U.S. patent application number 10/997492 was filed with the patent office on 2005-06-09 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 | 20050123907 10/997492 |
Document ID | / |
Family ID | 32045837 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050123907 |
Kind Code |
A1 |
Rava, Richard P. ; et
al. |
June 9, 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.
3380 Central Expressway
Santa Clara
CA
95051
|
Family ID: |
32045837 |
Appl. No.: |
10/997492 |
Filed: |
November 24, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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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: |
506/16 ;
435/287.2; 435/5; 435/6.12; 506/34; 702/20 |
Current CPC
Class: |
B01J 2219/00626
20130101; G01N 35/028 20130101; C40B 60/14 20130101; B01J
2219/00659 20130101; C40B 40/06 20130101; G01N 21/64 20130101; B01J
19/0046 20130101; B01J 2219/00432 20130101; B01J 2219/00317
20130101; B01J 2219/00619 20130101; C12Q 1/6837 20130101; B01L
3/5027 20130101; B01J 2219/00527 20130101; B01J 2219/00605
20130101; B01J 2219/00315 20130101; B01J 2219/00612 20130101; B01L
2400/0683 20130101; B01J 2219/00617 20130101; B01L 2300/041
20130101; B01L 2300/0819 20130101; B01J 2219/00621 20130101; B01J
2219/00722 20130101; B01J 2219/00608 20130101; G01N 2035/00158
20130101; B01J 2219/0061 20130101; B01J 2219/00662 20130101; B01J
2219/00576 20130101; B01L 2300/0636 20130101; B01L 3/5085 20130101;
B01L 2300/044 20130101; B01J 2219/00529 20130101; B01J 2219/00702
20130101; B01L 2300/0829 20130101; B01J 2219/00637 20130101; B01L
2300/046 20130101; B01J 2219/00286 20130101; B01J 2219/00639
20130101; B01J 2219/00707 20130101; B01L 3/50853 20130101 |
Class at
Publication: |
435/005 ;
435/006; 435/287.2; 702/020 |
International
Class: |
C12Q 001/70; C12Q
001/68; G06F 019/00; G01N 033/48; G01N 033/50; C12M 001/34 |
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. An array assay device, said device comprising: (a) a substrate
receiving element for receiving a substrate having at least on
array thereon, said substrate receiving element comprising a bottom
surface; and (b) a compression element for urging said bottom
surface in a direction towards a substrate when present in said
substrate receiving element so as to hold said bottom surface in a
fixed position relative to said substrate.
33. The array assay device according to claim 32, wherein, when
said bottom surface is held in said fixed position relative to a
substrate when present in said device, said bottom surface ranges
from about 0.1 mm to about 2 mm from said substrate.
34. The array assay device according to claim 32, wherein said
bottom surface further comprises a sealing element for producing a
seal around at least one array positioned on a substrate when held
in said fixed position.
35. The array assay device according to claim 34, wherein said seal
is substantially vapor and fluid tight.
36. The array assay device according to claim 34, wherein said
sealing element produces an assay volume of from about 10 .mu.1 to
about 1000 .mu.1.
37. The array assay device according to claim 34, wherein said
substrate comprises a plurality of arrays and said sealing element
produces a plurality of individual seals around each array.
38. The array assay device according to claim 37, wherein each of
said individual seals is substantially vapor and fluid tight.
39. The array assay device according to claim 32, wherein said
sealing element is a gasket
40. The array assay device according to claim 32, further
comprising at least one access port.
41. The array assay device according to claim 40, wherein said
device comprises a plurality of access ports.
42. The array assay device according to claim 40, wherein said
device comprises at least a first fluid introduction port and a
second venting port.
43. The array assay device according to claim 40, wherein said at
least one port is resealable.
44. The array assay device according to claim 32, further
comprising a removable array holder.
45. The array assay device according to claim 44, wherein said
array holder is configured to be used with an array scanner.
46. A system for performing array assays, said system comprising:
(a) an array assay device according to claim 32; and (b) a
substrate having at least one array.
47. A method for performing an array assay, said method comprising:
(a) providing an array assay device comprising: (i) a bottom
surface, (ii) a substrate receiving element for receiving a
substrate having at least on array thereon, and (iii) a compression
element for urging said bottom surface in a direction towards a
substrate when present in said substrate receiving element so as to
hold said bottom surface in a fixed position relative to said
substrate; (b) positioning a substrate comprising at least one
array in said substrate receiving element; (c) urging said bottom
surface in a direction towards said positioned substrate using said
compression element, whereby said bottom surface is fixed relative
to said substrate present in said receiving element; and (e)
contacting a sample to said at least one array.
48. The method according to claim 47, further comprising producing
a seal around said at least one array.
49. The method according to claim 48, wherein said seal is
substantially vapor and fluid tight.
50. The method according to claim 47, wherein said device comprises
at least one port and said sample is introduced through said
port.
51. The method according to claim 47, further comprising mixing
said sample with said at least one array.
52. The method according to claim 51, wherein said mixing is
accomplished by an air bubble.
53. The method according to claim 47, further comprising retaining
said substrate in an array holder.
54. A method comprising, following contacting said at least one
array to a sample according to claim 47, reading said at least one
array.
55. The method according to claim 54, where in said at least one
array is read while in the array holder of claim 53.
56. A method comprising forwarding data representing a result of a
reading obtained by the method of claim 54 from a first location to
a second location.
57. The method according to claim 56, wherein said second location
is remote from said first location.
58. A method comprising receiving data representing a result of a
reading obtained by the method of claim 54.
59. A method for performing an array assay, said method comprising:
(a) receiving a pre-packaged substrate having at least one array in
the array assay device of claim 32 from a remote site; (b)
performing an array assay using said received array assay device;
(c) removing said pre-packaged substrate from said array assay
device; and (d) reading said at least one array to obtain a
result.
60. The method according to claim 59, wherein said pre-packaged
substrate comprises a substrate retained in an array holder in said
array assay device.
61. A method for performing an array assay and reading a result of
said array assay, said method comprising: (a) performing an array
assay using the array assay device of claim 32 comprising a
substrate having at least on array retained in an array holder; (b)
removing said retained substrate having at least one array from
said array assay device; and (c) mounting said retained substrate
having at least one array on an array scanner so that said retained
substrate having at least one array may be read by said scanner
while retained in said array holder.
62. The method according to claim 61, further comprising reading
said mounted at least one array.
63. A kit for performing an assay, said kit comprising: (a) at
least one array assay device according to claim 32; and (b)
instructions for using said at least one array assay device in an
array based assay.
64. The kit according to claim 63, further comprising at least one
array holder.
65. The kit according to claim 63, further comprising at least one
array.
66. The kit according to claim 63, further comprising reagents for
generating a labeled sample.
67. The kit according to claim 63, wherein said kit further
comprises a buffer.
68. The kit according to claim 63, wherein said kit further
comprises a wash medium.
Description
BACKGROUND OF THE INVENTION
[0001] 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.
[0002] 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 have 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 has interacted with the
probe at that location.
[0003] 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.
[0004] 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.
[0005] 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 Project.
SUMMARY OF THE INVENTION
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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
[0013] FIG. 1 depicts a system of this invention having a
biological chip plate, fluid handling device, biological chip plate
reader and computer;
[0014] FIG. 2 depicts the scanning of a biological chip plate by a
biological chip plate reader;
[0015] FIG. 3 depicts a biological plate of this invention;
[0016] FIG. 4 depicts the mating of a wafer containing many
biological arrays with a body having channels to create a
biological chip plate;
[0017] 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;
[0018] FIG. 6 depicts a biological plate in cross section having a
body which has individual biological chips attached to the bottom
of the wells;
[0019] FIG. 7 is a top-down view of a test well containing a
biological array; and
[0020] 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
[0021] I. Definitions
[0022] The following terms are intended to have the following
general meanings as they are used herein:
[0023] A. Complementary:
[0024] 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:
[0026] 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.
[0027] C. Target:
[0028] 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.
[0029] D. Array:
[0030] A collection of probes, at least two of which are different,
arranged in a spacially defined and physically addressable
manner.
[0031] E. Biological Chip:
[0032] 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.
[0033] F. Wafer:
[0034] 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.
[0035] G. Biological Chip Plate:
[0036] 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.
[0037] II. General
[0038] 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.
[0039] 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.
[0040] 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.
[0041] A. Biological Chip Plate Reader
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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 Ser. No.
08/195,889, supra.)
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] The read time for a full microtiter plate will depend on the
photophysics of the fluorophore (i.e. 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.
[0055] 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.
[0056] 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.
[0057] B. Fluid Handling Instruments and Assay Automation
[0058] 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.
[0059] 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.
[0060] C. Biological Chip Plates
[0061] 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,
i.e., 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.
[0062] 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 weight of the
body on the wafer to resist the flow of fluids between test
wells.
[0063] 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.
[0064] In another embodiment, the biological chip plate has a wafer
having a plurality of probe 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.
[0065] 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 in 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.
[0066] 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.
[0067] 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.
[0068] D. Biological Chips
[0069] 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.
[0070] Referring to FIG. 8, in general, linker molecules,
.sup.--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.
[0071] 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.
[0072] A terminal end of the linker molecules is provided with a
reactive functional group protected with a photoremovable
protective group, 0-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 (.sup.--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.
[0073] 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 photorenovable
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.
[0074] The general process of synthesizing probes by removing
protective groups by exposure to light, coupling monomer units to
the exposed active sites, and tapping 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.
[0075] 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.
[0076] 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 oh 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] Useful light scattering labels include large colloids, and
especially the metal colloids such as those from gold, selenium and
titanium oxide.
[0082] 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.
[0083] E. Uses
[0084] 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 project.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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
* * * * *