal Functionality ACTIVATING AGENT (3' or 5')
(Homo or Heterobifunctional) NH.sub.2 (amino) NHS-NHS,
NHS-maleimide, iodoacetic anhydride, EDC (carbo-diimide) SH.sub.2
(thiol) maleimide-NHS COOH (carboxyl) EDC (carbodiimide) OH
(hydroxyl) carbodiimide (EDC) PO.sub.4 (phosphate)
N-methylimidazole (EDC) PO.sub.3S alpha-thiophosphate
maleimide-maleimide, maleimide-NHS
[0160] A specific example of the target probe domains that are
cognate to the capture probe. set of the Universal Array and can be
modified to allow for direct binding to a specifically modified
probe, nucleic acid or other molecule capable of selective binding
to the analyte of interest is illustrated in FIG. 5b. FIG. 5b is a
diagram showing direct binding for a target probe. As shown in FIG.
5b, the target probe is constructed from two parts; the first is a
presynthesized probe (TP1) complementary to a capture probe which
has a linkage element for attaching the second target complex
(TP2). Such embodiment yields a high degree of simplicity for the
customer since the first target component can be offered in a
ready-to-use format.
[0161] A sample protocol for the two piece approach is as follows:
[0162] 1) Obtain TP1 from commercial source, e.g., Genometrix
(synthesized as 3' amine, 5' thiol); [0163] 2) TP2 synthesized as
an amine; [0164] 3) TP2 is mixed with iodoacetic acid anhydride in
"Buffer A" to generate the iodoacetate derivative TP2*; [0165] 4)
Ethanol ppt, run over G25 spin column and collect the excluded
volume which contains TP2* only, with small molecule reactants
removed; [0166] 5) TP1+TP2* are mixed with "Buffer B"; [0167] 6)
Separate on G50 spin column.
[0168] For this invention, "Buffer A" consists of 10 mM sodium
citrate, pH 7.0, and "Buffer B" consists of 10 mM sodium
bicarbonate, pH 9.0.
3. Linker
[0169] In some instances, a chemical linker may be needed to
separate the two nucleic acid domains of the target probe, to
minimize stearic interaction between the target probe and the
solution state nucleic acid analyte. This linker may be constructed
from nucleic acid building blocks. For example, the sequence
T.sub.n (where n=1-5) is preferred because stretches of T are
readily synthesized and minimize the likelihood of sequence
dependent interactions with capture probe, other target probe
domains, or the solution phase nucleic acid analyte.
[0170] However, the linker is more preferably synthesized from an
inert polymer, such as oligo-ethylene glycolate linkages
(--O--CH2-CH2-O--).sub.n Linkages with n=3 are commercially
available as the phosphoramidate for ready synthesis into
oligonucleic acids via standard phosphodiester linkages. From one
to five linkers can be introduced as needed.
[0171] Detailed below is a specific example of the invention based
upon the capture probe set described above. Here, the linker domain
is listed as two repeats of a triethylene glycolate synthon, linked
by a phosphodiester linkage into the target oligonucleotide
backbone.
##STR00001##
X=--OPO.sub.2--(O--CH.sub.2--CH.sub.2--O--OPO.sub.2--).sub.2--O
DIETHYLENE GLYCOLOATE LINKAGE
TABLE-US-00004 [0172] TARGET PROBE 1 3'
AMINE-----------5'-OPO.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2-
--CH.sub.2--CH.sub.2--SH 3' PROPYL AMINE TARGET PROBE 2
NH2--CH.sub.2--CH(OH)--CH2--OPO.sub.2-1.sub.2-O-3'--------------
------5' IODO ACETATE DERIVATIVE TARGET PROBE 2
I--CH2--CO--NH--CH.sub.2--CH(OH)--CH2--OPO.sub.2-1.sub.2-O-3'--------------
--------5 COUPLED TP1 + TP2 PRODUCT 5' THIOL DERIVATIVE OF TP1 3'
ALKYL HALIDE OF TP2
3'-[TP1]-OPO.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C-
H.sub.2--S--CH.sub.2--CO--NH--CH.sub.2--CH(OH)--
CH2--OPO.sub.2-1.sub.2-O-[TP2]-5' TP1 = target probe 1 TP2 = target
probe 2
[0173] A Universal Array having 16 capture probes within a single
well of a 96 well microtiter plate is shown in FIG. 5c. FIG. 5c is
a printed computer image showing a multi-microtiter well proximal
CCD image of a 4.times.4 Universal Array. In FIG. 5c, target
specific hybridization is observed in 15 out of the 16 oligo
elements in the array. The results of 15 target specific
hybridizations conducted simultaneously in 3 separate reaction
chambers in a multiwell reaction vessel are quantitatively assessed
from the digital image obtained from the proximal CCD imager.
Hybrids are digoxigenin end-labeled oligonucleotide targets
detected using anti-digoxigenin antibody-alkaline phosphatase
conjugate and ELF.TM. fluorescence. In this assay (from Molecular
Probes, Inc) the antibody binds to the digoxigenin group,
delivering alkaline phosphatase to the bound target. The alkaline
phosphatase converts the non-fluorescent ELF precursor to a
fluorescent product which can be detected by UV irradiation.
[0174] FIG. 5d is a printed computer image showing a single
microtiter well proximal CCD image of a 4.times.4 Universal Array.
FIG. 5d shows the target specific hybridization of 4 of the 16
oligonucleotide elements in the array at positions A2, B2, C2, and
D2. Note the desirable absence of significant cross hybridization,
which has been specifically minimized by imposing the maximum
dissimilarly design constraints. Hybrids are digoxigenin
end-labeled oligonucleotide targets detected using anti-digoxigenin
alkaline phosphatase conjugate and ELF.TM. fluorescence as
described above.
4. Non-Nucleic Acid Embodiments
[0175] Small molecule Universal Arrays can be employed for rapid,
high throughput drug screening. In this format, surface bound
capture probes consist of small haptens or molecules arranged in
separated biosites attached to a solid support. Each biosite
consists of specifically-addressable, covalently immobilized small
molecules such as haptens, drugs and peptides. These organic
capture molecules are designed to have a high affinity association
with a bispecific ligand. These ligands contain both a domain
cognate to the small immobilized organic molecule (capture probe)
and cognate to the analyte of interest. The domain cognate to the
analyte can associate either directly to this target or to a label
on-the analyte.
[0176] Specific examples of bispecific ligands include, without
limitation, antibody: antibody, antibody:receptor, antibody:lectin,
receptor:receptor, bispecific antibodies. antibody:enzyme,
antibody:streptavidin, and antibody:peptide conjugates.
[0177] Analytes can include, but are not limited to, dsDNA, ssDiNA,
total RNA, mRNA, rRNA, peptides, antibodies, proteins, organic
enzyme substrates, drugs, pesticides, insecticides and small
organic molecules.
[0178] Conversely, the format for a small molecule Universal Array
can be inverted so that the macromolecular ligand becomes the
capture probe. Thus, a Universal, Array (Macromolecular Universal
Array) may contain large macromolecules such as, without
limitation, antibodies, proteins, polysaccachrides, peptides, or
receptors as the immobilized capture probe. In turn, unique small
molecule tags having a specific, high affinity association for the
macromolecular biosites are covalently attached to various probes
cognate to the analyte. These labeled probes now represent the
bispecific component cognate to both the capture macromolecule and
the target analyte. Some representative examples of small molecules
(haptens or drugs) are listed in Table 1 below. This is only a
partial list of commercially available antibodies to haptens,
steroid hormones and other small molecule drugs. Examples of these
bispecific, small molecule-labeled macromolecules include
antibodies, receptors, peptides, oligonucleotides, dsDNA, ssDNA,
RNA, polysaccharides, streptavidin, or lectins. A partial list of
48 representative compounds for which specific antibodies are
available include: fluorescein; dinitrophenol; amphetamine;
barbiturate; acetaminophen; acetohexamide; desipramine; lidocaine;
digitoxin; chloroquinine; quinine; ritalin; phenobarbital;
phenyloin; fentanyl; phencyclidine; methamphetamine; metaniphrine;
digoxin; penicillin; tetrahydrocannibinol; tobramycin; nitrazepam;
morphine; -Texas Red; TRITC; primaquine; progesterone; bendazac;
carbamazepine; estradiol; theophylline; methadone; methotrexate;
aldosterone; norethisterone; salicylate; warfarin; cortisol;
testosterone; nortriptyline; propanolol; estrone; androstenedione;
digoxigenin; biotin; thyroxine; and triiodothyronine.
[0179] The general concept of Universal Arrays. whether they be
DNA-based, small molecule-based, or protein-based allows for great
versatility and end-user friendliness. The various configurations
described allow for highly parallel, simultaneous, multiplexed,
high throughput screening and analysis of a wide variety of analyte
mixtures.
Step 3--Molecular Labeling Strategies
[0180] Molecular labeling strategies relate to versatile labeling
of the target molecules (fluorescence, chemiluminescence, etc.)
consistent with proximal large area detection/imaging.
1. Introduction--Conventional Labeling
[0181] Labeling can be achieved by one of the many different
methods known to those skilled in the art. In general, labeling and
detection of nucleic acid hybrids may be divided into two general
types: direct and indirect. Direct methods employ either covalent
attachment or direct enzymatic incorporation of the signal
generating moiety (e.g., isotope, fluorophore, or enzyme) to the
DNA probe. Indirect labeling uses a hapten (e.g., biotin or
digoxigenin) introduced into the nucleic acid probe (either
chemically or enzymatically), followed by detection of the hapten
with a secondary reagent such as streptavidin or antibody
conjugated to a signal generating moiety (e.g., fluorophore or
signal generating enzymes such as alkaline phosphatase or
horseradish peroxidase).
[0182] For example, methods of detecting the
association/hybridization include, without limitation, fluorescent
labeling, radioisotope labeling, chemiluminescence labeling,
bioluminescence labeling, calorimetric labeling and
electrochemiluminescence labeling. Many known labeling techniques
require a wash step to remove excess target from the
hybridization/association solution, e.g., fluorescent,
radioisotope, chemiluminescence, bioluminescence and calorimetric
labeling. Several of these will be described. below.
2. Fluorescent Labeling
[0183] Fluorescent labeling is suitable for this invention for
several reasons. First, potentially hazardous substances such as
radioisotopes are avoided. Furthermore, the fluorescent labeling
procedures are simpler than chemiluminescent methods since the
latter requires enzymatic reactions and detection in the solution
state. Finally, the fluorescent labeling approach can be modified
to achieve the highest signal-to-nose ratio SNR among the safest
labeling techniques by utilizing secondary linker chemistries that
enable the attachment of hundreds of fluorescent dye molecules per
target molecule.
[0184] The particular fluorescent dyes to be considered include
commercially available agents such as ethidium bromide, as well as
the novel dyes proposed in the affiliated chemistry component.
These labeling agents have intense absorption bands in the near UV
(300-350 nm) range while their principle emission band is in the
visible (500-650 nm) range of the spectrum. Hence, these
fluorescent labels appear optimal for the proposed proximal CCD
detection assay since the quantum efficiency of the device is
several orders of magnitude lower at the excitation wavelength (337
nm) than at the fluorescent signal wavelength (545 nm). Therefore,
from the perspective of detecting luminescence, the polysilicon CCD
gates have the built-in capacity to filter away the contribution of
incident light in the UV range, yet are very sensitive to the
visible luminescence generated by the proposed fluorescent reporter
groups. Such inherently large discrimination against UV excitation
enables large SNRs (greater than 100) to be achieved by the
CCDs.
3. Electrochemiluminescence Labeling
[0185] Electrochemiluminescence or electrical chemiluminescence
(ECL) labeling, e.g., ruthenium (Ru) does not require a wash step
to remove excess target from the solution and is highly sensitive.
Briefly, for electrochemiluminescence as a method of detection, the
internal surface of the reaction chamber is coated with a
conductive material, e.g., gold, and the biosite is attached to
this conductive surface (See FIG. 6). FIG. 6 is a diagram showing
ECL implementation in reaction vessel with proximal CCD imaging.
Using one microtiter well (of a 96 microtiter well plate) as a
reaction chamber, the biosites are deposited onto the internal
circumference of the microtiter well by one of several methods as
described above (ink-jet, capillary, or
photolithography/capillary).
[0186] This conductive surface acts as a cathode (positive lead),
and an anode (negative lead) is provided by inserting a metal cup
with an electrode protruding through its center into the reaction
chamber (microtiter well). The electrode is positioned such that it
is inserted into the hybridization solution. The voltage applied to
the anode induces an electrochemical event at the labeled molecule
surface which releases energy in the form of photons (light).
[0187] The specific ECL label, e.g., Ru, is attached to the target
molecule by the conventional means. The labeled target is added to
the hybridization solution and once hybridization occurs between
the Ru labeled target and biosite, e.g., after sufficient time has
passed for hybridization to be completed, a voltage is applied and
only Ru labeled target associated (hybridized) with the biosite
will emit light and be detected. In order for the Ru labeled target
to be detected, it must be in proximity to the cathode. The
residual excess Ru labeled target not associated with the biosite
will therefore not emit light.
[0188] The ECL reaction vessel is diagramed in FIG. 7. In FIG. 7,
the thin film substrate, e.g., plastic, glass, etc., is patterned
with a conductive metal, e.g., gold, platinum, etc., to form
electrodes within the reaction chambers. Next, the biosites are
deposited, with one of several methods described above (ink-jet,
capillary, photolithographic/capillary) onto the patterned
electrodes. Finally, the resulting thin film substrate is bonded
onto the reaction vessel which serves as the bottom of the reaction
chambers.
4. Lanthanide Chelate Labeling
[0189] As an alternative to ethidium-based fluorescent reporter
groups, which are known for their tendency to absorb
nonspecifically to surfaces causing increased signal background,
the use of aromatic lanthanide (Ln) chelators may be used in the
instant invention. Although the lanthanide ions (Tb and Eu
specifically) have luminescent yields near to one (1), and emission
lifetimes year to 100 uses, they absorb light weakly and are
therefore poor luminescent dyes. However, when chelated by an
appropriately chosen aromatic donor, energy transfer can occur
resulting in high overall luminescent yields. DPA (dipiccolimic
acid) is the prototype for such an aromatic Ln chelator, and has
excellent photophysical properties. However, its absorbance maximum
is near 260 nm, which overlaps the DNA absorption band and is
therefore inappropriate for the proximal CCD approach. Thus, the
synthesis of modified DPA derivatives with the correct absorption
properties and which have the capacity to be linked directly or
indirectly to the target molecules have been developed.
[0190] Since three DPA equivalents bind per Ln ion, the preferred
approach is to link the modified DPA to a polymeric lattice, which
provides for close spacing of chelators and can be designed to have
useful DNA or RNA binding properties. These results suggest that a
fused bicyclic DPA derivative is the candidate of choice.
[0191] FIG. 8 is a chemical drawing showing lanthanide chelators.
The two classes of polymeric lattice as illustrated in FIG. 8 can
be employed for-attachment of DPA derivatives, both based upon the
use of synthetic polypeptides in the 10.sup.4 MW range. Synthesis
can be conducted as described for simple DPA-peptide conjugates.
The first polymer is to be used for covalent attachment to RNA via
the transamination reaction to cytosine. This peptide lattice can
be simple poly-L-lys. The second approach involves the coupling of
modified DPA to a DNA binding peptide, which can be used to deliver
the Ln chelate to RNA by means of non-covalent nucleic acid
binding. For example, peptides can be synthesized in solution as a
Lys.sub.3Arg.sub.1 random co-polymer (average mw 10.sup.4).
Subsequent to the conversion of Lys residues to the modified DPA
conjugate, RNA binding can be driven by association with multiple
Arg equivalents, taking advantage of the known helix selectivity of
polyarginine. As for ethidium bromide (EB), addition of the
non-covalent chelator conjugate can be made after washing to retain
hybridization stringency.
Step 4--Detection System
[0192] A detection system relates to parallel detection and/or
imaging in the reaction vessel containing the reaction chambers
using a proximal large area detector/imager.
1. General Description
[0193] Following the hybridization process of the multiplexed
molecular analysis system, the amount of hybridized target
molecules bound to each biosite in the reaction chambers of the
reaction vessel must be quantitatively determined. The preferred
detection/imaging system for quantifying hybridization for the
instant invention is proximal charge-coupled device (CCD)
detection/imaging due to the inherent versatility (accommodates
chemiluminescence, fluorescent and radioisotope target molecule
reporter groups), high throughput, and high sensitivity as further
detailed below.
[0194] The detection/imaging apparatus used for the multiplexed
molecular analysis system is comprised of a lensless imaging array
comprising a plurality of solid state imaging devices, such as an
array of CCDs, photoconductor-on-MOS arrays, photoconductor-on-CMOS
arrays, charge injection devices (CIDs),
photoconductor-on-thin-film transistor arrays, amorphous silicon
sensors, photodiode arrays, or the like. The array is disposed in
proximity to the sample (target molecules hybridized to the
biosites) and is comparable in size to the reaction chambers. In
this manner, a relatively large format digital image of the spatial
distribution of the bound target molecules is produced without
requiring the use of one or more lenses between the sample and the
imaging array. This apparatus offers:
[0195] 1) high sensitivity (subattomole DNA detection);
[0196] 2) high throughput (seconds for complete image
acquisition);
[0197] 3) linear response over a wide dynamic range (3 to 5 orders
of magnitude);
[0198] 4) low noise;
[0199] 5) high quantum efficiency; and
[0200] 6) fast data acquisition.
[0201] Moreover by placing the imaging array in proximity to the
sample as illustrated in FIG. 1, the collection efficiency is
improved by a factor of at least ten (100 over any lens-based
technique such as found in conventional CCD cameras). Thus, the
sample (emitter or absorber) is in near contact with the detector
(imaging, array), thereby eliminating conventional imaging optics
such as lenses and mirrors. This apparatus can be used for
detecting and quantitatively imaging radioisotope, fluorescent, and
chemiluminescent labeled molecules, since a lensless CCD array
apparatus is highly sensitive to both photons and x-ray particles.
Hence a single imaging instrument can be used in conjunction with
numerous molecular labeling techniques, ranging from radioisotopes
to fluorescent dyes.
[0202] The detection/imaging apparatus invention as taught herein
can be divided into two subclasses. The first subclass entails a
static platform, whereby a plurality of imaging devices are
arranged in a relatively large format area comparable to the sample
size.
[0203] The second subclass entails a dynamic platform that enables
a smaller set of imaging devices to image a relatively large format
sample by moving either the array of imaging devises or sample,
relative to one another.
[0204] Thus, the dynamic embodiment of the detection/imager
invention generally concerns a method and apparatus for
ultrasensitive detection, high resolution quantitative digital
imaging and spectroscopy of the spatial and/or temporal
distribution of particle emissions or absorption from/by a sample
(target molecules) in a relatively large format. The apparatus of
this invention includes: [0205] a) a large area detector array for
producing a relatively large image of detected particle
distribution without the use of optical lenses; [0206] b) a scanner
for moving either the sensor array or the sample in a manner for
efficient imaging; and [0207] c) a source of energy for exciting
the sample or providing absorption by the sample.
[0208] Optimally, the ratio of detector array size to sample image
is one (1) for a static format and less than one (1) for a dynamic
format.
[0209] An electronic schematic of the proximal detector/imager to
be used with the multiplexed molecular analysis system is shown in
FIG. 9. FIG. 9 is a diagram showing a multiplexed molecular
analysis system electronics schematic. As illustrated in FIG. 9,
the reaction vessel is placed directly on the fiber optic faceplate
which is bonded to the sensor array. The faceplate provides sensor
isolation to accommodate routine cleaning as well as affording
thermal isolation for ultrasensitive detection under cooled sensor
operation. Also the optical faceplate can serve to filter
excitation radiation by employing selective coatings. The sensor
array is comprised of a plurality of smaller sensors such that the
composite array approaches the surface area of the reaction vessel.
The excitation source serves to excite the fluorescent reporter
groups attached to the target molecules. Depending on the chosen
reporter groups, the excitation source can be either a UV lamp,
laser, or other commonly used light source used by those skilled in
the art. The sensor array driver circuitry includes clocking,
biasing and gating the pixel electrodes within the sensors. The
cooling circuitry controls the thermoelectric cooler beneath the
sensor array to enable ultrasensitive detection by providing very
low thermal noise. Basically, the user selects the required
temperature of operation and through feedback circuitry, the sensor
array is held constant at such temperature. The image receive
circuitry is responsible for obtaining the digital image from the
sensor array and includes preamplification, amplification, analog
to digital conversion, filtering, multiplexing, sampling and
holding, and frame grabbing functions. Finally, the data processor
processes the quantitative imaging data to provide the required
parameters for the molecular analysis outcome. Also, a computer
display is included for displaying the digital image.
2. Sensor Array Implementations
[0210] A preferred embodiment of the detection/imaging sensor array
of the invention consists of a plurality of CCD arrays CCDI . . .
CCDN assembled in a large format module as illustrated in FIGS.
10A-10C. FIG. 10A depicts a CCD array with multiple pixels being
exposed to a labeled biological sample 32 which causes the
collection of electrons 34 beneath the respective pixel gate 16.
Individual CCD arrays are closely aligned and interconnected in
particular geometries to form a relatively large (greater than 1
cm.sup.2) format imaging sensors of the linear array type as shown
in FIG. 10B or the two dimensional row and column type as shown in
FIG. 10C.
[0211] Numerous CCD tiling strategies can be explored to determine
the best tradeoff analysis between detection throughput and
instrument cost. A large format tiled array with several wafer
scale CCDs would provide simultaneous detection of all biosites
within the reaction vessel within seconds. However, the cost of the
large (8.5.times.12.2 cm) CCD sensor array may be prohibitive. An
engineering compromise is therefore preferred, balancing the use of
smaller devices to significantly reduce the cost of the tiled
array, while also matching the throughput with the other processes
in the overall multiplexed molecular analysis system.
[0212] As shown in FIG. 10A, each CCD array CCDI . . . CCDN is
formed, in the conventional manner, by growing and patterning
various oxide layers 14 on a Si wafer/substrate 12. CCD gate
electrodes 10 are then formed by deposition of polysilicon or other
transparent gate material on the gate insulator or field oxide 14.
A dielectric or polymer layer 18, preferably of light transmissive
material such as silicon nitride or glass, SiO.sub.2 or polyamide
is then formed over the electrodes 16.
[0213] Preferably, in a labeled molecule embodiment, a filter shown
in dotted lines 17, which may be formed of an aluminum or tungsten
metal grating, or dielectric multilayer interference filter, or
absorption filter, is formed in the dielectric layer 18 between the
surface and the metal electrode 16. The filter is adapted to block
the excitation radiation and pass the secondary emission from the
sample 20. In a static platform embodiment, the sensor module
remains fixed with respect to the sample. Hence to achieve the
relatively large imaging format, a plurality of imaging devices
CCDI . . . CCDN should be arranged in a module as illustrated in
FIGS. 10B and 10C. The module can be packaged for easy installation
to facilitate multiple modules, each for specific applications.
Various tiling strategies have been documented and can be employed
to minimize the discontinuity between devices, such as described in
Burke, et al., "An Abuttable CCD Imager for Visible and X-Ray Focal
Plane Arrays," IEEE Trans On Electron Devices, 33(5):1069 (May,
1991).
[0214] As illustrated in FIG. 10A, a reaction vessel 20 is placed
in proximity to the CCD array sensor 10. The sample can be excited
by an external energy source or can be internally labeled with
radioisotopes emitting energetic particles or radiation, or photons
may be emitted by the sample when labeled with fluorescent and
chemiluminescent substances. Conversely, direct absorption may be
used to determine their presence. In this case, the absence of
illuminating radiation or the detector may constitute the presence
of a particular molecule structure. Preferably the sample can be
physically separated from the CCD detector by the faceplate which
is transparent to the particle emission.
[0215] The CCD detection and imaging arrays CCDI . . . CCDN
generate electron-hole pairs in the silicon 12 (see FIG. 10A) when
the charged particles or radiation of energy hv shown by the
asterisk 32 arising from or transmitted by the sample are incident
(arrows 30) on the CCD gates 16. Alternatively, the CCDs can be
constructed in a back illumination format whereby the charged
particles are incident in the bulk silicon 12 for increased
sensitivity. The liberated photoelectrons 34 are then collected
beneath adjacent CCD gates 16 and sequentially read out on a
display conventionally.
[0216] Silicon based CCDs are preferred as the solid state
detection and imaging sensor primarily due to the high sensitivity
of the devices over a wide wavelength range of from 1 to 10,000
{acute over (.ANG.)} wavelengths. That is, silicon is very
responsive to electromagnetic radiation from the visible spectrum
to soft x-rays. Specifically for silicon, only 1.1 eV of energy is
required to generate an electron-hole pair in the 3,000 to 11,000
{acute over (.ANG.)} wavelength range. Thus for visible light, a
single photon incident on the CCD gate 16 will result in a single
electron charge packet beneath the gate, whereas for soft x-rays, a
single beta particle (typically KeV to MeV range) will generate
thousands to tens of thousands of electrons. Hence the silicon CCD
device provides ultrasensitive detection and imaging for low energy
alpha or beta emitting isotopes (.sup.3H, .sup.14C, .sup.35S) as
well as high energy alpha or beta emitting isotopes (.sup.32P,
.sup.125I). Consequently, the CCD is both a visible imager
(applicable to fluorescent and chemiluminescent labeled molecular
samples) and a particle spectrometer (applicable to radioisotope
labeled samples as well as external x-ray radiated samples). Thus,
the CCD can provide simultaneous imaging and spectroscopy in the
same image.
[0217] In addition to the high sensitivity, the CCDs offer a wide
dynamic range (up-to 5 orders of magnitude) since the charge packet
collected beneath each pixel or gate 16 can range from a few to a
million electrons. Furthermore, the detection response is linear
over the wide dynamic range which facilitates the spectroscopy
function, since the amount of charge collected is directly
proportional to the incident photon energy. Hence, no reciprocity
breakdown occurs in CCDs, a well-known limitation in photographic
film.
3: Scanning Mechanics
[0218] To image the reaction vessels with a smaller sized and less
expensive sensor array, the reaction vessel can be imaged in a
column-by-column manner as it is moved across the sensor array with
a scanning mechanism. A plurality of imaging devices can be
arranged in a module of columns to minimize discontinuity. Also,
the scanning can be accomplished with intentional overlapping to
provide continuous high resolution imaging across the entire large
format sample area.
Example I
Differential Detection of Three NHS-Immobilized Haptens Using
Universal Arrays
[0219] This example demonstrates reduction to practice of small
molecule universal arrays as illustrated in FIGS. 18 and 19. FIG.
18 is a graphical schematic layout of a microarray that will be
printed on glass slides using the Hamilton 2200 Microlab robot.
This schematic layout illustrates the relative spatial
location/addresses of three separate covalently immobilized haptens
on to the glass substrate (e.g., digoxigenin, fluorescein, and
biotin). The robot will print the array by depositing 10 nL volumes
of each activated hapten (N-hydroxysuccinimide activated) on to an
amino-silanized glass surface thus creating a 4.times.4 matrix
microarray. Each hapten will be deposited by the robot 4 times as
illustrated by the schematic. For example, digoxigenin will be
deposited at array addresses indicated by address locations A1, B2,
C3, and D4. Similarly, fluorescin can be found at address locations
A4, B3, C2 and D1 and biotin at B2, B3, B4, and C3 as illustrated
in the schematic. A buffer blank (control) will be deposited at
locations A2, A3, D2, and D3. These buffer blanks should not
generate a signal on the CCD proximal detector in the presence of
hapten detecting conjugates.
[0220] Incubation of these covalently immobilized hapten
microarrays with an appropriate bispecific molecule (e.g., hapten
recognition site and enzyme reporter site) such as an
antibody/enzyme conjugate and subsequent detection of the
appropriate chemiluminescent substrate should generate an image
"pattern" on the CCD detector as predicted by the schematic
addresses shown in FIG. 18. In this example, specific light
generating substrate molecules are localized at
predictable/addressable biosites in the array either individually
or in a multiplexed fashion.
[0221] Briefly, in order to covalently immobilize the above
described hapten microarray the following protocol was developed.
First, several 22.times.22 mm square glass microscope cover slides
(150 .mu.m thick) were washed in a container containing ALCONOX
detergent solution, and subsequently transferred to a clean
container containing warm tap water to rinse off the detergent.
This rinse step was followed by two separate brief rinses. First,
in a container containing 100% acetone, then the slides were
transferred to a rinse in a solution of 100% methanol. The slides
were rinsed one final time in deionized H.sub.20 to remove traces
of organic solvent. The clean glass slides were then oven dried at
37.degree. C. After drying, the clean slides were then surface
derivitized by vacuum deposition of a solution of 3-amino
propyl-trimethoxysilane in a vacuum oven. The slides were laid down
in a metal tray on clean lint free paper towels. A 1:3 solution of
3-aminopropyltrimethoxysilane and xylene was freshly prepared by
mixing 1 ml of 3-aminopropyltrimethoxysilane (Aldrich) with 3 ml of
dry p-xylene solvent in a small glass petri dish. The dish was
covered with aluminum foil and a small needle puncture was made in
the foil. This solution was placed in the tray with the glass
slides. The tray was subsequently covered with aluminum foil and
placed in a NAPCO vacuum oven at 75.degree. C. under 25'' of Hg
vacuum overnight. The next day the amino-silanized glass slides
were removed from the vacuum oven and stored in a dry place until
used.
[0222] In order to robotically dispense and print hapten
microarrays, four separate activated hapten solutions were made as
follows. First, approximately 1 mg of the following compounds were
weighed out into separate weigh boats:
fluorescein-5-(and-6)-carbixamido)hexanoic acid, succinimidyl
ester, followed by 1 mg of sulfosuccinimidyl 6-(biotinamido)
hexanoate and then 1 mg of
digoxigenin-3-O-methylcarbonyl-.gamma.-amino-caproic
acid-N-hydroxysuccinimide ester. Each activated hapten was
dissolved in 100 .mu.L DMSO. Subsequently 50 .mu.l of each hapten
was mixed into separate tubes containing 950 .mu.l of 0.1 M
Na.sub.2HCO.sub.3/NaCO.sub.3, buffer at pH 8.05. A blank solution
containing 50 .mu.L of DMSO into this buffer was also made as a
control for dispensing on to the array as described above. Each of
the four solutions (100 .mu.L) was placed into 16 wells of a
microtiter plate. The microtiter plate was then placed on the
Hamilton 2200 Microlab robot and 10 mL aliquots were collected and
dispensed by the robotic dispensing needle onto the amino-silanized
glass cover slides at known address locations illustrated by the
schematic layout in FIG. 18.
[0223] Following microfluid dispensing of four separate (identical)
glass cover slides by the computer controlled robot needle the
arrays were air dried for 15 minutes. To detect the immobilized
haptens the glass slides were rinsed for 10 minutes in 10 ml of
1.times.TBS divided by 0.1% Tween.RTM. 20 (Tris-Buffered Saline,
100 mM Tris-HCl, 150 mM NaCl, pH 7.5). Individual slides war then
incubated with appropriate conjugate dilutions. Image A was
generated by incubating one of the slides in 10 ml of a 1:5000
dilution of streptavidin: horseradish peroxidase conjugate in
1.times.TBS divided by 0.1% Tween.RTM. 20. Image B was generated by
incubating one of the slides in a 1:5000 dilution of
anti-digoxigenin:horseradish peroxidase conjugate. Image C was
generated by incubating in a 1:1000 dilution of
antitluorescin:horseradish peroxidase conjugate. Finally, Image D
was generated by incubating a fourth slide simultaneously with all
three horseradish peroxidase conjugates at the above dilutions.
Following ebnjugate incubation all slides were washed by a 10
minute rinse on a platform shaker in 10 ml 1.times.TBS divided by
0.1% Tween.RTM. 20 to remove excess conjugates. The slides were
then imaged by adding 200 .mu.L of freshly made chemiluminescent
substrate (SuperSignal.TM. Substrate from Pierce Chemical) as
recommended by the manufacturer. The slides containing substrate
were imaged by a 10 second integration time at room temperature on
the proximal CCD detector described above.
[0224] FIG. 19A is a printed computer image showing specific
imaging of biotin-addressable biosites detected using
streptavidin:HRP conjugate (4.times.4 single well microarray). In
FIG. 19A, Image A was generated by incubating the small molecule
4.times.4 universal array with a streptavidin:HRP conjugate
specific for biotin. As seen in this image, only biosites with
addresses B1, C1, B4, and C4 known to contain biotin (refer to FIG.
18) are detected using proximal CCD imaging of chemiluminescent
signals. Specific addressing of these biosites generates a "box"
image pattern.
[0225] As shown in FIGS. 19B and 19C, Image B and Image C are two
additional 4.times.4 microarrays incubated with the indicated
antibody conjugate. FIG. 19B is a printed computer image showing
specific imaging of digoxigenin-addressable biosites detected using
anti-digoxigenin:HRP conjugate (4.times.4 single well microarray).
As seen in 19B, only biosites with addresses A1, B2, C3, and D4
known to contain digoxigenin (refer to FIG. 18) are detected using
proximal CCD imaging of chemiluminescent signals. FIG. 19C is a
printed computer image showing specific imaging of
fluorescein-addressable biosites detected using
anti-fluorescein:HRP conjugate (4.times.4 single well microarray).
As seen in 19C, only biosites with addresses A4, B3, C2, and D1
known to contain fluorescein (refer to FIG. 18) are detected using
proximal CCD imaging of chemiluminescent signals. Thus, the signals
from these two small molecules generate the predicted "diagonals"
as illustrated in FIG. 18.
[0226] Additionally, in FIG. 19D, Image D illustrates simultaneous
detection of all three haptens in a single well by simultaneously
incubating a single 4.times.4 array with all three conjugates. FIG.
19D is a printed computer image showing simultaneous imagings of
fluorescein, biotin, and digoxigenin biosites detected using
anti-fluorescein, anti-digoxigenin and streptavidin:HRP conjugates
(4.times.4 single well microarray). This image generates the
predicted "H" patter as expected because wells A2, A3, D2 and D3
were blank (see FIG. 18).
Example II
Use
Microarrays in a Microplate
[0227] Several applications of the multiplexed molecular analysis
system are detailed below which can be accommodated with a multiple
well microplate serving as the particular reaction vessel. The
novelty, however, is the plurality of biosites within each well.
That is, each well in the multiple well microtiter plate contains N
biosites where N ranges from 2 through 1,000. The upper bound is
based on the resolution limitations posed by the bottom substrate
of the microtiter plate used in conjunction with the proximal CCD
detector/imager.
[0228] For example, each well or reaction chamber can contain 96
biosites as shown in FIG. 11. FIG. 11 is a printed computer image
showing microarrays within a microplate reaction vessel. One single
reaction chamber is shown as an insert. Thus, the reaction vessel
essentially consists of microarrays within a microplate which
cumulatively affords 9,216 (96.times.96) hybridization experiments
per microtiter plate--a 100 to 1 multiplexing capacity.
[0229] The specific multiplexed microtiter plate reaction vessels
to be used with proximal imaging are constructed by bonding thin
films (typically glass or plastics) to conventional bottomless
microtiter plates. All commercially available microtiter plates
tested to date are incompatible to proximal imaging due to the
thickness and composition of the bottom substrates.
[0230] The biosites are deposited by one of the several methods
disclosed, either before or after the bottoms are bonded to the
plate. In both situations, the probe molecules comprising the
individual biosites must be attached to the glass or plastic
surfaces.
[0231] In a preferred embodiment, thin (50-300 .mu.m) vinyl
substrates are amino or epoxy functionalized with silanes similar
to glass substrates. Thin vinyl substrates are immersed in a 1-2%
aqueous solution of polyvinyl alcohol at 65.degree. C. The adsorbed
polyvinyl alcohol is then reacted with either epoxy, silane or
amino silane, thus functionalizing the polymeric hydroxyl groups.
Such optically clear vinyl substrates have the distinct advantage
of blocking a large amount of the UV excitation source incident on
the proximal CCD detector, but allowing the longer wavelengths
(e.g., 500-650 nm) to pass through efficiently. This allows for
greater sensitivity of labeled detector molecules that emit in such
wavelength region.
[0232] Nucleic acid probe attachment to glass employs well-known
epoxy silane methods (see FIG. 12) described by Southern and others
(U. Maskos et al., Nucleic Acids Res (1992) 20:1679-84; S. C.
Case-Green et al., Nucleic Acids Res (1994) 22:131-36; and Z. Guo
et al., Nucleic Acids Res (1994) 22:5456-65). FIG. 12 is a diagram
showing glass and polypropylene surface coupling chemistries. With
3' amine-modified probes, covalent surface densities can be
obtained having 10.sup.11 molecules/mm.sup.2 which is near the
theoretical packing density limit. Amino-modified polypropylene is
a convenient alternative to a glass substrate since it is
inexpensive and optically clear above 300 nm. Amine-modified
polypropylene can be converted to a carboxylic acid-modified
surface by treatment with concentrated succinic anhydride in
acetonitrile. Amine-modified probe is then coupled to this surface
by standard carbodiimide chemistry in H.sub.2O to yield probes at
densities near 10.sup.9/mm.sup.2 (see FIG. 12).
Example III
Use
Multiplexed Diagnostics
[0233] The multiplexed molecular analysis system can be employed
for immunoassay and probe based diagnostics. For immunoassays, the
throughput of conventional ELISA assays can be increased with the
multiplexed microplate format wherein a patient sample can be
simultaneously interrogated by numerous antigens/antibodies within
a single reaction chamber (well).
[0234] Similarly for probe-based diagnostics, target molecules
derived from a patient sample can be dispensed into a single well
containing numerous biosites for diagnosing genetic or infectious
diseases. For example, single-stranded nucleic acid probes which
are complementary to 96 known mutations of cystic fibrosis are
arranged within a single well in a microplate. Upon hybridization
with the patient's DNA sample, the resulting binding pattern
obtained from the proximal CCD detector/imager indicates the
presence of such known mutations.
[0235] The system can also be employed for high throughput,
probe-based infectious disease diagnostics. Here the array of
biosites within a single well in the microtiter plate can comprise
DNA probes complementary to known viral strains. For example; a.
panel of probes (biosites) is arranged to diagnose a number of
sexually transmittable diseases within a single well (reaction
chamber). Consequently for a single microtiter plate, numerous
patient samples can be simultaneously interrogated each against a
panel of numerous probes to provide a very rapid, cost effective
diagnostic testing platform.
[0236] Universal Arrays are perfectly suited for analysis and
detection of multiple point mutations within a single PCR template.
Often technical constraints are encountered when attempting to
analyze multiple point mutations from a single PCR amplicon
reaction. Most point mutation analysis techniques such as
ribonuclease protection assay, SSCP, or CLEAVASE.TM. are well
suited for detecting a single point mutation pet amplicon or DNA
template and require lengthy gel-based separation techniques. The
simultaneous, rapid detection of numerous point mutations within a
single PCR amplicon without an expensive; lengthy gel separation
step is welt beyond the capability of these technologies. Other
newer, non-gel based technologies such as TAQMAN.TM. are also
poorly suited for multiplexed analysis within a single reaction
vessel. FIG. 13 illustrates the concept of using Universal Arrays
for point mutation analysis (genotyping) at a single loci. FIG. 13
is a diagram showing genotyping by universal point mutation
scanning. For example purposes only, FIG. 13 uses a single point
mutation biosite to illustrate this type of analysis but could just
as easily be simultaneously carried out on 25 different loci on a
single PCR template as illustrated in FIG. 14.
[0237] Briefly, as shown in FIG. 13, the PCR template is aliquoted
into 4 separate tubes (one for each dNTP) containing a standard
sequencing mix, with the exception that dideoxynucleotides are not
included. Instead, a single, alpha-thio dNTP is substituted in each
of the four separate mixes. Each mix also contains a single labeled
primer with a universal sequence or "handle" at the 5' end which
anneals just one nucleotide away from the mutation site an the PCR
template (note: multiple primers each with unique universal
sequences and complementary to different loci on the template is
readily accomplished). After standard thermal cycle extension
reactions are complete each tube is briefly incubated with snake
venom phosphodiesterase. Only primers and templates that were not
extended during the sequencing reaction are vulnerable to digestion
by this 3'-specific exonuclease. Mutation primers containing a 3'
thiophosphate ester linkage are highly resistant to digestion.
[0238] In this specific example, only the A reaction extended since
a T was the next complementary base on the PCR template. Each
digested, sequencing reaction mix in turn is then hybridized to
four microtiter wells each containing. identical immobilized
microarrays complementary to the universal primer sequences. In
this case, only the microtiter well hybridized to the A reaction
mix gives a positive signal at a biosite loci complementary to the
universal handle. In this fashion, up to 96 loci could be probed
for point mutations on a single PCR template. Both strands in the
PCR amplicon could be "scanned" in this manner simultaneously to
allow more room for many primers to anneal without competition for
the same hybridization loci on the template. In FIG. 13, "5-DIG"
means 5' digoxigenin labeled.
[0239] For probe based diagnostics where both multiplexing within a
single target molecule and low target concentrations are a problem,
amplification with either PCR or LCR using the microtiter plate in
a microtiter well concept conjoined to the Universal Array has
distinct advantages. In a preferred embodiment, universal "handles"
can be synthesized directly on the 5' end of Polymerase Chain
Reaction or Ligase Chain Reaction primers and following in situ
thermal cycling the amplified products can be simultaneously
hybridized to 96 separate biosites. This format has other
diagnostic advantages such as homogeneous detection of amplified
products without having to open or expose the sample well to the
ambient environment.
[0240] FIG. 14 is a diagram showing microtiter-based throughput
genotyping. Briefly. FIG. 14 illustrates the concept of high
throughput genotyping using microarrays. In practice, 96 separate
PCR amplification reactions would be carried out using genomic DNA
templates isolated from 96 different patient samples. The figure
illustrates the concept of genotyping starting with 96 previously
robotically purified PCR templates from these reactions. Each
purified PCR product from each of the 96 wells is split into 4
separate aliquots/wells of a 384 well plate. Each well in this new
plate would contain a pre-made sequencing buffer mixture, 25
individual primers, a thermostable DNA polymerase, and only one of
the four athio-dNTP's. The primers would anneal in a juxtaposed
fashion to the PCR template just one nucleotide away from the
nucleotide locus being genotyped. In each of the four wells, those
primers juxtaposed next to the included nucleotide in the
sequencing mix would be extended. Following the simultaneous
extension of 334 reactions, each of the 384 wells is in situ
digested with snake venom phosphodiesterase. Only primers in each
reaction that had been extended by a single base are protected from
digestion. All other DNA is degraded to mononucleotides. Following
a brief thermal denaturation of the exonuclease, the contents of
all the wells is robotically transferred to a new 334-well
microtiter plate containing sequence complements microarrayed in a
5.times.5 microarray attached to the bottom of each well. Each of
the 25 primers that had not been digested would hybridize to its
corresponding complement in the array and imaged on the CCD
detector to define the genotype at each loci.
[0241] FIG. 15 illustrates this homogenous multiplexed approach for
the Polymerase Chain Reaction (PCR) simultaneously at 3 different
loci. FIG. 15 is a diagram showing homogeneous in situ microarray
detection of multiplexed PCR amplicons. FIG. 15 illustrates
specific multiplex hybridization detection of PCR products using
microtiter based microarrays. Briefly, in this figure three
separate amplification loci are being detected simultaneously. Each
locus (e.g. PCR LOCUS 1) is defined by two specially modified
amplification primers that define the ends of the amplified PCR
product. One primer in the pair, contains a fluorescently
detectable label such as fluorescein. The other primer in the pair
contains two domains, one is a unique universal sequence
complementary to a capture probe arrayed at the bottom of a single
microtiter well and the other domain specific for template
amplification. The universal sequence is attached to the
amplification primer in a 5'.times.5' linkage so that when the
polymerase is amplifying the region of interest it does not jump
over this specialized juncture, leaving the universal sequence as a
single stranded motif. If a particular template in a sample well
being amplified contains both primer loci (i.e., detection and
capture sites), then a PCR product will be generated that can
simultaneously hybridize and be detected to a complementary member
of a universal capture array by the CCD proximal detector. Since
only PCR amplicons hybridized to members of the universal array at
the bottom of each well are proximal to the detector, the assay
requires no special separation step to detect hybridized amplicons
and thus becomes homogenous in nature.
[0242] Similarly, FIG. 16 illustrates this multiplexed concept with
Gap-Ligase Chain Reaction (G-LCR). FIG. 16 is a diagram showing
homogeneous in situ microarray detection of multiplexed gap-ligase
chain reaction products. The ability to detect hybridization events
homogeneously is provided by the fact that only molecules
proximally associated with specific biosites can be imaged by the
detector. FIG. 16 illustrates specific multiplex hybridization
detection of Gap-Ligase Chan Reaction products using
microtiter-based microassays. Similarly, as described previously
for PCR products (see FIG. 15), this figure illustrates the assay
at three separate ligation-dependent amplification loci
simultaneously. Each locus (e.g., LOCUS 1) is defined by two
specially modified primers that define the ends of the gap ligase
chain reaction product. One primer in the pair, contains a
fluorescently detectable label such as fluorescein. The other
primer in the pair contains two domains, one is a unique universal
sequence complementary to a capture probe arrayed at the bottom of
a single microtiter well and the other domain is specific for a
region on the template being detected. The universal sequence
attached to this primer serves as a sequence specific single
stranded handle. When the template is present in the sample then
sequence directed ligation will join both the label and the
universal handle into a single product. After many cycles this
amplified ligated product can be simultaneously hybridized and
detected to its complementary member on a universal capture array
immobilized to the bottom of a microtiter well and imaged by the
CCD proximal detector. Since only ligated products hybridized to
members of the universal array at the bottom of each well are
proximal to the detector, the assay requires no special separation
step to detect hybridized amplicons and thus becomes homogenous in
nature.
Example IV
Drug Discovery/Screening Analysis
[0243] In this example, a small molecule Universal Array could use
high affinity commercially available antibodies to numerous
haptens, steroids, or small molecule drugs. A partial list of 48
representative compounds are enumerated in Table 1 for which
specific antibodies are available. This table is only a partial
list of commercially available antibodies to haptens, steroid
hormones and other small molecule drugs.
TABLE-US-00005 TABLE 1 fluorescein dinitrophenol amphetamine
barbiturate acetaminophen acetohexamide desipramine lidocaine
digitoxin chloroquinine quinine ritalin phenobartibal phenytoin
fentanyl phencyclidine methamphetamine metaniphrine digoxin
penicillin tetrahydrocannibinol tobramycin nitrazepam morphine
Texas Red TRITC primaquine progesterone bendazac carbamazepine
estradiol theophylline methadone methotrexate aldosterone
norethisterone salicylate warfarin cortisol testerone nortryptyline
propanolol estrone androstenedione digoxigenin biotin thyroxine
triidothyronine
[0244] Small molecule Universal Arrays are made by covalent
attachment of small molecules such as those found in Table 1 to
substrate surfaces. Immobilization of haptens, steroids, or drugs
is accomplished by introducing a functionalized moiety at one end
of the small molecule. These moieties are well known to those
skilled in the art (e.g., N-hydroxy-succinimide, maleimide,
isothiocyanate, iodoacetamide or other amine or sulfur reactive
moieties). Small functionalized molecules or drugs can then be
reacted with NH.sub.2 or SH.sub.2 derivitized plastic or glass
substrates. Some specific examples of such commercially available
activated haptens include NHS-fluorescein, NHS-biotin,
NHS-digoxigenin, maleimide-biotin, and
maleimide-tetramethylrhodamine.
[0245] Following deposition of the individual small molecule
biosites, a bispecific ligand can be used to spatially localize
specific binding events to given biosites. The bispecific ligand
can comprise, but is not limited to, antibody-antibody conjugates,
antibody receptor, antibody-streptavidin, antibody-peptide,
antibody-small molecule conjugates or bispecific antibodies.
[0246] The bispecific ligand is specific to both the immobilized
hapten or drug on the substrate surface (biosite) and the analyte
being screened. Examples of Universal Array screening are diagramed
in FIG. 17. FIG. 17 is a diagram showing small molecule Universal
Array (drug screening/discovery). FIG. 17 illustrates the basic
small molecule Universal Array concept using four different
immobilized haptens in a single well. Various bispecific molecules
are diagramed for illustration purposes. FIG. 17 illustrates four
separate and distinct haptens immobilized at the bottom of each of
96 wells of a microtiter plate. Each locus or biosite in the array
is defined by four unique immobilized haptens illustrated in this
example by fluorescein, digoxigenin, 2.4 dinitrophenol, and TRITC.
Bispecific molecules uniquely specific for both the immobilized
hapten and another labeled analyte in the sample are added to each
well. In this fashion, different multiple analytes can be
simultaneously detected and their presence indicated by signals at
specific hapten biosites. In this example, 96 individual samples
can be assayed for four different analytes simultaneously. As
shown, the fluorescein biosite detects a labeled receptor (protein)
analyte, both the 2,4 dinitrophenol and digoxigenin haptens allow
for the simultaneous detection or presence of two additional types
of protein receptors in the sample. Finally, the TRITC hapten
allows for detection and presence of a specific enzyme substrate
via an intervening enzyme conjugate. Once again, the proximal mode
of detection allows for homogenous imaging of only those binding
events at the surface of the array. The advantages of such a
multiplexed immunological approach is the exquisite specificity and
variety of small molecules that comprise such a Universal Array
using non-DNA based recognition of biosites.
[0247] Actual reduction to practice of small molecule Universal
Arrays is illustrated in FIGS. 18 and 19 and described in Example 1
above.
Example V
Use
Gene Expression Analysis
[0248] The multiplexed molecular analysis system is also useful for
analyzing the expression of hundreds of different mRNA species in a
single tissue sample within a single well of a microtiter plate.
Here synthetic nucleic acids form the distinct biosites which
constitute numerous highly sensitive and selective hybridization
analyses per sample, employing only 50 .mu.L of sample extract.
Such massive hybridization analyses enables the discovery and
employment of numerous biomarkers for specific diseases such as
cancer. Essentially, the search for biomarkers of early phase lung
cancer becomes an iterative, combinatorial process. For lung cancer
and other epithelial disease, several hundred mRNAs are analyzed
for their value as biomarkers at relatively low cost. In such an
iterative process, the biostatistician becomes the end-user of the
technology and a central component in the development of the final
set of mRNA biomarkers. Once an mRNA biomarker set is discovered by
this iterative approach, the technology is naturally suited for low
cost, high throughput screening of large patient populations with
the mRNA biomarker set of choice.
Example VI
Use
Cell Sorting
[0249] Conversely, intact cells are analyzed utilizing the
multiplexed format of this invention. Specifically, most "cell
enrichment" protocols involve either double label flow cytometry,
or physical separation of cells via affinity chromatography of some
kind. Both require access to an antibody which is specific to the
cell type of interest.
[0250] In a multiplexed microplate former, the cell-specific
antibodies are arranged in a matrix fashion within the reaction
chamber (single well in the 96 well microplate). The key to making
the cellular analysis work is creating a situation wherein such
antibody arrays retain the capacity for high affinity and high
selectivity binding of intact cells.
[0251] The procedure is to add a complex cellular mixture, e.g., a
biological sample (for example, blood or sputum), to such an
antibody matrix, then with some local mixing, allowing the cells to
bind to the surface. If cells bind to such a matrix with good
affinity and selectivity, they are then fixed to the matrix,
permeabilized, and subjected to labeled probe hybridization (or
PCR) in a fashion which is nearly identical to the methods which
are currently used to analyze DNA or RNA in cells for microscopy or
flow cytometry. The principle benefit of the multiplexed format is
that many different cell types are separated in a single well of a
microtiter plate.
Example VII
Use
Microorganic Monitoring
[0252] Microorganism monitoring applications can also be addressed
by the multiplexed molecular analysis system. In particular for
monitoring air, water, and food for microorganisms, the system can
rapidly and cost effectively provide detection and quantification
of complex biological mixtures. An example would be a ribosomal RNA
probe-based assay in which nucleic acid probes serving as the
biosites are chosen to selectively capture RNA of characteristic
microorganisms.
[0253] Basically, the procedure is initiated by preparing the
microbial rRNA sample for hybridization to the biosite array within
the reaction chamber. Following specific binding of the
fluorescently labeled microbial RNA to the probe array, a two
dimensional image results that uniquely characterizes the sample.
The analyzer output is the microbial spectrum, consisting of the
amount and type of microorganisms present in the sample. The
rationale for the proposed approach to simultaneous monitoring of
microorganisms includes:
[0254] 1) Fast microbial analysis can be achieved due to the
avoidance of standard cell cultivation procedures which require
days to perform. Moreover, the proposed highly sensitive proximal
CCD detection procedure, combined with the inherent amplification
property of rRNA, reduces the combined sample preparation, assay,
and detection time from days to hours.
[0255] 2) Simultaneous microbial monitoring can be achieved due to
the high density arrays that support hundreds of immobilized probes
per cm.sup.2 to facilitate multiple microorganism detection and
identification in a high throughput manner.
[0256] 3) Minimal labor and training is required since no cell
culturing or gel-based sequencing is required. Instead, an operator
merely subjects the prepared sample to automated hybridization,
washing, and drying processes to obtain the microbial spectrum.
[0257] 4) Minimal equipment is necessary since the probe-based
assay is integrated with the proximal CCD detection device, thereby
alleviating traditional macro-detection techniques such as
epifluorescent and confocal microscopy.
[0258] The following references may facilitate the understanding or
practice of the certain aspects and/or embodiments of this
invention. Inclusion of a reference in this list is not intended to
and does not constitute an admission that the reference represents
prior art with respect to the present invention. [0259] Hansell,
U.S. Pat. No. 2,512,743 [0260] D. Bogg, F. Talke, IBM Jour. Res.
Develop. (1984) 29:214-321 [0261] Burke, et al., "An Abuttable CCD
Imager for Visible and X-Ray Focal Plane Arrays," IEEE Trans. On
Electron Devices, 38(5):1069 (May, 1991). [0262] Maskos, U., et
al., Nucleic Acids Res. 20:1679-1684 (1992). [0263] Stephen C.
Case-Green, et al., Nucleic Acids Res. 22:131-136 (1994). [0264]
Guo, Z., et al., Nucleic Acids Res. 22:5456-5465 (1994).
[0265] A number of embodiments of the present invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
35116DNAArtificial SequenceSynthesized Capture Probe 1tgattcagac
cggccg 16216DNAArtificial SequenceSynthesized Capture Probe
2cccggggcgt cttaac 16316DNAArtificial SequenceSynthesized Capture
Probe 3ggacgccata tgcgct 16416DNAArtificial SequenceSynthesized
Capture Probe 4tgagggctcc gccata 16516DNAArtificial
SequenceSynthesized Capture Probe 5aacccgtgac gtgtgc
16616DNAArtificial SequenceSynthesized Capture Probe 6agcatcgccg
gtcctg 16716DNAArtificial SequenceSynthesized Capture Probe
7cctgcaaggc tgacgt 16816DNAArtificial SequenceSynthesized Capture
Probe 8cagttgtcga ccccgg 16916DNAArtificial SequenceSynthesized
Capture Probe 9cggcgcgtcc aattcg 161016DNAArtificial
SequenceSynthesized Capture Probe 10atcgatctga gggccc
161116DNAArtificial SequenceSynthesized Capture Probe 11gtacatgcgg
cctgca 161216DNAArtificial SequenceSynthesized Capture Probe
12tagccgctcg ctagag 161316DNAArtificial SequenceSynthesized Capture
Probe 13cctagtgatg accggc 161416DNAArtificial SequenceSynthesized
Capture Probe 14gtctgagggc aacctc 161516DNAArtificial
SequenceSynthesized Capture Probe 15ctagctggct acgcag
161616DNAArtificial SequenceSynthesized Capture Probe 16gccatccgct
tggagc 161718DNAArtificial SequenceSynthesized Elemental Target
Probe 17ttactaagtc tggccggc 181818DNAArtificial SequenceSynthesized
Elemental Target Probe 18ttgggccccg cagaattg 181918DNAArtificial
SequenceSynthesized Elemental Target Probe 19ttcctgcggt atacgcga
182018DNAArtificial SequenceSynthesized Elemental Target Probe
20ttactcccga ggcggtat 182118DNAArtificial SequenceSynthesized
Elemental Target Probe 21ttttgggcac tgcacacg 182218DNAArtificial
SequenceSynthesized Elemental Target Probe 22tttcgtagcg gccaggac
182318DNAArtificial SequenceSynthesized Elemental Target Probe
23ttggacgttc cgactgca 182418DNAArtificial SequenceSynthesized
Elemental Target Probe 24ttgtcaacag ctggggcc 182518DNAArtificial
SequenceSynthesized Elemental Target Probe 25ttgccgcgca ggttaagc
182618DNAArtificial SequenceSynthesized Elemental Target Probe
26tttagctaga ctcccggg 182718DNAArtificial SequenceSynthesized
Elemental Target Probe 27ttcatgtacg ccggacgt 182818DNAArtificial
SequenceSynthesized Elemental Target Probe 28ttatcggcga gcgatctc
182918DNAArtificial SequenceSynthesized Elemental Target Probe
29ttggatcact actggccg 183018DNAArtificial SequenceSynthesized
Elemental Target Probe 30ttcagactcc cgttggag 183118DNAArtificial
SequenceSynthesized Elemental Target Probe 31ttgatcgacc gatgcgtc
183218DNAArtificial SequenceSynthesized Elemental Target Probe
32ttcggtaggc gaacctcg 183317DNAArtificial SequenceSynthesized as
and Amine 33ccacactgga actgaga 173417DNAArtificial
SequenceSynthesized Elemental Target Probe 34ggtgtgacct tgactct
173516DNAArtificial SequenceSynthesized Elemental Target Probe
35actaagtctg gccggc 16
* * * * *