U.S. patent application number 11/044834 was filed with the patent office on 2005-08-18 for methods for making a device for concurrently processing multiple biological chip assays.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Fodor, Stephen P.A., Norviel, Vernon A., Rava, Richard P., Trulson, Mark.
Application Number | 20050181403 11/044834 |
Document ID | / |
Family ID | 32045837 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181403 |
Kind Code |
A1 |
Rava, Richard P. ; et
al. |
August 18, 2005 |
Methods for making a device for concurrently processing multiple
biological chip assays
Abstract
Methods for concurrently processing multiple biological chip
assays by providing a biological chip plate comprising a plurality
of test wells, each test well having a biological chip having a
molecular probe array; introducing samples into the test wells,
subjecting the biological chip plate to manipulation by a fluid
handling device that automatically performs steps to carry out
reactions between target molecules in the samples and probes; and
subjecting the biological chip plate to a biological chip plate
reader that interrogates the probe arrays to detect any reactions
between target molecules and probes.
Inventors: |
Rava, Richard P.; (San Jose,
CA) ; Fodor, Stephen P.A.; (Palo Alto, CA) ;
Trulson, Mark; (San Jose, CA) ; Norviel, Vernon
A.; (San Jose, CA) |
Correspondence
Address: |
AFFYMETRIX, INC
ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3380 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
|
Family ID: |
32045837 |
Appl. No.: |
11/044834 |
Filed: |
January 26, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11044834 |
Jan 26, 2005 |
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10997492 |
Nov 24, 2004 |
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10997492 |
Nov 24, 2004 |
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10795086 |
Mar 5, 2004 |
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10795086 |
Mar 5, 2004 |
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10157252 |
May 28, 2002 |
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6720149 |
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10157252 |
May 28, 2002 |
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09247430 |
Feb 10, 1999 |
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09247430 |
Feb 10, 1999 |
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08630051 |
Apr 9, 1996 |
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5874219 |
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08630051 |
Apr 9, 1996 |
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08476850 |
Jun 7, 1995 |
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5545531 |
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Current U.S.
Class: |
435/6.12 ;
702/20 |
Current CPC
Class: |
B01J 2219/00432
20130101; B01L 2300/041 20130101; B01J 2219/00317 20130101; B01J
2219/00639 20130101; B01L 2300/0636 20130101; B01L 3/5085 20130101;
B01J 2219/00529 20130101; B01J 2219/00707 20130101; G01N 35/028
20130101; B01L 2300/0829 20130101; B01L 2300/0819 20130101; B01J
2219/00619 20130101; B01J 2219/00617 20130101; B01J 2219/00662
20130101; C12Q 1/6837 20130101; B01J 2219/00576 20130101; B01J
2219/00612 20130101; B01J 2219/00722 20130101; B01L 3/50853
20130101; C40B 60/14 20130101; B01J 19/0046 20130101; B01J
2219/00626 20130101; B01J 2219/00605 20130101; B01L 2400/0683
20130101; B01J 2219/00315 20130101; B01J 2219/00637 20130101; G01N
21/64 20130101; C40B 40/06 20130101; B01J 2219/00659 20130101; B01J
2219/00286 20130101; B01L 2300/046 20130101; G01N 2035/00158
20130101; B01J 2219/00608 20130101; B01L 2300/044 20130101; B01J
2219/00527 20130101; B01J 2219/00621 20130101; B01L 3/5027
20130101; B01J 2219/0061 20130101; B01J 2219/00702 20130101 |
Class at
Publication: |
435/006 ;
702/020 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Claims
1. A method for concurrently processing multiple biological chip
assays comprising the steps of: (a) providing a biological chip
plate comprising a plurality of probe arrays and, surrounding the
probe arrays, material resistant to the flow of liquid, thereby
forming a plurality of test wells, each test well defining a space
for the introduction of a sample; (b) introducing into each test
well test samples from a plurality of different patients, wherein
each test sample contains target molecules; (c) manipulating the
biological chip plate with a fluid handling device that
automatically performs steps to carry out reactions between target
molecules in the test samples and probes in a plurality of the test
wells, wherein the fluid handling device controls temperature,
sample handling, substrate handling and washing of the test wells;
and (d) interrogating the probe arrays of the biological chip plate
with a biological chip plate reader to detect reactions between
target molecules and probes in a plurality of the test wells to
generate assay results.
2-31. (canceled)
32. A method of simultaneously conducting multiple chemical
reactions in a reaction assembly that comprises a microtiter plate
of wells containing test samples and an array of sets of chemical
reactants comprising the steps of: assembling the array of sets of
chemical reactants to the microtiter plate of test samples such
that the array covers open ends in the test sample wells of the
microtiter plate to form a plurality of closed cells, each closed
cell comprising a set of chemical reactants and a respective test
sample; sealing the microtiter plate to the array to create one or
more of a gas tight, a liquid tight, and a fluid tight seal; and
mechanically agitating the sealed reaction assembly to contact the
test samples with the chemical reactants in each closed cell
simultaneously.
33. The method of claim 32, wherein the step of assembling
comprises the step of placing a pliable gasket between the
microtiter plate and the array, the gasket having an arrangement of
through holes that align with the test sample wells and the sets of
chemical reactants; and the step of sealing comprises the step of
applying one or more of mechanical clamps radiation, heat, external
fluid pressure, vacuum and an adhesive to the reaction
assembly.
34. The method of claim 33, wherein the gasket further comprises an
adhesive on at least one gasket surface that interfaces with the
microtiter plate or the array, and wherein the step of scaling
further comprises removing the one or more of mechanical clamps,
radiation, heat, external fluid pressure, and vacuum after a period
of time.
35. The method of claim 34, wherein the adhesive is selected from
an ultraviolet (UV) light curable adhesive that has increased
adhesion with the application of UV light to the adhesive, and a
releasable adhesive that has reduced adhesion with the application
of one or more of heat, cold and radiation to the adhesive.
36. The method of claim 33, wherein in the step of placing the
gasket, the gasket further has at least one channel that
interconnects at least two through holes, such that in the step of
assembling, the reaction assembly has at least two closed cells
that are interconnected, and wherein in the step of mechanically
agitating, the agitation has an acceleration, and the step of
mechanically agitating comprises the step of incrementally
increasing the acceleration to sequentially mix the test samples of
the interconnected closed cells.
37. The method of claim 36, wherein the interconnected closed cells
are located adjacent to each other.
38. The method of claim 32, wherein in the step of assembling, the
array is made of a flexible material, and wherein in the step of
sealing, the array is placed against the microtiter plate using one
or more of mechanical clamps, radiation, heat, external fluid
pressure, vacuum and an adhesive to seal the reaction assembly.
39. The method of claim 32, wherein in the step of assembling, the
array is made of an optically transparent flexible film having an
adhesive surface that surrounds the sets of chemical reactants, the
adhesive surface being contacted with the microtiter plate.
40. The method of claim 32, wherein in the step of assembling, the
array is made of a flexible material having an adhesive on a
surface that comprises the sets of chemical reactants, and the
adhesive surface is contacted with the microtiter plate, and
wherein the step of sealing comprises applying one or more of
mechanical clamps, radiation, heat, external fluid pressure, and
vacuum to the reaction assembly for a period of time until the
adhesive adheres the array to the plate.
41. The method of claim 40, wherein the adhesive is selected from
an ultraviolet (UV) light curable adhesive that has increased
adhesion with the application of UV light to the adhesive, and a
releasable adhesive that has reduced adhesion with the application
of one or more of heat, cold and radiation to the adhesive.
42. The method of claim 32, wherein the test sample wells are
spatially arranged in a surface of the microtiter plate, each well
having a side wall adjacent to a closed end that together enclose
the well except for an open end at the surface of the microtiter
plate, and wherein the ray comprises an array substrate having the
sets of chemical reactants bound to an ray surface of the array
substrate in an array pattern of features, the array pattern being
similar to the spatial arrangement of test sample wells on the
microtiter plate.
43. The method of claim 32, wherein in the step of mechanically
agitating a difference in mass densities between the test sample
and gas filling any space between the test sample and the set of
chemical reactants in each closed cell causes mixing of the test
sample with the chemical reactants in each closed cell.
44. The method of claim 32, further comprising the step of
analyzing reaction products in the closed cells after the step of
mechanically agitating.
45. The method of claim 44, wherein one or both of the microtiter
plate and the array is optically transparent.
46. The method of claim 32, further comprising the step of
analyzing reaction products after the step of mechanically
agitating comprising the steps of: disassembling the reaction
assembly; rinsing the array; and interrogating the array.
47. The method of claim 32, wherein the microtiter plate is
selected from a 96, 234, 384, and 1536 well microtiter plate and
the number of sets of chemical reactants on the array match the
selected microtiter plate.
48. The method of claim 32, wherein each set of chemical reactants
is an array feature that comprises a subarray having the chemical
reactants arranged in a subarray pattern of subfeatures, and
wherein the chemical reactant is different in at least one feature
or in at least one subfeature on the array.
49. The method of claim 32, wherein the test sample is different in
at least one well of the microtiter plate.
50. A method of simultaneously conducting multiple chemical
reactions between a first chemical sample and a second chemical
sample comprising the steps of: providing a plate having a
plurality of wells spatially arranged in a surface of the plate in
a well array pattern, each well having a side wall adjacent to a
closed end that enclose the well except for an open end that is
opposite the closed end and that is adjacent to the plate surface,
the plurality of wells for receiving the first chemical sample via
the open end; providing an array of the second chemical sample, the
ray comprising sets of the second chemical sample bound to and
spatially arranged on a surface of an array substrate in an array
pattern of features, the well array pattern being spatially similar
to the feature array pattern; assembling the array onto the plate
to form a sealed reaction assembly, such that the surface of the
array faces the surface of the plate and encloses the open ends of
the plurality of wells to form closed cells, each closed cell
comprising the first chemical sample and a respective set of the
second chemical sample features, wherein the sealed reaction
assembly is one or more of gas tight, liquid tight, and fluid
tight; and contacting the first chemical sample with the second
chemical sample in each closed cell of the sealed reaction
assembly.
51. The method of claim 50, wherein the array substrate is made of
a flexible material, and wherein in the step of assembling, the
array substrate is contacted with the plate using one or more of
mechanical clamps, radiation, heat, external fluid pressure, vacuum
and an adhesive to seal the reaction assembly.
52. The method of claim 50, wherein the array substrate is made of
an optically transparent flexible film having an adhesive on the
surface to which the second chemical samples are bound, the
adhesive surrounding the features, and wherein in the step of
assembling, the adhesive is contacted with the plate surface to
seal the reaction assembly.
53. The method of claim 51, wherein the flexible array substrate
further comprises the adhesive on the surface to which the second
chemical samples are bound, and wherein in the step of assembling,
the adhesive surface of the array is contacted with the plate
surface, and the reaction assembly is sealed using one or more of
beat, radiation, and pressure.
54. The method of claim 53, wherein the adhesive is selected from a
releasable adhesive, such that adhesion is reduced with the
application of heat, cold or radiation to the adhesive, and an
ultraviolet light (UV) curable adhesive, such that adhesion is
increased with the application of UV light to the adhesive.
55. The method of claim 50, wherein the plate further comprises a
pliable gasket material integral with the surface of the plate, the
gasket comprising a plurality of through holes spatially arranged
trough a thickness of the gasket material to correspond with
arrangement of the plurality of wells, and wherein in the step of
assembling, the surface of the ray is contacted with the integral
gasket with one or more of mechanical clamps, radiation, heat,
external fluid pressure, vacuum and an adhesive to seal the
reaction assembly.
56. The method of claim 55, wherein the integral gasket further has
at least one channel that interconnects at least two through holes,
such that in the step of assembling, the reaction assembly has at
least two closed cells that are interconnected, and wherein in the
step of contacting, the first chemical samples in the
interconnected closed cells mix with each other and with the second
chemical samples in each interconnected closed cell via the
channel.
57. The method of claim 50, further comprising the step of
providing a gasket having a plurality of through holes spatially
ranged through a thickness of the gasket in a through hole array
pattern, wherein the well pattern, the array pattern and the
through hole pattern are dimensionally and spatially similar, the
gasket being made of a pliable material, and wherein the step of
assembling comprises placing the pliable gasket between the plate
surface and the array surface, such that the plurality of through
holes are aligned with the features of the second chemical sample
and the wells, and sealing the gasket to the array and the plate
using one or more of mechanical clamps, radiation, heat, external
fluid pressure, vacuum and an adhesive to seal the reaction
assembly.
58. The method of claim 57, wherein the pliable gasket comprises
the adhesive on at least one surface adjacent either the plate
surface or the array surface.
59. The method of claim 58, wherein the adhesive is selected from a
releasable adhesive, such that adhesion is reduced with the
application of heat, cold or radiation to the adhesive, and an
ultraviolet light (UV) curable adhesive, such that adhesion is
increased with the application of UV light to the adhesive.
60. The method of claim 57, wherein in the step of providing the
gasket, the gasket further has at least one channel that
interconnects at least two through holes, such that in the step of
assembling, the reaction assembly has at least two closed cells
that are interconnected, and wherein in the step of contacting, the
first chemical samples in the interconnected closed cells mix with
each other and with the second chemical samples in each
interconnected closed cell via the channel.
61. The method of claim 50, wherein the step of contacting
comprises one or more of mechanically agitating the reaction
assembly, controlling the reaction temperature of the reaction
assembly, directing radiation into the assembly, and inverting the
reaction assembly to cause mixing between the first chemical sample
and the second chemical sample.
62. The method of claim 50, before the step of assembling, further
comprising the steps of: providing a gasket having a plurality of
spatially arranged through holes, the arrangement of through holes
being similar to the arrangement of the wells and of the array
pattern, the gasket being made of a pliable material; and
introducing an aliquot of the first chemical sample into each well
of the plate, the first chemical samples being fluid and partially
filling the wells, and introducing a volume of a second fluid to
the wells, the second fluid having a mass density that is different
from a mass density of the first chemical sample and the second
fluid being non-reactive with the first chemical sample and second
chemical sample, wherein the step of assembling comprises the steps
of: placing the gasket on the surface of the plate, placing the
array on the gasket, such that the array features are aligned with
the through holes and the wells, and sealing the plate, the gasket
and the array together using one or more of mechanical clamps,
radiation, heat, external fluid pressure, vacuum and an adhesive,
and wherein the step of contacting comprises mechanically agitating
the reaction assembly to mix the first chemical sample and the
second chemical sample in each closed reaction cell.
63. The method of claim 62, wherein in the step of mechanically
agitating, the difference in the mass densities of the first
chemical sample and the second fluid causes mixing of the first
chemical sample with the second chemical sample in each closed
cell.
64. The method of claim 62, wherein in the step of providing the
gasket, the gasket further has at least one channel that
interconnects at least two through holes, such that in the step of
assembling, the reaction assembly has at least two closed cells
that are interconnected, and wherein in the step of mechanically
agitating, the agitation has an acceleration, and the step of
mechanically agitating comprises incrementally increasing the
acceleration in magnitude to sequentially mix the first chemical
samples of the interconnected closed cells.
65. The method of claim 64, wherein the interconnected closed cells
are located adjacent to each other.
66. The method of claim 62, wherein the gasket further comprises
the adhesive on surfaces that interface with the plate and the
array, and wherein the step of sealing further comprises removing
the one or more of mechanical clamps, radiation, heat, external
fluid pressure, and vacuum after a period of time.
67. The method of claim 66, wherein the gasket is made of a
flexible adhesive film.
68. An apparatus for simultaneously conducting multiple chemical
reactions comprising: a plate having a plurality of wells spatially
arranged in a surface of the plate in a well array pattern, each
well having a side wall adjacent to a closed end that enclose the
well except for an open end that is opposite the closed end and
that is adjacent to the plate surface, the plurality of wells for
receiving a test sample via the open end; an array of sets of
chemical reactants, the sets of chemical reactants being bound to
and spatially arranged on a surface of an array substrate in an
array pattern of features, the well array pattern being spatially
similar to the feature array pattern, wherein the array surface
faces the plate surface and covers the open ends of the wells to
form closed cells, each closed cell comprising a respective test
sample and a respective set of the chemical reactants; and a seal
between the plate and the array that is one or more of gas tight,
liquid tight, and fluid tight.
69. The apparatus of claim 68, wherein the seal comprises a pliable
gasket and one or more of mechanical clamps, radiation, heat,
external fluid pressure, vacuum and an adhesive, the gasket having
a plurality of through holes spatially arranged through a thickness
of the gasket in a through hole pattern that is dimensionally and
spatially similar to the well pattern and the array pattern.
70. The apparatus of claim 69, wherein the pliable gasket is a
flexible adhesive film.
71. The apparatus of claim 69, wherein the pliable gasket further
has at least one channel that interconnects at least two through
holes, such that the respective test samples in the interconnected
closed cells can be mixed via the channel.
72. The apparatus of claim 69, wherein the pliable gasket is
integral with the surface of the plate.
73. The apparatus of claim 68, wherein the seal comprises the array
substrate being made of a flexible material and one or more of
mechanical clamps, radiation, heat, external fluid pressure, vacuum
and an adhesive.
74. The apparatus of claim 68, wherein the seal comprises the array
substrate being made of an optically transparent flexible film
having an adhesive that surrounds the features on the array
surface, the adhesive being contacted with the plate surface.
75. The apparatus of claim 68, wherein the seal comprises using an
adhesive selected from a releasable adhesive, such that adhesion is
reduced with the application of heat, cold or radiation to the
adhesive, and an ultraviolet light curable adhesive, such that
adhesion is increased with the application of ultraviolet light to
the adhesive.
76. The apparatus of claim 75, wherein the releasable adhesive is
an ultraviolet light-releasable adhesive.
77. The apparatus of claim 72, wherein in the plate is a microtiter
plate selected from a 96, 234, 384, and 1536 well plate, and
wherein the array has the array pattern that matches the selected
microtiter plate.
78. A kit for simultaneously conducting multiple different assays
of biological materials comprising: an array having a plurality of
sets of chemical reactants spatially arranged on an may substrate;
and a plate having a plurality of spatially arranged wells in the
plate, the wells being closed at one end and open at an opposite
end for receiving a sample under test, wherein the array and the
plate form a multiple closed cell reaction assembly when the array
is assembled to the plate, such that the array covers the open ends
of the wells to form closed cells, each closed cell comprising the
test sample and a respective set of the chemical reactants, the
reaction assembly comprising a seal between the plate and the array
that is one or more of gas tight, liquid tight, and fluid tight
when assembled.
79. The kit of claim 78, further comprising one or more of: a
pliable gasket having a plurality of spatially arranged through
holes similar to the spatial arrangement of the wells and the sets
of reactants, the pliable gasket providing the seal between the
plate and the array when combined with one or more of mechanical
clamps, radiation, heat, external fluid pressure, vacuum and an
adhesive; an adhesive for sealing at least the array and the plate;
a sample biological material for a control experiment; instructions
for simultaneously conducting multiple reactions; and instructions
for assembling the array to the plate.
80. A method of simultaneously conducting multiple chemical
reactions in a reaction assembly that comprises a microtiter plate
of wells containing test samples and an may of sets of chemical
reactants comprising the steps of: assembling the array of sets of
chemical reactants to the microtiter plate of test samples such
that the may covers open ends in the test sample wells of the
microtiter plate to form a plurality of closed cells, each closed
cell comprising a set of chemical reactants and a respective test
sample, the sets of chemical reactants being bound to an array
surface of the array; sealing the microtiter plate to the array to
create one or more of a gas tight, a liquid tight, and a fluid
tight seal; and mechanically agitating the sealed reaction assembly
to contact the test samples with the array-bound chemical reactants
in each closed cell simultaneously.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/157,252, filed May 28, 2002 which is a continuation of Ser.
No. 09/247,430, filed Feb. 10, 1999 (now abandoned), which is a
continuation of U.S. application Ser. No. 08/630,051, filed Apr. 9,
1996, now U.S. Pat. No. 5,874,219, which is a continuation of U.S.
application Ser. No. 08/476,850, filed Jun. 7, 1995, now U.S. Pat.
No. 5,545,531 The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods for concurrently
performing multiple biological chip assays. The invention therefore
relates to diverse fields impacted by the nature of molecular
interaction, including chemistry, biology, medicine and
diagnostics.
[0003] New technology, called VLSIPS.TM., has enabled the
production of chips smaller than a thumbnail that contain hundreds
of thousands or more of different molecular probes. These
biological chips or arrays have probes arranged in arrays, each
probe assigned a specific location. Biological chips nave been
produced in which each location has a scale of, for example, ten
microns. The chips can be used to determine whether target
molecules interact with any of the probes on the chip After
exposing the array to target molecules under selected test
conditions, scanning devices can examine each location in the array
and determine whether a target molecule nas interacted with the
probe at that location.
[0004] Biological chips or arrays are useful in a variety of
screening techniques for obtaining information about either the
probes or the target molecules For example, a library of peptides
can be used as probes to screen for drugs. The peptides can be
exposed to a receptor, and those probes that bind to the receptor
can be identified.
[0005] Arrays of nucleic acid probes can be used to extract
sequence information from, for example, nucleic acid samples The
samples are exposed to the probes under conditions that allow
hybridization. The arrays are then scanned to determine to which
probes the sample molecules have hybridized. One can obtain
sequence information by careful probe selection and using
algorithms to compare patterns of hybridization and
non-hybridization This method is useful for sequencing nucleic
acids, as well as sequence checking. For example, the method is
useful in diagnostic screening for genetic diseases or for the
presence and/or identity of a particular pathogen or a strain of
pathogen. For example, there are various strains of HIV, the virus
that causes AIDS. Some of them have become resistant to current
AIDS therapies. Diagnosticians can use DNA arrays to examine a
nucleic acid sample from the virus to determine what strain it
belongs to.
[0006] Currently, chips are treated individually, from the step of
exposure to the target molecules to scanning. These methods yield
exquisitely detailed information However, they are not adapted for
handling multiple samples simultaneously. The ability to do so
would be advantageous in settings in which large amounts of
information are required quickly, such as in clinical diagnostic
laboratories or in large-scale undertakings such as the Human
Genome Protect.
SUMMARY OF THE INVENTION
[0007] This invention provides methods for concurrently processing
multiple biological chip assays According to the methods, a
biological chip plate comprising a plurality of test wells is
provided. Each test well defines a space for the introduction of a
sample and contains a biological array The array is formed on a
surface of the substrate, with the probes exposed to the space. A
fluid handling device manipulates the plates to perform steps to
carry out reactions between the target molecules in samples and the
probes in a plurality of test wells The biological chip plate is
then interrogated by a biological chip plate reader to detect any
reactions between target molecules and probes in a plurality of the
test wells, thereby generating results of the assay. In a further
embodiment of the invention, the method also includes processing
the results of the assay with a computer. Such analysis is useful
when sequencing a gene by a method that uses an algorithm to
process the results of many hybridization assays to provide the
nucleotide sequence of the gene.
[0008] The methods of the invention can involve the binding of
tagged target molecules to the probes. The tags can be, for
example, fluorescent markers, chemiluminescent markers, light
scattering markers or radioactive markers. In certain embodiments,
the probes are nucleic acids, such as DNA or RNA molecules. The
methods can be used to detect or identify a pathogenic organism,
such as HIV, or to detect a human gene variant, such a the gene for
a genetic disease such as cystic fibrosis, diabetes, muscular
dystrophy or predisposition to certain cancers.
[0009] This invention also provides systems for performing the
methods of this invention. The systems include a biological chip
plate, a fluid handling device that automatically performs steps to
carry out assays on samples introduced into a plurality of the test
wells, a biological chip plate reader that determines in a
plurality of the test wells the results of the assay and,
optionally, a computer comprising a program for processing the
results. The fluid handling device and plate reader can have a
heater/cooler controlled by a thermostat for controlling the
temperature of the samples in the test wells and robotically
controlled pipets for adding or removing fluids from the test wells
at predetermined times.
[0010] In certain embodiments, the probes are attached by
light-directed probe synthesis The biological chip plates can have
96 wells arranged in 8 rows and 12 columns, such as a standard
microtiter plate The probe arrays can each have at least about 100,
1000, 100,000 or 1,000,000 addressable features (e.g., probes). A
variety of probes can be used on the plates, including, for
example, various polymers such as peptides or nucleic acids.
[0011] The plates can have wells in which the probe array in each
test well is the same Alternatively, when each of several samples
are to be subjected to several tests, each row can have the same
probe array and each column can have a different array.
Alternatively, all the wells can have different arrays.
[0012] Several methods of making biological chip plates are
contemplated. In one method, a wafer and a body are provided. The
wafer includes a substrate and a surface to which is attached a
plurality of arrays of probes. The body has a plurality of
channels. The body is attached to the surface of the wafer whereby
the channels each cover an array of probes and the wafer closes one
end of a plurality of the channels, thereby forming test wells
defining spaces for receiving samples. In a second method, a body
having a plurality of wells defining spaces is provided and
biological chips are provided. The chips are attached to the wells
so that the probe arrays are exposed to the space Another
embodiment involves providing a wafer having a plurality of probe
arrays; and applying a material resistant to the flow of a liquid
sample so as to surround the probe arrays, thereby creating test
wells.
[0013] This invention also provides a wafer for making a biological
chip plate The wafer has a substrate and a surface to which are
attached a plurality of probe arrays The probe arrays are arranged
on the wafer surface in rows and columns, wherein the probe arrays
in each row are the same and the probe arrays in each column are
different.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 depicts a system of this invention having a
biological chip plate, fluid handling device, biological chip plate
reader and computer;
[0015] FIG. 2 depicts the scanning of a biological chip plate by a
biological chip plate reader,
[0016] FIG. 3 depicts a biological plate of this invention,
[0017] FIG. 4 depicts the mating of a wafer containing many
biological arrays with a body having channels to create a
biological chip plate,
[0018] FIG. 5 depicts a biological chip plate in cross section
having a body attached to a wafer to create closed test wells in
which a probe array is exposed to the space in the test well;
[0019] FIG. 6 depicts a biological plate in cross section having a
body which has individual biological chips attached to the bottom
of the wells;
[0020] FIG. 7 is a top-down view of a test well containing a
biological array; and
[0021] FIG. 8 depicts a method of producing an array of
oligonucleotide probes on the surface of a substrate by using a
mask to expose certain parts of the surface to light, thereby
removing photoremovable protective groups, and attaching
nucleotides to the exposed reactive groups.
DETAILED DESCRIPTION OF THE INVENTION
[0022] I Definitions
[0023] The following terms are intended to have the following
general meanings as they are used herein
[0024] A Complementary: Refers to the topological compatibility or
matching together of interacting surfaces of a probe molecule and
its target Thus, the target and its probe can be described as
complementary, and furthermore, the contact surface characteristics
are complementary to each other
[0025] B. Probe A probe is a surface-immobilized molecule that can
be recognized by a particular target Examples of probes that can be
investigated by this invention include, but are not restricted to,
agonists and antagonists for cell membrane receptors, toxins and
venoms, viral epitopes, hormones (e.g., opioid peptides, steroids,
etc), hormone receptors, peptides, enzymes, enzyme substrates,
cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids,
oligosaccharides proteins, and monoclonal antibodies
[0026] C. Target: A molecule that has an affinity for a given
probe. Targets may be naturally-occurring or man-made molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Targets may be attached, covalently
or noncovalently, to a binding member, either directly or via a
specific binding substance. Examples of targets which can be
employed by this invention include, but are not restricted to,
antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, oligonucleotides,
nucleic acids, peptides, cofactors, lectins, sugars,
polysaccharides, cells, cellular membranes, and organelles. Targets
are sometimes referred to in the art as anti-probes. As the term
targets is used herein, no difference in meaning is intended A
"Probe Target Pair" is formed when two macromolecules have combined
through molecular recognition to form a complex.
[0027] D. Array A collection of probes, at least two of which are
different, arranged in a spacially defined and physically
addressable manner
[0028] E. Biological Chip A substrate having a surface to which one
or more arrays of probes is attached. The substrate can be, merely
by way of example, silicon or glass and can have the thickness of a
glass microscope slide or a glass cover slip. Substrates that are
transparent to light are useful when the method of performing an
assay on the chip involves optical detection. As used herein, the
term also refers to a probe array and the substrate to which it is
attached that form part of a wafer
[0029] F. Wafer: A substrate having a surface to which a plurality
of probe arrays are attached. On a wafer, the arrays are physically
separated by a distance of at least about a millimeter, so that
individual chips can be made by dicing a wafer or otherwise
physically separating the array into units having a probe
array.
[0030] G. Biological Chip Plate: A device having an array of
biological chips in which the probe array of each chip is separated
from the probe array of other chips by a physical barrier resistant
to the passage of liquids and forming an area or space, referred to
as a "test well," capable of containing liquids in contact with the
probe array.
[0031] II General
[0032] This invention provides automated methods for concurrently
processing multiple biological chip assays Currently available
methods utilize each biological chip assay individually. The
methods of this invention allow many tests to be set up and
processed together Because they allow much higher throughput of
test samples, these methods greatly improve the efficiency of
performing assays on biological chips.
[0033] In the methods of this invention, a biological chip plate is
provided having a plurality of test wells Each test well includes a
biological chip Test samples, which may contain target molecules,
are introduced into the test wells A fluid handling device exposes
the test wells to a chosen set of reaction conditions by, for
example, adding or removing fluid from the wells, maintaining the
liquid in the wells at predetermined temperatures, and agitating
the wells as required, thereby performing the test. Then, a
biological chip reader interrogates the probe arrays in the test
wells, thereby obtaining the results of the tests A computer having
an appropriate program can further analyze the results from the
tests.
[0034] Referring to FIG. 1, one embodiment of the invention is a
system for concurrently processing biological chip assays. The
system includes a biological chip plate reader 100, a fluid
handling device 110, a biological chip plate 120 and, optionally, a
computer 130. In operation, samples are placed in wells on the chip
plate 120 with fluid handling device 110 The plate optionally can
be moved with a stage translation device 140. Reader 100 is used to
identify where targets in the wells have bound to complementary
probes. The system operates under control of computer 130 which may
optionally interpret the results of the assay.
[0035] A. Biological Chip Plate Reader
[0036] In assays performed on biological chips, detectably labeled
target molecules bind to probe molecules Reading the results of an
assay involves detecting a signal produced by the detectable label.
Reading assays on a biological chip plate requires a biological
chip reader Accordingly, locations at which target(s) bind with
complementary probes can be identified by detecting the location of
the label Through knowledge of the characteristics/sequence of the
probe versus location, characteristics of the target can be
determined. The nature of the biological chip reader depends upon
the particular type of label attached to the target molecules.
[0037] The interaction between targets and probes can be
characterized in terms of kinetics and thermodynamics As such, it
may be necessary to interrogate the array while in contact with a
solution of labeled targets. In such systems, the detection system
must be extremely selective, with the capacity to discriminate
between surface-bound and solution-born targets. Also, in order to
perform a quantitative analysis, the high-density of the probe
sequences requires the system to have the capacity to distinguish
between each feature site. The system also should have sensitivity
to low signal and a large dynamic range.
[0038] In one embodiment, the chip plate reader includes a confocal
detection device having a monochromatic or polychromatic light
source, a focusing system for directing an excitation light from
the light source to the substrate, a temperature controller for
controlling the substrate temperature during a reaction, and a
detector for detecting fluorescence emitted by the targets in
response to the excitation light. The detector for detecting the
fluorescent emissions from the substrate, in some embodiments,
includes a photomultiplier tube. The location to which light is
directed may be controlled by, for example, an x-y-z translation
table. Translation of the x-y-z table, temperature control, and
data collection are managed and recorded by an appropriately
programmed digital computer.
[0039] Further details for methods of detecting fluorescently
labelled materials on biological chips are provided in U.S. patent
application Ser. No. 08/195,889, filed Feb. 10, 1994 and
incorporated herein by reference.
[0040] FIG. 2 illustrates the reader according to one specific
embodiment. The chip plate reader comprises a body 200 for
immobilizing the biological chip plate Excitation radiation, from
an excitation source 210 having a first wavelength, passes through
excitation optics 220 from below the array. The light passes
through the chip plate since it is transparent to at least this
wavelength of light The excitation radiation excites a region of a
probe array on the biological chip plate 230 In response, labeled
material on the sample emits radiation which has a wavelength that
is different from the excitation wavelength Collection optics 240,
also below the array, then collect the emission from the sample and
image it onto a detector 250, which can house a CCD array, as
described below The detector generates a signal proportional to the
amount of radiation sensed thereon. The signals can be assembled to
represent an image associated with the plurality of regions from
which the emission originated.
[0041] According to one embodiment, a multi-axis translation stage
260 moves the biological chip plate to position different wells to
be scanned, and to allow different probe portions of a probe array
to be interrogated. As a result, a 2-dimensional image of the probe
arrays in each well is obtained.
[0042] The biological chip reader can include auto-focusing feature
to maintain the sample in the focal plane of the excitation light
throughout the scanning process. Further, a temperature controller
may be employed to maintain the sample at a specific temperature
while it is being scanned. The multi-axis translation stage,
temperature controller, auto-focusing feature, and electronics
associated with imaging and data collection are managed by an
appropriately programmed digital computer 270.
[0043] In one embodiment, a beam is focused onto a spot of about 2
.mu.m in diameter on the surface of the plate using, for example,
the objective lens of a microscope or other optical means to
control beam diameter. (See, e.g., U.S. patent application
08/195,889, supra).
[0044] In another embodiment, fluorescent probes are employed in
combination with CCD imaging systems Details of this method are
described in U.S. application Ser. No. 08/301,051, incorporated
herein by reference in its entirely In many commercially available
microplate readers, typically the light source is placed above a
well, and a photodiode detector is below the well. In the present
invention, the light source can be replaced with a higher power
lamp or laser In one embodiment, the standard absorption geometry
is used, but the photodiode detector is replaced with a CCD camera
and imaging optics to allow rapid imaging of the well. A series of
Raman holographic or notch filters can be used in the optical path
to eliminate the excitation light while allowing the emission to
pass to the detector. In a variation of this method, a fiber optic
imaging bundle is utilized to bring the light to the CCD detector.
In another embodiment, the laser is placed below the biological
chip plate and light directed through the transparent wafer or base
that forms the bottom of the biological chip plate In another
embodiment, the CCD array is built into the wafer of the biological
chip plate.
[0045] The choice of the CCD array will depend on the number of
probes in each biological array. If 2500 probes nominally arranged
in a square (50.times.50) are examined, and 6 lines in each feature
are sampled to obtain a good image, then a CCD array of
300.times.300 pixels is desirable in this area. However, if an
individual well has 48,400 probes (220.times.220) then a CCD array
with 1320.times.1320 pixels is desirable. CCD detectors are
commercially available from, e.g., Princeton Instruments, which can
meet either of these requirements.
[0046] In another embodiment, the detection device comprises a line
scanner, as described in U.S. patent application Ser. No.
08/301,051, filed Sep. 2, 1994, incorporated herein by reference.
Excitation optics focuses excitation light to a line at a sample,
simultaneously scanning or imaging a strip of the sample Surface
bound labeled targets from the sample fluoresce in response to the
light Collection optics image the emission onto a linear array of
light detectors. By employing confocal techniques, substantially
only emission from the light's focal plane is imaged. Once a strip
has been scanned, the data representing the 1-dimensional image are
stored in the memory of a computer. According to one embodiment, a
multi-axis translation stage moves the device at a constant
velocity to continuously integrate and process data Alternatively,
galvometric scanners or rotating polyhedral mirrors may be employed
to scan the excitation light across the sample. As a result, a
2-dimensional image of the sample is obtained.
[0047] In another embodiment, collection optics direct the emission
to a spectrograph which images an emission spectrum onto a
2-dimensional array of light detectors By using a spectrograph, a
full spectrally resolved image of the sample is obtained.
[0048] The read time for a full microtiter plate will depend on the
photophysics of the fluorophore (ie fluorescence quantum yield and
photodestruction yield) as well as the sensitivity of the detector
For fluorescein, sufficient signal-to-noise to read a chip image
with a CCD detector can be obtained in about 30 seconds using 3
mW/cm.sup.2 and 488 nm excitation from an Ar ion laser or lamp By
increasing the laser power, and switching to dyes such as CY3 or
CY5 which have lower photodestruction yields and whose emission
more closely matches the sensitivity maximum of the CCD detector,
one easily is able to read each well in less than 5 seconds. Thus,
an entire plate could be examined quantitatively in less than 10
minutes, even if the whole plate has over 4.5 million probes.
[0049] A computer can transform the data into another format for
presentation. Data analysis can include the steps of determining,
e.g., fluorescent intensity as a function of substrate position
from the data collected, removing "outliers" (data deviating from a
predetermined statistical distribution), and calculating the
relative binding affinity of the targets from the remaining data.
The resulting data can be displayed as an image with color in each
region varying according to the light emission or binding affinity
between targets and probes therein.
[0050] One application of this system when coupled with the CCD
imaging system that speeds performance of the tests is to obtain
results of the assay by examining the on- or off-rates of the
hybridization In one embodiment of this method, the amount of
binding at each address is determined at several time points after
the probes are contacted with the sample. The amount of total
hybridization can be determined as a function of the kinetics of
binding based on the amount of binding at each time point. Thus, it
is not necessary to wait for equilibrium to be reached. The
dependence of the hybridization rate for different oligonucleotides
on temperature, sample agitation, washing conditions (e.g. pH,
solvent characteristics, temperature) can easily be determined in
order to maximize the conditions for rate and signal-to-noise
Alternative methods are described in Fodor et al., U.S. Pat. No.
5,324,633, incorporated herein by reference.
[0051] B. Fluid Handling Instruments and Assay Automation
[0052] Assays on biological arrays generally include contacting a
probe array with a sample under the selected reaction conditions,
optionally washing the well to remove unreacted molecules, and
analyzing the biological array for evidence of reaction between
target molecules the probes. These steps involve handling fluids.
The methods of this invention automate these steps so as to allow
multiple assays to be performed concurrently. Accordingly, this
invention employs automated fluid handling systems for concurrently
performing the assay steps in each of the test wells. Fluid
handling allows uniform treatment of samples in the wells.
Microtiter robotic and fluid-handling devices are available
commercially, for example, from Tecan AG.
[0053] The plate is introduced into a holder in the fluid-handling
device. This robotic device is programmed to set appropriate
reaction conditions, such as temperature, add samples to the test
wells, incubate the test samples for an appropriate time, remove
unreacted samples, wash the wells, add substrates as appropriate
and perform detection assays The particulars of the reaction
conditions depends upon the purpose of the assay For example, in a
sequencing assay involving DNA hybridization, standard
hybridization conditions are chosen However, the assay may involve
testing whether a sample contains target molecules that react to a
probe under a specified set of reaction conditions. In this case,
the reaction conditions are chosen accordingly.
[0054] C Biological Chip Plates
[0055] FIG. 3 depicts an example of a biological chip plate 300
used in the methods of this invention based on the standard 96-well
microtiter plate in which the chips are located at the bottom of
the wells. Biological chip plates include a plurality of test wells
310, each test well defining an area or space for the introduction
of a sample, and each test well comprising a biological chip 320,
ie, a substrate and a surface to which an array of probes is
attached, the probes being exposed to the space. FIG. 7 shows a
top-down view of a well of a biological chip plate of this
invention containing a biological chip on the bottom surface of the
well.
[0056] This invention contemplates a number of embodiments of the
biological chip plate. In a preferred embodiment, depicted in FIG.
4, the biological chip plate includes two parts. One part is a
wafer 410 that includes a plurality of biological arrays 420 The
other part is the body of the plate 430 that contains channels 440
that form the walls of the well, but that are open at the bottom.
The body is attached to the surface of the wafer so as to close one
end of the channels, thereby creating wells. The walls of the
channels are placed on the wafer so that each surrounds and
encloses the probe array of a biological array. FIG. 5 depicts a
cross-section of this embodiment, showing the wafer 510 having a
substrate 520 (preferably transparent to light) and a surface 530
to which is attached an array of probes 540. A channel wall 550
covers a probe array on the wafer, thereby creating well spaces
560. The wafer can be attached to the body by any attachment means
known in the art, for example, gluing (e.g, by ultraviolet-curing
epoxy or various sticking tapes), acoustic welding, sealing such as
vacuum or suction sealing, or even by relying on the height of the
body on the wafer to resist the flow of fluids between test
wells.
[0057] In another preferred embodiment, depicted in cross section
in FIG. 6, the plates include a body 610 having pre-formed wells
620, usually flat-bottomed. Individual biological chips 630 are
attached to the bottom of the wells so that the surface containing
the array of probes 640 is exposed to the well space where the
sample is to be placed.
[0058] In another embodiment, the biological chip plate has a wafer
having a plurality of prone arrays and a material resistant to the
flow of a liquid sample that surrounds each probe array. For
example, in an embodiment useful for testing aqueous-based samples,
the wafer can be scored with waxes, tapes or other hydrophobic
materials in the spaces between the arrays, forming cells that act
as test wells. The cells thus contain liquid applied to an array by
resisting spillage over the barrier and into another cell. If the
sample contains a non-aqueous solvent, such as an alcohol, the
material is selected to be resistant to corrosion by the
solvent.
[0059] The microplates of this invention have a plurality of test
wells that can be arrayed in a variety of ways. In one embodiment,
the plates have the general size and shape of standard-sized
microtiter plates having 96 wells arranged an an 8.times.12 format.
One advantage of this format is that instrumentation already exists
for handling and reading assays on microtiter plates. Therefore,
using such plates in biological chip assays does not involve
extensive re-engineering of commercially available fluid handling
devices. However, the plates can have other formats as well.
[0060] The material from which the body of the biological chip
plate is made depends upon the use to which it is to be put. In
particular, this invention contemplates a variety of polymers
already used for microtiter plates including, for example,
(poly)tetrafluoroethylene, (poly)vinylidenedifluoride,
polypropylene, polystyrene, polycarbonate, or combinations thereof.
When the assay is to be performed by sending an excitation beam
through the bottom of the plate collecting data through the bottom
of the plate, the body of the plate and the substrate of the chip
should be transparent to the wavelengths of light being used.
[0061] The arrangement of probe arrays in the wells of a microplate
depends on the particular application contemplated. For example,
for diagnostic uses involving performing the same test on many
samples, every well can have the same array of probes If several
different tests are to be performed on each sample, each row of the
plate can have the same array of probes and each column can contain
a different array Samples from a single patient are introduced into
the wells of a particular column Samples from a different patient
are introduced into the wells of a different column In still
another embodiment, multiple patient samples are introduced into a
single well. If a well indicates a "positive" result for a
particular characteristic, the samples from each patient are then
rerun, each in a different well, to determine which patient sample
gave a positive result.
[0062] D. Biological Chips
[0063] The biological chip plates used in the methods of this
invention include biological chips. The array of probe sequences
can be fabricated on the biological chip according to the
pioneering techniques disclosed in U.S. Pat. No. 5,143,854, PCT WO
92/10092, PCT WO 90/15070, or U.S. application Ser. Nos.
08/249,188, 07/624,120, and 08/082,937, incorporated herein by
reference for all purposes. The combination of photolithographic
and fabrication techniques may, for example, enable each probe
sequence ("feature") to occupy a very small area ("site" or
"location") on the support. In some embodiments, this feature site
may be as small as a few microns or even a single molecule. For
example, a probe array of 0.25 mm.sup.2 (about the size that would
fit in a well of a typical 96-well microtiter plate) could have at
least 10, 100, 1000, 10.sup.4, 10.sup.5 or 10.sup.6 features. In an
alternative embodiment, such synthesis is performed according to
the mechanical techniques disclosed in U.S. Pat. No. 5,384,261,
incorporated herein by reference.
[0064] Referring to FIG. 8, in general, linker molecules, "O--X,"
are provided on a substrate. The substrate is preferably flat but
may take on a variety of alternative surface configurations For
example, the substrate may contain raised or depressed regions on
which the probes are located The substrate and its surface
preferably form a rigid support on which the sample can be formed.
The substrate and its surface are also chosen to provide
appropriate light-absorbing characteristics For instance, the
substrate may be functionalized glass, Si, Ge, GaAs, GaP,
SiO.sub.2, SiN.sub.4, modified silicon, or any one of a wide
variety of gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene, polycarbonate,
polypropylene, or combinations thereof. Other substrate materials
will be readily apparent to those of skill in the art upon review
of this disclosure In a preferred embodiment the substrate is flat
glass or silica.
[0065] Surfaces on the solid substrate usually, though not always,
are composed of the same material as the substrate. Thus, the
surface may be composed of any of a wide variety of materials, for
example, polymers, plastics, resins, polysaccharides, silica or
silica-based materials, carbon, metals, inorganic glasses,
membranes, or any of the above-listed substrate materials. In one
embodiment, the surface will be optically transparent and will have
surface Si--OH functionalities, such as those found on silica
surfaces.
[0066] A terminal end of the linker molecules is provided with a
reactive functional group protected with a photoremovable
protective group, O--X. Using lithographic methods, the
photoremovable protective group is exposed to light, hv, through a
mask, M.sub.1, that exposes a selected portion of the surface, and
removed from the linker molecules in first selected regions. The
substrate is then washed or otherwise contacted with a first
monomer that reacts with exposed functional groups on the linker
molecules ("T-X). In the case of nucleic acids, the monomer can be
a phosphoramidite activated nucleoside protected at the 5'-hydroxyl
with a photolabile protecting group.
[0067] A second set of selected regions, thereafter, exposed to
light through a mask, M.sub.2, and photoremovable protective group
on the linker molecule/protected amino acid or nucleotide is
removed at the second set of regions The substrate is then
contacted with a second monomer containing a photoremovable
protective group for reaction with exposed functional groups. This
process is repeated to selectively apply monomers until polymers of
a desired length and desired chemical sequence are obtained
Photolabile groups are then optionally removed and the sequence is,
thereafter, optionally capped Side chain protective groups, if
present, are also removed.
[0068] The general process of synthesizing probes by removing
protective groups by exposure to light, coupling monomer units to
the exposed active sites, and capping unreacted sites is referred
to herein as "light-directed probe synthesis." If the probe is an
oligonucleotide, the process is referred to as "light-directed
oligonucleotide synthesis" and so forth.
[0069] The probes can be made of any molecules whose synthesis
involves sequential addition of units. This includes polymers
composed of a series of attached units and molecules bearing a
common skeleton to which various functional groups are added.
Polymers useful as probes in this invention include, for example,
both linear and cyclic polymers of nucleic acids, polysaccharides,
phospholipids, and peptides having either .alpha.-, .beta.-, or
.omega.-amino acids, heteropolymers in which a known drug is
covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or
other polymers which will be apparent upon review of this
disclosure. Molecules bearing a common skeleton include
benzodiazepines and other small molecules, such as described in
U.S. Pat. No. 5,288,514, incorporated herein by reference.
[0070] Preferably, probes are arrayed on a chip in addressable rows
and columns in which the dimensions of the chip conform to the
dimension of the plate test well. Technologies already have been
developed to read information from such arrays The amount of
information that can be stored on each plate of chips depends on
the lithographic density which is used to synthesize the wafer. For
example, if each feature size is about 100 microns on a side, each
array can have about 10,000 probe addresses in a 1 cm.sup.2 area. A
plate having 96 wells would contain about 192,000 probes However,
if the arrays have a feature size of 20 microns on a side, each
array can have close to 50,000 probes and the plate would have over
4,800,000 probes.
[0071] The selection of probes and their organization in an array
depends upon the use to which the biological chip will be put. In
one embodiment, the chips are used to sequence or re-sequence
nucleic acid molecules, or compare their sequence to a referent
molecule. Re-sequencing nucleic acid molecules involves determining
whether a particular molecule has any deviations from the sequence
of reference molecule. For example, in one embodiment, the plates
are used to identify in a particular type of HIV in a set of
patient samples. Tiling strategies for sequence checking of nucleic
acids are described in U.S. patent application Ser. No. 08/284,064
(PCT/US94/12305), incorporated herein by reference.
[0072] In typical diagnostic applications, a solution containing
one or more targets to be identified (i.e., samples from patients)
contacts the probe array. The targets will bind or hybridize with
complementary probe sequences. Accordingly, the probes will be
selected to have sequences directed to (i.e., having at least some
complementarity with) the target sequences to be detected, e.g.,
human or pathogen sequences. Generally, the targets are tagged with
a detectable label. The detectable label can be, for example, a
luminescent label, a light scattering label or a radioactive label.
Accordingly, locations at which targets hybridize with
complimentary probes can be identified by locating the markers.
Based on the locations where hybridization occurs, information
regarding the target sequences can be extracted. The existence of a
mutation may be determined by comparing the target sequence with
the wild type.
[0073] In a preferred embodiment, the detectable label is a
luminescent label Useful luminescent labels include fluorescent
labels, chemi-luminescent labels, bio-luminescent labels, and
calorimetric labels, among others Most preferably, the label is a
fluorescent label such as fluorescein, rhodamine, cyanine and so
forth. Fluorescent labels include, inter alia, the commercially
available fluorescein phosphoramidites such as Fluoreprime
(Pharmacia), Fluoredite (Millipore) and FAM (ABI) For example, the
entire surface of the substrate is exposed to the activated
fluorescent phosphoramidite, which reacts with all of the
deprotected 5'-hydroxyl groups. Then the entire substrate is
exposed to an alkaline solution (eg, 50% ethylenediamine in ethanol
for 1-2 hours at room temperature). This is necessary to remove the
protecting groups from the fluorescein tag.
[0074] To avoid self-quenching interactions between fluorophores on
the surface of a biological chip, the fluorescent tag monomer
should be diluted with a non-fluorescent analog of equivalent
reactivity. For example, in the case of the fluorescein
phosphoramidites noted above, a 1:20 dilution of the reagent with a
non-fluorescent phosphoramidite such as the standard
5'-DMT-nucleoside phosphoramidites, has been found to be suitable.
Correction for background non-specific binding of the fluorescent
reagent and other such effects can be determined by routine
testing.
[0075] Useful light scattering labels include large colloids, and
especially the metal colloids such as those from gold, selenium and
titanium oxide.
[0076] Radioactive labels include, for example, .sup.32P. This
label can be detected by a phosphoimager. Detection of course,
depends on the resolution of the imager. Phosophoimagers are
available having resolution of 50 microns. Accordingly, this label
is currently useful with chips having features of that size.
[0077] E Uses
[0078] The methods of this invention will find particular use
wherever high through-put of samples is required. In particular,
this invention is useful in clinical settings and for sequencing
large quantities of DNA, for example in connection with the Human
Genome protect.
[0079] The clinical setting requires performing the same test on
many patient samples The automated methods of this invention lend
themselves to these uses when the test is one appropriately
performed on a biological chip. For example, a DNA array can
determine the particular strain of a pathogenic organism based on
characteristic DNA sequences of the strain The advanced techniques
based on these assays now can be introduced into the clinic. Fluid
samples from several patients are introduced into the test wells of
a biological chip plate and the assays are performed
concurrently.
[0080] In some embodiments, it may be desirable to perform multiple
tests on multiple patient samples concurrently. According to such
embodiments, rows (or columns) of the microtiter plate will contain
probe arrays for diagnosis of a particular disease or trait. For
example, one row might contain probe arrays designed for a
particular cancer, while other rows contain probe arrays for
another cancer. Patient samples are then introduced into respective
columns (or rows) of the microtiter plate. For example, one column
may be used to introduce samples from patient "one," another column
for patient "two" etc. Accordingly, multiple diagnostic tests may
be performed on multiple patients in parallel. In still further
embodiments, multiple patient samples are introduced into a single
well. In a particular well indicator the presence of a genetic
disease or other characteristic, each patient sample is then
individually processed to identify which patient exhibits that
disease or trait. For relatively rarely occurring characteristics,
further order-of-magnitude efficiency may be obtained according to
this embodiment.
[0081] Particular assays that will find use in automation include
those designed specifically to detect or identify particular
variants of a pathogenic organism, such as HIV Assays to detect or
identify a human or animal gene are also contemplated In one
embodiment, the assay is the detection of a human gene variant that
indicates existence of or predisposition to a genetic disease,
either from acquired or inherited mutations in an individual DNA
These include genetic diseases such as cystic fibrosis, diabetes,
and muscular dystrophy, as well as diseases such as cancer (the P53
gene is relevant to some cancers), as disclosed in U.S. patent
application Ser. No. 08/143,312, already incorporated by
reference.
[0082] The present invention provides a substantially novel method
for performing assays on biological arrays While specific examples
have been provided, the above description is illustrative and not
restrictive Many variations of the invention will become apparent
to those of skill in the art upon review of this specification The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0083] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
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