U.S. patent application number 10/359463 was filed with the patent office on 2004-01-01 for apparatus, systems and method for assaying biological materials using an annular format.
Invention is credited to Schembri, Carol T., Schleifer, Arthur.
Application Number | 20040002085 10/359463 |
Document ID | / |
Family ID | 23165870 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002085 |
Kind Code |
A1 |
Schembri, Carol T. ; et
al. |
January 1, 2004 |
Apparatus, systems and method for assaying biological materials
using an annular format
Abstract
An apparatus, systems and methods use an r, .theta. format for
the manufacture and analysis of biological materials. The apparatus
is an array of discrete features of biological material in an
annular region on a substrate. The apparatus is formed with a
system for synthesizing arrays that includes a spinner assembly for
rotating the substrate during the synthesis of the biological
material thereon. The spinner assembly provides efficient means for
printing an annular array pattern, spreading and removing ancillary
fluids used in the synthesis process. The apparatus is hybridized
with complementary biological material using a system for
hybridizing that includes a spinner assembly to spin the apparatus
after the complementary biological material is added. The spinning
motion spreads the complementary biological material efficiently
over the annular array such that a much smaller amount of
complementary biological material is needed for the assay. The
spinning motion effectively removes unhybridized material and
ancillary wash fluids and moves any bubbles that form out of the
array region on the substrate. The hybridized apparatus is
optically interrogated with a system for interrogation that
includes a light source, optics, and a scanning assembly that holds
and rotates the apparatus so that the light source can remain
stationary. The hybridized apparatus is interrogated in an r,
.theta. format using the rotating scanning assembly. The
interrogation system further comprises a linear stage to move the
apparatus or the optics radially to efficiently expose all features
in the annular region of the apparatus to the light source.
Rotation of the substrate in the synthesis, hybridization and
optical interrogation of the array provides many advantages to the
process of assaying biological materials that are not found in
conventional systems using an x, y format. The method of assaying
biological materials uses the systems for synthesizing, hybridizing
and optically interrogating the apparatus in accordance with the
invention.
Inventors: |
Schembri, Carol T.; (San
Mateo, CA) ; Schleifer, Arthur; (Portola Valley,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
Legal Department, DL429
Intellectual Property Administration
P.O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
23165870 |
Appl. No.: |
10/359463 |
Filed: |
February 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10359463 |
Feb 5, 2003 |
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09302011 |
Apr 27, 1999 |
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6518056 |
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Current U.S.
Class: |
506/16 ;
435/287.2; 435/6.1; 435/6.12; 506/37 |
Current CPC
Class: |
B01J 2219/00612
20130101; B01J 2219/0061 20130101; G01N 2035/00504 20130101; B01J
2219/00421 20130101; G01N 33/54366 20130101; B01J 2219/00637
20130101; B01J 2219/00675 20130101; C12Q 1/6837 20130101; G01N
35/1083 20130101; B01J 2219/00689 20130101; B82Y 30/00 20130101;
B01J 19/0046 20130101; G01N 35/00069 20130101; B01L 3/5085
20130101; C40B 40/06 20130101; B01J 2219/00596 20130101; C40B 60/14
20130101; B01J 2219/00605 20130101; C12Q 1/6832 20130101; B01J
2219/00585 20130101; C40B 50/14 20130101; G01N 2035/00158 20130101;
G01N 35/025 20130101; B01J 2219/00702 20130101; B01J 2219/00536
20130101; B01J 2219/00626 20130101; B01J 2219/00659 20130101; B01J
2219/00722 20130101; G01N 1/2813 20130101; B01J 2219/0059 20130101;
B01J 2219/00378 20130101; C12Q 1/6832 20130101; C12Q 2523/32
20130101; C12Q 1/6837 20130101; C12Q 2523/32 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. An apparatus for assaying biological materials comprising: a
substrate having a surface, an outer edge and a center; and a
plurality of discrete features bound to an annular region on the
surface of the substrate, the plurality of discrete features
comprising a first biological material.
2. The apparatus of claim 1, wherein the plurality of discrete
features further comprises: a second biological material chemically
associated with the first biological material; and a signal
producing system chemically associated with the plurality of
discrete features.
3. The apparatus of claim 2, wherein the second biological material
is complementary to the first biological material.
4. The apparatus of claim 3, wherein the first biological material
is an oligonucleotide probe and the second biological material is a
target nucleotide sequence.
5. The apparatus of claim 1, wherein the plurality of discrete
features are in an annular pattern in the annular region of the
substrate surface.
6. The apparatus of claim 5, wherein the annular pattern is a
plurality of annular rings.
7. The apparatus of claim 6, wherein the plurality of annular rings
are concentric annular rings of decreasing diameter from the outer
edge of the substrate to the center.
8. The apparatus of claim 6, wherein the plurality of annular rings
are segmented.
9. The apparatus of claim 5, wherein the annular pattern is a
spiral pattern having one end adjacent to the outer edge of the
substrate and an opposite end adjacent to the center of the
substrate.
10. The apparatus of claim 1, further comprising a housing for
holding the substrate and the plurality of discrete features bound
to the substrate surface.
11. The apparatus of claim 10, wherein the housing comprises: a
plate having an outer edge, a first side and a second side opposite
to the first side, the plate comprising: a port extending from the
first side to the second side of the plate, a first recess in the
first side coaxial with the port, a second recess in the first side
having an annular shape and being coaxial with the first recess, a
third recess in the first side having an annular shape and being
coaxial with the second recess, the third recess for receiving the
substrate, wherein the first recess and the second recess in the
plate and the substrate define a chamber in the housing for
receiving the second biological material and ancillary materials
through the port, the plurality of discrete features being adjacent
to a region in the chamber corresponding to the second recess and
being visible from the first side of the plate; and a recess
extending radially from the outer edge of the plate inward being in
communication with the second recess; and a valve assembly in the
radially extending recess, the valve assembly comprising an
actuator, a biasing means and a cap, the cap being attached to the
outer edge of the plate such that the radially extending recess
forms a valve chamber inside the plate.
12. The apparatus of claim 11, wherein the port comprises a septum
for allowing the second biological material and ancillary materials
to pass into the chamber, the septum preventing the materials from
exiting the chamber through the port.
13. The apparatus of claim 11, wherein the housing further
comprises: a second radially extending recess located diametrically
opposite to the first mentioned radially extending recess; a second
valve assembly similar to the first mentioned valve assembly
enclosed in the second radially extending recess; a fourth recess
in the second side of the plate having an annular shape; and a ring
frame attached to the second side of the plate, the ring frame
enclosing the fourth recess, the fourth recess and the ring frame
forming an annular cavity within the housing, wherein the first
radially extending recess and the second radially extending recess
intersect with the fourth recess and are in communication with the
annular cavity, and wherein at least one of the first and second
radially extending recesses being in communication with the
exterior of the plate adjacent to the second side.
14. A system for synthesizing an annular array of biological
materials comprising: a holder for holding a substrate; a spinner
assembly for rotating the holder; an enclosure for enclosing at
least the holder and substrate; a deposition assembly for
depositing biological material onto a surface of the substrate; a
dispenser assembly for providing ancillary materials to the surface
of the substrate; and a controller for automatically controlling
the deposition assembly, the spinner assembly and the dispenser
assembly.
15. The system of claim 14, wherein the spinner assembly comprises
a spindle and a motor, the spindle being attached to the holder,
the motor imparting rotational motion to the spindle and the holder
incrementally and continuously, and providing variable rotational
speed.
16. The system of claim 14, wherein the deposition assembly
comprises: a deposition tool having a plurality of nozzles to
deposit a plurality of monomers of the biological material as
discrete features in an annular region on the surface of the
substrate as the substrate is rotated in the holder by the spinner
assembly, wherein there is one nozzle for each different monomer of
the biological material to be deposited; a plurality of reservoirs
of biological materials to be deposited, wherein there is one
reservoir for each different monomer to be deposited; and a conduit
for each reservoir of the plurality of reservoirs for carrying a
respective monomer to a respective nozzle.
17. The system of claim 14, wherein the deposition assembly
comprises: a plurality of deposition tools to deposit a plurality
of monomers of the biological material as discrete features in an
annular pattern on the surface of the substrate as the substrate is
rotated in the holder by the spinner assembly, wherein there is one
deposition tool of the plurality of deposition tools for each
different monomer of the biological material to be deposited,
wherein each deposition tool has a plurality of nozzles aligned in
a row on the deposition tool radially over the substrate to provide
the annular pattern; a plurality of reservoirs of biological
materials to be deposited, wherein there is one reservoir for each
different monomer to be deposited; and a conduit for each reservoir
of the plurality of reservoirs for carrying a respective monomer to
a respective deposition tool.
18. A system for hybridizing biological materials in an annular
array pattern comprising: a holder for holding a substrate, the
substrate having an array of first biological material bound to an
annular region on a surface of the substrate; a spinner assembly
for rotating the holder at a plurality of rotational speeds, the
holder being connected to the assembly; a deposition tool for
providing a second biological material onto the surface of the
substrate; and a dispenser tool for dispensing ancillary materials
into the chamber, wherein the spinner assembly rotates the holder
and the substrate at least one of the plurality of rotational
speeds after the second biological material is provided and at
least another one of the plurality of rotational speeds after the
ancillary materials are dispensed.
19. The hybridization system of claim 18, wherein the plurality of
rotational speeds comprises: a first rotational speed sufficient to
spread the second biological material into contact with the first
biological material for hybridization, and a second rotational
speed sufficient to remove unhybridized second biological material
from the substrate surface after hybridization, wherein the first
rotational speed is sufficient to spread the ancillary materials
into contact with hybridized biological materials and the second
rotational speed is sufficient to remove the ancillary materials
from the substrate surface.
20. The hybridization system of claim 18, wherein the substrate is
enclosed in a housing and the holder is adapted to hold the housed
substrate.
21. The hybridization system of claim 20, wherein the housing
comprises: a cavity adjacent to the first biological material on
the substrate, a port for receiving the second biological material
into the cavity; and an exit valve assembly in communication with
the cavity and in communication with an exterior of the housing,
wherein the second biological material is deposited by the
deposition tool through the port and into the cavity, and wherein
the ancillary materials are dispensed by the dispenser assembly
through the port and into the cavity, and wherein the valve
assembly receives unhybridized second biological material and the
ancillary materials when sufficient centrifugal force is created by
one of the plurality of rotational speeds.
22. A system for optically interrogating an annular array of
hybridized biological material comprising: a light source for
emitting a light beam; a first optics subsystem for directing the
light beam onto the array; a scanning subsystem for providing
rotational movement to the array during an optical scan; a detector
subsystem for detecting a signal from the array in response to the
light beam; and an analysis subsystem for analyzing the detected
signals gathered by the detection subsystem and for automatically
controlling the light source, the scanning subsystem and the
detector subsystem.
23. The optical interrogation system of claim 22, wherein the
scanning subsystem comprises: a holder for holding the hybridized
annular array; and an annular subassembly for providing rotational
movement to the holder and the array.
24. The optical interrogation system of claim 23, wherein the
annular subassembly comprises: a motor having incremental and
variable rotational speeds; and a spindle which is connected to the
motor at one end, the holder being connected to the spindle at
another end opposite to the one end.
25. The optical interrogation system of claim 23, wherein the
scanning subsystem further comprises a linear movement subassembly
for further providing linear movement during the optical scan.
26. The optical interrogation system of claim 25, wherein the
linear movement subassembly provides linear movement to the annular
subassembly during the optical scan.
27. The optical interrogation system of claim 25, wherein the
linear movement subassembly provides linear movement to the optics
subsystem during the optical scan.
28. A method of assaying biological material in an annular array
comprising the steps of: providing a plurality of discrete features
comprising a first biological material in an annular region on a
surface of a substrate; hybridizing a second biological material
with the first biological material on the substrate, the hybridized
discrete feature having a signal producing system; optically
interrogating the hybridized discrete features on the substrate;
and determining characteristics about the first biological material
and the second biological material from the optical
interrogation.
29. The method of claim 28, wherein the step of providing a first
biological material comprises the steps of: a plurality of steps of
depositing monomers of the first biological material; rotating the
substrate incrementally to each discrete feature location between
each deposition step of the plurality of steps of depositing to
deposit the monomers in the annular region; and dispensing
ancillary materials on the substrate after each different monomer
is deposited in the annular region; and spinning the substrate at a
speed high enough to remove the ancillary materials from the
substrate after each step of dispensing.
30. The method of claim 28, wherein the step of hybridizing
comprises the steps of: depositing the second biological material
on the substrate; spinning the substrate at a first speed
sufficient to spread the second biological material into contact
with the first biological material; providing an environment for
hybridization to occur; spinning the substrate at a second speed
high enough to remove any unhybridized second biological material
from the substrate; and dispensing ancillary materials on the
substrate after the step of spinning at the second speed to wash
the substrate; and spinning the substrate at the second speed to
remove the ancillary materials from the substrate.
31. The method of claim 30, further comprising the step of
enclosing the substrate in a housing before the step of depositing,
wherein the step of enclosing comprises the steps of: providing a
base plate with a central recess, a first annular recess and a
second annular recess on a first side thereof, the central recess
having a port providing communication between the first side and a
second side of the plate; assembling a valve assembly in a radially
extending recess in the base plate, wherein the step of assembling
comprises the step of attaching a cover over one opening in the
radial recess adjacent to an outer edge of the base plate to
enclose the valve assembly and form a valve chamber, wherein the
valve chamber is in communication with an exterior of the base
plate; attaching a cover over the port, the port having a septum,
the port for receiving biological material and ancillary materials,
the septum for preventing the materials from escaping from the
port; and attaching the substrate to the second annular recess in
the base plate, wherein the substrate surface is adjacent to the
first annular recess and wherein the substrate, the central recess
and the first annular recess form a chamber for receiving the
materials through the port, wherein the chamber is in communication
with the valve chamber.
32. The method of claim 31, wherein the step of depositing
comprises the step of depositing the second biological material
into the chamber of the housing through the port, and the step of
dispensing comprises the step of dispensing the ancillary materials
into the chamber through the port, and wherein the method further
comprises the step of spinning the enclosed substrate at the second
speed to generate sufficient centrifugal force to activate the
valve assembly to release the unhybridized second biological
material and the ancillary materials into the valve chamber.
33. The method of claim 28, wherein the step of optical
interrogation comprises the steps of: mounting the hybridized array
substrate into a rotating holder; impinging a light beam on the
array substrate in a scan line; and rotating the substrate at speed
sufficient for the beam to impinge on the discrete features on the
array, such that the hybridized discrete features emit a signal;
moving the scan line incrementally in a radial direction after each
step of rotating until the beam impinges on all of the discrete
features on the array; detecting the signal from the hybridized
discrete features; and analyzing the signals that were detected to
determine information about the first biological material and the
second biological material.
34. The method of claim 28 further comprising the step of
controlling each of the steps of providing, hybridizing,
interrogating and determining with a computer subsystem to automate
the method of assaying.
Description
TECHNICAL FIELD
[0001] This invention relates to analytical tools and methods for
assaying biological materials in a variety of applications. In
particular, the invention relates to an apparatus, systems and a
method for assaying biological materials for monitoring levels of
gene expression and mutations in gene sequences using an annular
format.
BACKGROUND ART
[0002] Conventional analysis of biological materials, such as DNA,
RNA, proteins and the like, employs an apparatus having biological
material on a substrate in an array pattern of discrete features.
The features are typically chemically bound to the substrate. The
features may be either "probes" of known molecular make-up or
"targets" of unknown molecular make-up. For the purposes of
simplicity, hereinafter the features bound to the substrate will be
referred to as probes and the samples under test will be referred
to as "targets". Arrays of biological probes are quickly becoming a
powerful method of simultaneously assaying thousands of targets
within a single biological sample. The surface bound probes are
typically formed of DNA oligonucleotides, cDNA's, PCR products,
antibodies, antigens, and the like.
[0003] The arrays are manufactured using automated equipment, such
that the spatial location on the substrate of each type of surface
bound probe is known within a certain margin of error. The sample
containing the unknown quantities of the targets is modified so
that each potential target molecule is labeled with a fluorescent
label. The sample is applied to the array surface so that the
targets may hybridize or bind to their complementary surface bound
probe. After the reaction is complete, the surface of the array is
washed. The hybridized array is interrogated optically to determine
the level of hybridization and the locations and therefore, the
identity of the hybridized targets. Optionally, the array substrate
may be put into a package for handling, processing and optical
interrogation. The array can be held in the package with an
appropriate adhesive or glue.
[0004] Optical interrogation is typically performed with
commercially available optical scanning systems, examples of which
are described in U.S. Pat. No. 5,837,475, U.S. Pat. No. 5,760,951
(confocal scanner) and U.S. Pat. No. 5,585,639 (off axis scanner),
all incorporated herein by reference. Typical scanning fluorometers
are commercially available from different sources, such as
Molecular Dynamics of Sunnyvale, Calif., General Scanning of
Watertown, Mass., Hewlett Packard of Palo Alto, Calif. and Hitachi
USA of So. San Francisco, Calif. Analysis of the data, (i.e.,
collection, reconstruction of image, comparison and interpretation
of data) is performed with associated computer systems and
commercially available software, such as IMAGEQUANT.TM. by
Molecular Dynamics or GENECHIP.TM. by Affymetrix of Santa Clara,
Calif. Typically, a laser beam is scanned across the array surface.
The laser beam excites the fluorescent labels on the hybridized
targets and the fluorescent signal is detected by a detector and
processed by a computer. The intensity of the signal at each
physical location in the array is a measure of the hybridization
efficiency of a target with a known chemical probe. The intensity
relates directly to the concentration of that target within the
sample. The signal can be used to simply identify the targets
within the unknown or quantitate the targets. The identity of the
target is known since it is the complement of the probe.
[0005] While there are many methods known in the art for forming
and analyzing such arrays, all of them assume that the array will
be created and, subsequently analyzed or "read" in "an x, y
format", where "x" and "y" are the coordinate axes of a two
dimensional Cartesian coordinate system. "Reading" an array in an
x, y format typically requires raster scanning a laser beam across
the array surface. Typically, either the laser beam and the
fluorescence detection system must be moved or the array must be
moved in relation to the optics to scan in a raster pattern or x, y
format. In either case, the system typically requires some kind of
x, y table or a single axis motion table and a galvanometer.
[0006] Typical specifications of a biological array require a very
sensitive optical assay. Therefore, the size of the laser beam is
normally focussed to about 5 to 10 microns to meet the sensitivity
requirements of the assay. In order to scan an array surface in
increments of 5 or 10 microns, the x, y table or galvanometer must
be very precise. Such precision is normally expensive. The price of
an optical scanner is typically above $50,000, which can be cost
prohibitive for small analytical laboratories.
[0007] Moreover, if an x, y table is employed to move either the
optics or the array in a raster scan fashion, the x, y table must
be moved in a precise way. In particular, the table must be moved:
(i) very quickly in one direction; (ii) it must accelerate up to
"reading velocity" prior to the beam touching the array; (iii) then
it must move across the array at a constant speed; (iv) decelerate
outside of the array area; (v) reverse direction; and then repeat
steps (i)-(v) until the surface is completely scanned. A
significant portion of the total time required to scan the array is
spent in making changes in the direction during the raster
scan.
[0008] Moreover, in the x, y format, the laser beam must be scanned
beyond the region of interest on the array to allow space for the
deceleration of the beam and for the beam to reverse direction.
Since the array under test is normally glued into a package, the
beam may hit a glue edge or line. Conventionally, adhesive glues
are often fluorescent and are likely to be more fluorescent than
the signals from the very sensitive array. If the laser beam
encounters the glue line, the resulting signal can overload the
detection system. Overload can result in either temporary
degradation of the detection channel or permanent damage.
[0009] If a galvanometer is employed, the collection optics must be
much larger to collect signal from the entire swept line. Larger
optics are more expensive and more likely to have aberrations.
[0010] Therefore, it would be advantageous to have an assay system
that does not require the complexities and expense of, and avoids
the problems associated with, the traditional x, y raster scan
format.
[0011] U.S. Pat. No. 5,508,200 Tiffany et al., discloses an
automated chemical analysis system for high volume chemical testing
that creates arrays of chemical reactions on an absorbent media or
a solid substrate patterned with microwells. The system includes a
dispensing mechanism for dispensing multiple reagents or test
samples within a common test area on the media. The common test
area has microcuvettes or microwells to hold the samples and
reagents and prevent commingling of chemicals between each sample.
Mixing of the samples with the reagents is either performed before
dispensing or on the media. Tiffany employs a CCD camera to
simultaneously monitor the individual reactions at the discrete
locations within the common area.
[0012] In one embodiment, Tiffany discloses using a rotatable
circular disk of an absorbent matrix instead of a continuous strip
for the purpose of accommodating multiple sample spots. As a
result, photometric measurements with the CCD camera can be made at
a single station. The circular disk is rotated to bring the set of
microcuvettes into position for photometric measurement. The
circular disk format facilitates analyses requiring measurement at
variable time intervals and also multipoint rate analyses because
of the ease of returning to the single camera station.
[0013] However for several reasons, the chemical analysis system
described by Tiffany et al. is not conducive to the conventional
analysis of biological materials using arrays, as described above.
The arrays of biological materials require that each feature be
exposed to the entire quantity of the target sample since the
concentration of the analytes is typically orders of magnitude less
than that of chemistries Tiffany et al. use. The analysis system of
Tiffany et al. requires fluid isolation between each location. This
is achieved by physical isolation (microwells) or by using such
small fluid aliquots on absorbent media that the fluid spots do not
connect.
[0014] In the manufacture of conventional biological arrays, the
read probes are chemically bound to the substrate and the targets
are hybridized to the read probes. Tiffany et al. disclose using
reagents and samples that are dried onto the absorbent media or
captured within a microwell. Therefore, the system of Tiffany et
al. would not be conducive to the conventional hybridization and
subsequent wash steps.
[0015] Moreover, Tiffany et al. disclose using CCD cameras for
optical interrogation of the individual chemical reactions. Current
technology in CCD cameras is not as sensitive to fluorescent signal
as a laser beam with a photomultiplier tube detector used in
conventional biological array assays. The conventional biological
array assay described above typically scans the entire surface in a
systematic pattern rather than taking simultaneous camera shots of
an entire area.
[0016] The methodologies disclosed by Tiffany et al. dictate a
"dispense and immediately read" process. These methodologies are
not useful to conventional biological array assays, which require
reaction times of at least 30 minutes to overnight to complete.
Furthermore, Tiffany et al. disclose using homogeneous chemistries
in the chemical analysis system. The conventional biological array
allows for heterogeneous assays.
[0017] U.S. Pat. No. 4,940,322 Miwa et al. discloses a fluorescent
analysis apparatus for measuring the presence of microorganisms
using fluorescent substances. Each sample is placed in a discrete
container with reactive liquids. The containers are placed in a
rotatable holder or carousel in a reaction section of the system.
After a specified time, the containers are rotated into position by
means of a motor and gearing arrangement at a measuring section of
the system, where they are interrogated with a fluorescent
excitation light.
[0018] The analysis apparatus disclosed by Miwa et al. is also not
conducive to biological array assays for many of the same reasons
mentioned above for Tiffany et al. In particular, the apparatus of
Miwa et al. does not allow for conventional manufacture and
hybridization processes used in conventional biological array
assays.
[0019] U.S. Pat. No. 5,812,272 issued to King et al. and assigned
to the assignee of the present invention, discloses an apparatus
and method for analyzing target chemicals with a tiled light source
array. At column 9, line 28, it is disclosed that the light source
tiles can be arranged in a circular pattern and held in place by
grooved channels formed within a support. However, there is no
disclosure of why or how the circular pattern of tiles is used and
if there are any advantages to using a circular pattern of light
source tiles.
[0020] Thus, it would be advantageous to have an apparatus and
method for assaying biological materials that follows much of the
conventional array manufacturing and analysis methodologies, but
employs a less complex interrogation scheme.
SUMMARY OF THE INVENTION
[0021] The present invention provides an apparatus, systems and
methods for assaying biological materials which use an r, .theta.
format, where "r" and ".theta." are the coordinate axes of a two
dimensional polar coordinate system. In accordance with the present
invention, the complexities and expense of conventional optical
interrogation equipment and methods are overcome, without
compromising assay sensitivity, precision and speed.
[0022] The apparatus of the present invention is an array of
discrete features of biological material in a circular or annular
array pattern. The present array is manufactured using conventional
materials, manufacturing equipment and methods, except that the
biological materials are deposited onto a substrate in the circular
or annular array pattern.
[0023] One system of the present invention is a system for
synthesizing arrays that includes a spinner assembly for rotating
the apparatus during the synthesis and deposition of the biological
material on the substrate to form the circular or annular pattern
of biological features. The spinner assembly provides efficient
means for annular deposition and spreading and removing ancillary
materials used in the synthesis process.
[0024] Another system of the present invention is a system for
hybridizing the array apparatus of the invention with a
complementary biological material. The hybridization system uses
well-established methods and materials for hybridization. However,
the hybridization system further includes a spinner assembly to
rotate the apparatus after the complementary biological material is
added. The spinning motion spreads the complementary biological
material efficiently over the annular array such that less
complementary biological material is needed for the assay. Further
the spinning motion effectively removes unhybridized material and
ancillary materials and moves any bubbles that form out of the
array area. In the systems of synthesizing and hybridizing, the
spinner assemblies are used to spin the array such that the
advantages of centrifugal force on array processing are
realized.
[0025] Still another system of the present invention is a system
for optically interrogating the apparatus of the present invention.
The interrogation system includes a conventional light source to
emit a light beam, optics, detection and analysis subsystems.
Moreover, the interrogation system of the present invention employs
a scanning assembly that holds and rotates the apparatus so that
the light source and optics remain stationary, and a galvanometer
is not necessary. The hybridized apparatus is scanned and read in a
r, .theta. format using a spinner subassembly. The optical
interrogation further comprises a linear stage to move the
apparatus or the optics radially to efficiently expose all features
in the annular array pattern on the apparatus to the light
source.
[0026] The r, .theta. format is simpler to use and implement than
the conventional x, y format. Detection of the fluorescent signals
from the hybridized targets is accomplished with detection systems
employing conventional photomultiplier tubes, so that sensitivity
is not compromised. The signal information gathered by the
conventional detection system is stored and processed by
conventional analysis systems that are adapted to process r,
.theta. formatted data and provide information about the target
sample under investigation.
[0027] In accordance with the invention, the optics in the optical
scanner advantageously remain focused on a single point. The array
is scanned in a spiral pattern or by sweeping around a circle at a
specific radius and then stepping to a next radius and sweeping in
a circle again. The scan profile is scanned either with a constant
angular and radial speed or with a constant rate of area scanned.
The entire scan time is dedicated to reading active array rather
than accelerating and decelerating after every scan line, as in the
conventional x, y format. The present system requires a motor to
rotate the array and a linear system to move the array radially. In
another embodiment, the linear system is adapted to move the read
optics instead of the array. Therefore, the present invention
avoids the expense of a precision x, y table in the optical
scanning equipment.
[0028] The present system allows the optics to be smaller, lower
weight and less expensive. Optics with small field of view can be
made with high light collection efficiency (high numerical
aperture) since aberrations are more easily avoided. A "high"
numerical aperture starts at approximately 0.5-0.6. The optics only
need to be corrected for a field of view covering the alignment
tolerances, as in conventional systems. In addition, the present
system avoids using large galvanometer mirrors, which are otherwise
associated with high numerical aperture scanners.
[0029] Moreover, in accordance with the invention, the present
system avoids the risk of detector overload found in the optical
scanners using the x, y format discussed above. The array may be
packaged in some applications. Advantageously, the array is adhered
using an annular glue line, rendering the glue line concentric with
the scan pattern. Therefore, there is no risk of the laser beam
crossing the glue line and overloading the detection system.
[0030] The present invention also includes a method for assaying
biological materials. The method includes the steps of providing an
array of biological material on a substrate in an annular array of
discrete features, hybridizing the array with a another biological
material, optically interrogating the hybridized array and
determining information about the biological materials from the
results of the interrogation. The steps of the method use the
respective systems for synthesizing, hybridizing and optically
interrogating the apparatus. The method takes advantage of the use
of the spinner assemblies to implement the r, .theta. format in
accordance with the invention. Rotating and spinning the substrate
in the synthesis, the hybridization and the optical interrogation
of the array provide many advantages to the process of assaying
biological materials that are not found in conventional systems
using an x, y format.
[0031] The present invention provides additional advantages. For
example, during the manufacture of the hybridized array apparatus,
centrifugal force is applied to the fluids on the array. Quite
advantageously, the centrifugal force simplifies the washing
procedure after hybridization because the fluids are easily removed
by spinning the substrate. Moreover, the centrifugal force
advantageously minimizes the quantity of target sample required for
analysis in several ways: First, the target sample is spread
efficiently over the array features by the spinning motion, so that
little target sample is needed for hybridization. In particular, in
a preferred embodiment, the apparatus is packaged in a housing,
where little target sample is needed to fill the fluid channel in
the package leading to the array surface. Second, the surface
energy of the package array requires less control since capillary
action is not needed to fill the fluid channel in the package.
Third, the filling and emptying of the fluid cavity in the package
can be handled automatically. Moreover, any bubbles in the fluid,
which form during the sample loading or hybridization steps, are
advantageously spun out of the active read area.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The various features and advantages of the present invention
may be more readily understood with reference to the following
detailed description taken in conjunction with the accompanying
drawings, where like reference numerals designate like structural
elements, and in which:
[0033] FIG. 1A illustrates a top view of one embodiment of an array
apparatus according to the present invention;
[0034] FIG. 1B illustrates a magnified view of a portion of the
array in FIG. 1A according to the present invention;
[0035] FIGS. 1C-1F illustrate top views of other embodiments of the
array apparatus according to the present invention;
[0036] FIG. 2A illustrates a schematic view of the array
synthesizer in accordance with the present invention;
[0037] FIGS. 2B and 2C illustrate side views of two embodiments of
the deposition tool used in the array synthesizer of FIG. 2A.
[0038] FIG. 3A illustrates a perspective view of a housing in
accordance with a preferred embodiment, for holding the array of
the present invention;
[0039] FIG. 3B illustrates a cross sectional view of the array
housing taken along lines 3B-3B of FIG. 3A;
[0040] FIG. 3C illustrates an exploded view of the array and a
preferred embodiment of the housing assembly in FIG. 3D;
[0041] FIG. 3D illustrates a cross sectional view of the preferred
embodiment of the array package taken along lines 3D-3D of FIG.
3A;
[0042] FIG. 4 illustrates a block diagram of the optical scanning
system in accordance with the present invention;
[0043] FIG. 5 illustrates a schematic view of the preferred
embodiment of the subsystems of FIG. 4 in more detail;
[0044] FIG. 6 illustrates an exploded view of the holder that holds
the array in accordance to the invention;
[0045] FIG. 7 illustrates an output from the optical scanner of a
hybridized annular array in accordance with the invention; and
[0046] FIGS. 8A-8D illustrate flow charts of the methods of the
present invention. FIG. 8A illustrates the method of assaying
biological materials; FIG. 8B illustrates the method of
synthesizing the array; FIG. 8C illustrates the method of
hybridizing the array with another biological material; and FIG. 8D
illustrates the method of optically interrogating the hybridized
array, all in accordance with the present invention.
MODES FOR CARRYING OUT THE INVENTION
[0047] Definitions
[0048] The following terms are intended to have the following
general meanings as they are used herein:
[0049] Polynucleotide--a compound or composition that is a
polymeric nucleotide or nucleic acid polymer. The polynucleotide
may be a natural compound or a synthetic compound. In the context
of an assay, the polynucleotide can have from about 5 to 5,000,000
or more nucleotides. The larger polynucleotides are generally found
in the natural state. In an isolated state the polynucleotide can
have about 30 to 50,000 or more nucleotides, usually about 100 to
20,000 nucleotides, more frequently 500 to 10,000 nucleotides. It
is thus obvious that isolation of a polynucleotide from the natural
state often results in fragmentation. The polynucleotides include
nucleic acids, and fragments thereof, from any source in purified
or unpurified form including DNA, double-stranded or single
stranded (dsDNA and ssDNA), and RNA, including t-RNA, m-RNA, r-RNA,
mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNA
hybrids, or mixtures thereof, genes, chromosomes, plasmids, the
genomes of biological materials such as microorganisms, e.g.
bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,
humans, and the like. The polynucleotide can be only a minor
fraction of a complex mixture such as a biological sample. Also
included are genes, such as hemoglobin gene for sickle-cell anemia,
cystic fibrosis gene, oncogenes, cDNA, and the like.
[0050] The polynucleotide can be obtained from various biological
materials by procedures well known in the art. The polynucleotide,
where appropriate, may be cleaved to obtain a fragment that
contains a target nucleotide sequence, for example, by shearing or
by treatment with a restriction endonuclease or other site specific
chemical cleavage method.
[0051] For purposes of this invention, the polynucleotide, or a
cleaved fragment obtained from the polynucleotide, will usually be
at least partially denatured or single stranded or treated to
render it denatured or single stranded. Such treatments are well
known in the art and include, for instance, heat or alkali
treatment, or enzymatic digestion of one strand. For example,
double stranded DNA (dsDNA) can be heated at 90-100.degree. C. for
a period of about 1 to 10 minutes to produce denatured material,
while RNA produced via transcription from a dsDNA template is
already single stranded.
[0052] Target nucleotide sequence--a sequence of nucleotides to be
identified, usually existing within a portion or all of a
polynucleotide, usually a polynucleotide analyte. The identity of
the target nucleotide sequence generally is known to a extent
sufficient to allow preparation of various probe sequences
hybridizable with the target nucleotide sequence.
[0053] The target sequence usually contains from about 30 to 5,000
or more nucleotides, preferably 50 to 1,000 nucleotides. The target
nucleotide sequence is generally a fraction of a larger molecule or
it may be substantially the entire molecule such as a
polynucleotide as described above. The minimum number of
nucleotides in the target nucleotide sequence is selected to assure
that the presence of a target polynucleotide in a sample is a
specific indicator of the presence of polynucleotide in a sample.
The maximum number of nucleotides in the target nucleotide sequence
is normally governed by several factors: the length of the
polynucleotide from which it is derived, the tendency of such
polynucleotide to be broken by shearing or other processes during
isolation, the efficiency of any procedures required to prepare the
sample for analysis (e.g. transcription of a DNA template into RNA)
and the efficiency of detection and/or amplification of the target
nucleotide sequence, where appropriate.
[0054] Oligonucleotide--a polynucleotide, usually single stranded,
usually a synthetic polynucleotide but may be a naturally occurring
polynucleotide. The oligonucleotide(s) are usually comprised of a
sequence of at least 5 nucleotides, usually, 10 to 100 nucleotides,
more usually, 20 to 50 nucleotides, preferably, 10 to 30
nucleotides, more preferably, 20 to 30 nucleotides, and desirably
about 25 nucleotides in length.
[0055] Various techniques can be employed for preparing an
oligonucleotide. Such oligonucleotides can be obtained by
biological synthesis or by chemical synthesis. For short sequences
(up to about 100 nucleotides), chemical synthesis will frequently
be more economical as compared to the biological synthesis. In
addition to economy, chemical synthesis provides a convenient way
of incorporating low molecular weight compounds and/or modified
bases during specific synthesis steps. Furthermore, chemical
synthesis is very flexible in the choice of length and region of
target polynucleotides binding sequence. The oligonucleotide can be
synthesized by standard methods such as those used in commercial
automated nucleic acid synthesizers. Chemical synthesis of DNA on a
suitably modified glass or resin can result in DNA covalently
attached to the surface. This may offer advantages in washing and
sample handling. For longer sequences standard replication methods
employed in molecular biology can be used such as the use of M13
for single stranded DNA as described in J. Messing (1983) Methods
Enzymol. 101:20-78.
[0056] In situ synthesis of oligonucleotide or polynucleotide
probes on the substrate is performed in accordance with well-known
chemical processes, such as sequential addition of nucleotide
phosphoramidites to surface-linked hydroxyl groups, as described by
T. Brown and Dorcas J. S. Brown in Oligonucleotides and Analogues A
Practical Approach, F. Eckstein, editor, Oxford University Press,
Oxford, pp. 1-24 (1991), and incorporated herein by reference.
Indirect synthesis may be performed in accordance biosynthetic
techniques (e.g. polymerase chain reaction "PCR"), as described in
Sambrook, J. et al., "Molecular Cloning, A Laboratory Manual",
2.sup.nd edition 1989, incorporated herein by this reference.
[0057] Other methods of oligonucleotide synthesis include
phosphotriester and phosphodiester methods (Narang, et al., (1979)
Meth. Enzymol 68:90) and synthesis on a support (Beaucage, et al.
(1981) Tetrahedron Letters 22:1859-1862) as well as phosphoramidate
techniques (Caruthers, M. H., et al., "Methods in Enzymology," Vol.
154, pp.287-314 (1988) and others described in "Synthesis and
Applications of DNA and RNA," S. A. Narang, editor, Academic Press,
New York, 1987, and the references contained therein. The chemical
synthesis via a photolithographic method of spatially addressable
arrays of oligonucleotides bound to glass surfaces is described by
A. C. Pease, et al., Proc. Nat. Aca. Sci. USA (1994) 91:5022-5026,
all incorporated herein by reference.
[0058] Oligonucleotide probe--an oligonucleotide employed to bind
to a portion of a polynucleotide such as another oligonucleotide or
a target nucleotide sequence. The design and preparation of the
oligonucleotide probes are generally dependent upon the sensitivity
and specificity required, the sequence of the target polynucleotide
and, in certain cases, the biological significance of certain
portions of the target polynucleotide sequence.
[0059] Monomer--A member of the set of small molecules which can be
joined together to form a polymer. The set of monomers includes but
is not restricted to, for example, the set of common L-amino acids,
the set of D-amino acids, the set of synthetic amino acids, the set
of nucleotides and the set of pentoses and hexoses. As used herein,
monomers refers to any member of a basis set for synthesis of a
polymer. For example, dimers of the 20 naturally occurring L-amino
acids form a basis set of 400 monomers for synthesis of
polypeptides. Different basis sets of monomers may be used at
successive steps in the synthesis of a polymer. Furthermore, each
of the sets may include protected members that are modified after
synthesis.
[0060] Nucleotide--a base-sugar--phosphate combination that is the
monomeric unit of nucleic acid polymers, i.e., DNA and RNA.
[0061] Hybridization (hybridizing) and binding--in the context of
nucleotide sequences these terms are used interchangeably herein.
The ability of two nucleotide sequences to hybridize with each
other is based on the degree of complementarity of the two
nucleotide sequences, which in turn is based on the fraction of
matched complementary nucleotide pairs. The more nucleotides in a
given sequence that are complementary to another sequence, the more
stringent the conditions can be for hybridization and the more
specific will be the binding of the two sequences. Increased
stringency is achieved by elevating the temperature, increasing the
ratio of co-solvents, lowering the salt concentration, and the
like.
[0062] In accordance with the invention, the conventional
hybridization solutions and processes for hybridization can be
used, such as those described in J. Sambrook, E. F. Fritsch, T.
Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, Ed. 2.sup.nd, 1989,
vol. 1-3, incorporated herein by reference. Conditions for
hybridization typically include (1) high ionic strength solution,
(2) at a controlled temperature, and (3) in the presence of carrier
DNA and detergents and divalent cation chelators, all of which are
well known in the art.
[0063] Hybridization efficiency--the productivity of a
hybridization reaction measured as either the absolute or relative
yield of oligonucleotide probe/polynucleotide target duplex formed
under a given set of conditions in a given amount of time.
[0064] Complementary--Two sequences are complementary when the
sequence of one can bind to the sequence of the other in an
anti-parallel sense wherein the 3'-end of each sequence binds to
the 5'-end of the other sequence and each A, T(U), G, and C of one
sequence is then aligned with a T(U), A, C, and G, respectively, of
the other sequence. RNA sequences can also include complementary
G=U or U=G base pairs.
[0065] Substrate or surface--a porous or non-porous water insoluble
material. The surface can have any one of a number of shapes, such
as strip, plate, disk, rod, particle, including bead, and the like.
The substrate can be hydrophobic or hydrophilic or capable of being
rendered hydrophobic or hydrophilic and includes inorganic powders
such as silica, magnesium sulfate, and alumina; natural polymeric
materials, particularly cellulosic materials and materials derived
from cellulose, such as fiber containing papers, e.g., filter
paper, chromatographic paper, etc.; synthetic or modified naturally
occurring polymers, such as nitrocellulose, cellulose acetate, poly
(vinyl chloride), polyacrylamide, cross linked dextran, agarose,
polyacrylate, polyethylene, polypropylene, poly (4-methylbutene),
polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,
poly(vinyl butyrate), etc.; either used by themselves or in
conjunction with other materials; glass available as Bioglass,
ceramics, metals, and the like. Natural or synthetic assemblies
such as liposomes, phospholipid vesicles, and cells can also be
employed. Common substrates used for arrays are surface-derivatized
glass or silica, or polymer membrane surfaces, as described in Z.
Guo et al. (cited above) and U. Maskos, E. M. Southern, Nucleic
Acids Res 20, 1679-84 (1992) and E. M. Southern et al., Nucleic
Acids Res 22, 1368-73 (1994), both incorporated herein by
reference.
[0066] Immobilization of oligonucleotides on a substrate or surface
may be accomplished by well-known techniques, commonly available in
the literature. See, for example, A. C. Pease, et al., Proc. Nat.
Acad. Sci. USA, 91:5022-5026 (1994); Z. Guo, R. A. Guilfoyle, A. J.
Thiel, R. Wang, L. M. Smith, Nucleic Acids Res 22, 5456-65 (1994);
and M. Schena, D. Shalon, R. W. Davis, P. O. Brown, Science, 270,
467-70 (1995), each incorporated herein by reference.
[0067] Label--a member of a signal producing system. Usually the
label is part of a target nucleotide sequence or an oligonucleotide
probe, either being conjugated thereto or otherwise bound thereto
or associated therewith. The label is capable of being detected
directly or indirectly. Labels include (i) reporter molecules that
can be detected directly by virtue of generating a signal, (ii)
specific binding pair members that may be detected indirectly by
subsequent binding to a cognate that contains a reporter molecule,
(iii) oligonucleotide primers that can provide a template for
amplification or ligation or (iv) a specific polynucleotide
sequence or recognition sequence that can act as a ligand such as
for a repressor protein, wherein in the latter two instances the
oligonucleotide primer or repressor protein will have, or be
capable of having, a reporter molecule. In general, any reporter
molecule that is detectable can be used.
[0068] The reporter molecule can be isotopic or nonisotopic,
usually non-isotopic, and can be a catalyst, such as an enzyme, a
polynucleotide coding for a catalyst, promoter, dye, fluorescent
molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive
group, a small organic molecule, amplifiable polynucleotide
sequence, a particle such as latex or carbon particle, metal sol,
crystallite, liposome, cell, etc., which may or may not be further
labeled with a dye, catalyst or other detectable group, and the
like. The reporter molecule can be a fluorescent group such as
fluorescein, a chemiluminescent group such as luminol, a terbium
chelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid
that is capable of detection by delayed fluorescence, and the
like.
[0069] The label can generate a detectable signal either alone or
together with other members of the signal producing system. As
mentioned above, a reporter molecule can be bound directly to a
nucleotide sequence or can become bound thereto by being bound to
an specific binding pair (sbp) member complementary to an sbp
member that is bound to a nucleotide sequence. Examples of
particular labels or reporter molecules and their detection can be
found in U.S. Pat. No. 5,508,178, the relevant disclosure of which
is incorporated herein by reference. When a reporter molecule is
not conjugated to a nucleotide sequence, the reporter molecule may
be bound to an sbp member complementary to an sbp member that is
bound to or part of a nucleotide sequence.
[0070] Signal producing system--the signal producing system may
have one or more components, at least one component being the
label. The signal producing system generates a signal that
typically relates to the presence or amount of a target
polynucleotide in a medium. The signal producing system includes
all of the reagents required to produce a measurable signal. Other
components of the signal producing system may be included in the
developer solution and can include substrates, enhancers,
activators, chemiluminescent compounds, cofactors, inhibitors,
scavengers, metal ions, specific binding substances required for
binding of signal generating substances, and the like. Other
components of the signal producing system may be coenzymes,
substances that react with enzymic products, other enzymes and
catalysts, and the like. The signal producing system provides a
signal detectable by external means, by use of electromagnetic
radiation, desirably by visual examination. Signal-producing
systems that may be employed in the present invention are those
described more fully in U.S. Pat. No. 5,508,178, the relevant
disclosure of which is incorporated herein by reference.
[0071] Ancillary materials--Various ancillary materials will
frequently be employed in the methods and assays utilizing
biological materials designed in accordance with the present
invention. For example, conventional ancillary materials, such as
reagents and wash fluids for the synthesis and hybridization of
biological materials, including but not limited to acetonitrile,
buffers and salts, will normally be used in an assay medium, as
well as stabilizers for the assay medium and the assay components.
Frequently, in addition to these additives, ancillary materials
include but are not limited to proteins, such as albumins, organic
solvents, such as formamide, quaternary ammonium salts,
polycations, such as spermine, surfactants, particularly non-ionic
surfactants, binding enhancers, e.g., polyalkylene glycols, or the
like.
[0072] Biological material--nucleic acids, such as polynucleotides,
oligonucleotides, oligonucleotide probes, target nucleotide
sequences, proteins, amino acids, antibodies, antigens, enzymes,
coenzymes, ligands, receptors, hormones and labels, and monomers
thereof, and genes that specify any of the above, and any other
materials from any form of life.
[0073] Member of a specific binding pair ("sbp member")--one of two
different molecules, having an area on the surface or in a cavity
that specifically binds to and is thereby defined as complementary
with a particular spatial and polar organization of the other
molecule. The members of the specific binding pair are referred to
as cognates or as ligand and receptor (antiligand). These may be
members of an immunological pair such as antigen-antibody, or may
be operator-repressor, nuclease-nucleotide, biotin-avidin,
hormones-hormone receptors, nucleic acid duplexes, IgG-protein A,
DNA-DNA, DNA-RNA, and the like.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The apparatus 10 of the present invention comprises a
substrate 11 having a surface 12 and biological material 13, either
a biological probe 13a or a target sample 13b, bound in an annular
region on the surface 12 of the substrate in the form of discrete
features 14. In the preferred embodiment, the biological probes 13a
are bound to the substrate surface 12. The features 14 may be
patterned in the annular region on the substrate 11 in any number
of annular patterns. FIGS. 1A and 1C-1F illustrate examples of five
different potential array patterns realized by the invention. FIG.
1A illustrates one embodiment of the array apparatus 10 of the
present invention. In this embodiment, the array 10 includes a
substrate 11, which is preferably circular in shape, and has an
array pattern comprised of a plurality of annular rings positioned
in concentric circles 15, the outermost ring 15a being positioned
adjacent to the edge 11a of the substrate and having the largest
diameter, wherein the concentric rings 15 decrease in diameter as
they approach the center 11b of the substrate 11.
[0075] FIG. 1B is a magnified view of the deposits of biological
material 13 as discrete features 14 on the substrate 11, which
better illustrates a portion of each concentric ring 15a-15e of the
preferred embodiment in FIG. 1A, and the spacing between features
14 and rings 15. The discrete features 14 within each ring 15 are
spaced apart by approximately 30-200 microns, and preferably at
least approximately 50 microns, in order to ensure separation of
the features 14 during deposition. The concentric rings 15 are
spaced apart by approximately 30-200 microns, and preferably at
least approximately 50 microns, in order to ensure separation of
the features 14 in each ring 15. There are typically 5-50
concentric rings 15 in each array 10 and preferably, 20 rings 15 of
discrete features 14 are used.
[0076] FIGS. 1C-1F illustrate examples of alternative annular array
patterns that are within the scope of the invention. FIG. 1C
represents an annular array pattern of features 14 arranged in
ordered rows such that the features are close packed. FIG. 1D
represents an annular array pattern of features 14 arranged in
ordered rows and columns. There is also a lack of features 14 in
particular locations in each row and column, respectively, thereby
providing the distinctive annular array patterns. FIG. 1E
illustrates a spiral pattern of features 14 in the annular region
of the substrate. In FIG. 1F, the features 14 are patterned in
segmented annular rings. FIG. 1F illustrates segments containing
three columns of six features 14 each. The feature 14 locations on
the left side of each array pattern in FIGS. 1C, 1D and 1F are
mirror images of the feature locations on the right side of the
array pattern. It should be noted that FIGS. 1A, 1C-1F are merely
illustrative and not intended to limit the scope of the invention.
As long as the pattern is located in an annular region of the
substrate, or has an annular array pattern, it is within the scope
of the invention. It should also be noted that although a circular
substrate is illustrated in FIGS. 1A-1F, substrates that are
substantially elliptical, rectangular, square, or other shapes are
within the scope of the present invention.
[0077] In accordance with the invention, the substrates are made of
glass, fused silica or clear plastics, and preferably water white
glass is used in the invention because of its low intrinsic
fluorescence. The water white glass can be obtained from Erie
Scientific or Corning.
[0078] The processes and materials used for the manufacture of the
array apparatus 10 will depend on the biological material that will
be analyzed. For the purposes of simplifying the discussion,
hereinafter the present invention will be described with respect to
analysis of nucleic acids, polynucleotides, oligonucleotides or
nucleotides, and the like, and the biological material 13 bound to
the substrate 11 will hereinafter be referred to as the probes 13a,
or oligonucleotide probes 13a. The biological material introduced
for hybridization shall be referred to as the target sample 13b or
target nucleotide sequences 13b. The target samples 13b will be
labeled with a signal producing system, such as a fluorophore
label, which will fluoresce when illuminated. It is not the intent
of the inventors to be limited to this particular class of
biological materials for application of the invention; to the
surface-bound "probe" configuration; or to the target sample 13b
having the signal producing system. It should be understood by
those skilled in the art that the present invention has broad
application to biological material analysis and that the present
apparatus 10 is configurable to accommodate either member of
complementary biological materials under evaluation as the surface
bound material and that either the probes or the targets may
contain the signal producing system. For information regarding the
processes and materials used for the manufacture of arrays of
biological materials, such as proteins, antibodies, or the like in
accordance with the invention, see for example U.S. Pat. Nos.
4,591,570; 5,143,854; and 5,252,743 and the following articles:
Ekins, R. et. al., "Development of microspot multi-analyte
ratiometric immunoassay using dual fluorescent-labeled antibodies"
Analytica Chimica Acta, (1989), 227:73-96; and Ekins, R P and F W
Chu. "Multianalyte microspot immunoassay--microanalytical `compact
disc` of the future" (1991).
[0079] In accordance with the invention, the oligonucleotide probes
13a are either synthesized directly onto the substrate 11 (in situ)
using conventional chemical processes or synthesized indirectly
using biological processes and deposited onto the substrate 11. In
the preferred embodiment, the oligonucleotide probes 13a are
synthesized in-situ using the conventional methods referenced
above.
[0080] The present invention applies nucleotide bases to the array
substrate 11 using the technology concepts from the thermal ink jet
printing systems made by Hewlett-Packard of Palo Alto, Calif., or
piezoelectric printing systems, made by Epson of Japan. According
to a preferred embodiment, in situ synthesis of oligonucleotide
probes 13a is accomplished with a thermal ink jet array writer 20
illustrated in FIG. 2. The array writer 20 of the present invention
is an automated system that is computer controlled and comprises an
enclosure 21, a stage 22 for holding substrate 11 while probes 13a
are synthesized on the surface 12 of the substrate 11, and a
spinner assembly 23 for spinning the stage 22.
[0081] The stage 22 is removably attached to the spinning assembly
23. The stage 22 comprises a top plate 22a and a pedestal portion
22b. The top plate 22a has a recess 22c machined into one surface
for receiving the array substrate 11. The array substrate 11 is
held in place in the recess 22c either with a clamping mechanism
22e or the stage 22 is plumbed for vacuum to hold the substrate 11
(not shown). The pedestal portion 22b of the stage 22 has a
receptacle 22d.
[0082] The enclosure 21 comprises a bottom portion 21a, a wall
portion 21b and a drain 24 and an orifice 25 in a bottom portion
21a of the enclosure 21. The orifice 25 is preferably centered in
the middle of the bottom portion 21a. The drain 24 may be placed
adjacent to the wall portion 21b of the enclosure 21.
[0083] The spinner assembly 23 comprises a rotating rod or spindle
portion 26 that fits through the orifice 25 and is received by the
receptacle 22d in the pedestal portion 22b of stage 22 and a motor
23a with a variable rotational speed control. When the spinning
motor 23a is activated, the rotating rod 26 rotates the stage 22
incrementally and/or continuously 360 degrees. The spinner assembly
23 is a commercially available assembly available from Compumotor
Div. of Parker Hannifin, Rohnert Park, Calif., for example. In the
preferred embodiment, a Model No. ZETA67-51 Compumotor spinner
assembly 23 was used.
[0084] The array writer 20 further comprises a plurality of ink jet
heads 27. The ink jet heads 27 are each connected via tubing 28a to
a separate reservoir 28 containing a different one of the four
nucleotide bases and a respective conventional activator solution.
Only one of each of the ink jet heads 27, reservoirs 28 and
connector tubing 28a is illustrated in FIG. 2 for simplicity. The
ink jet heads 27 deposit nucleotide bases in the form of droplets
of biological material 13 into each feature 14 location on to the
surface 12 of the substrate 11 to form probes 13a. The nucleotide
base droplets 13 are placed accurately and repeatably on the
feature locations 14 and such that the nucleotide base make up of
probes 13a in each location 14 is known because it is preprogrammed
into the computer (not shown). The spinner motor 23 rotates the
substrate 11 incrementally during the printing of the nucleotide
bases 13 to form the annular pattern of droplets 14 on the surface
12.
[0085] In one embodiment, four inkjet heads 27a, one for each
nucleotide base type, or alternatively, a single inkjet head 27a
with four nozzles 27c and four reservoirs 28, one for each
nucleotide base type, may be used, as illustrated in FIG. 2B. The
inkjet head 27a moves radially, while the substrate is rotated, to
place the appropriate nucleotide base in the appropriate location
in the annular pattern in accordance with the preprogrammed
nucleotide base make-up of the probes 13a. A linear movement
assembly is necessary to move the inkjet heads 27a radially across
the substrate 11. For the invention, a linear assembly created
using a THK Co., Inc. (Japan) linear ball slide and a generic rack
and pinion motor was used. The substrate is rotated incrementally
through 360 degrees during the printing process.
[0086] In another embodiment, each inkjet head or pen 27b has a
plurality of nozzles arranged in a straight line along the radius
of the circular substrate 11. Each nozzle 27c provides monomer to a
location in the annular pattern on the surface 12 of the substrate
11. FIG. 2C illustrates the annular pattern of FIG. 1A, for
example. There is one nozzle for each concentric annular ring 15 in
each inkjet pen 27b located at the appropriate radius of the
respective annular ring 15. There are four of the above described
inkjet pens 27b and four reservoirs 28, one for each nucleotide
base type. Each of the four inkjet heads 27b is located at a
different angle, but all have their series of nozzles aligned with
the same concentric rings 15. A nucleotide base may be printed onto
the substrate surface 12 from a particular nozzle on a particular
inkjet head 27b in accordance with the preprogrammed nucleotide
base make up of the probes 13a. In this embodiment, no radially
movement of the inkjet heads 27b is needed to place a nucleotide
base in the proper location on the substrate 11. Only the substrate
moves, in accordance with the invention, by rotating incrementally
360 degrees.
[0087] The array writer 20 further comprises a plurality of
reagent/wash dispensers 29 to provide conventional ancillary
materials, such as reagents and washing fluids, during the
synthesis of the array 10 of the oligonucleotide probes 13a. Only
one dispenser 29 is illustrated in FIG. 2 for simplicity. Four wash
fluids are typically used during in situ synthesis, one for each of
the conventional capping, oxidizing, deblocking and washing steps.
The dispensers 29 are essentially stationary in the writer 20 and
positioned above the surface 12 of the substrate 11. When the
reagents or wash solutions are dispensed during the synthesis
process, the spinner motor 23 rotates the stage 22 and the array
substrate 11 so that the solutions are dispensed completely over
the substrate surface 12. The reagents or wash solutions are
removed from the surface 12 of the substrate 11 by spinning the
substrate 11 at high speed within the enclosure 21. Excess reagents
or wash solutions are drained from the enclosure 21 via drain
24.
[0088] In the conventional in situ synthesis, the surface 12 of the
substrate 11 is prepared chemically, according to conventional
methods, to receive and bind a phosphoramidite (i.e., a single
nucleotide base). Referring to FIG. 8B, the method 60 of in situ
synthesis using the array writer 20 in accordance with the present
invention include depositing 5 (step 61) a nucleotide base onto the
prepared substrate 11 in a feature location 14 in the annular
region or in an annular pattern and rotating (step 62) the
substrate 12 incrementally to a next feature location 14 for
deposition. The deposition and rotation steps are repeated (step
63) until all feature locations 14 in annular pattern have a
nucleotide base deposited thereon.
[0089] The bases bind to the array surface 12, but not to each
other since the second binding site on each phosphoramidite has
been blocked by a dimethoxytrityl. The array surface 12 is washed
(step 64a) with acetonitrile solvent or equivalent from dispensers
29, as described in the literature, and then the substrate is spun
(step 66a) at high speed to remove the solvent. A capping reagent
is then dispensed (step 65) from dispenser 29 and flooded across
the array substrate 11. In the preferred embodiment, the binding
sites that did not bind a phosphoramidite are capped off rather
than allow the sites to bind to another base at the next step to
improve synthesis efficiency. The capping reagents are spun off
(step 66b) the substrate 11 at a high speed. The substrate 12 is
then washed (step 64b) in with conventional solutions of ancillary
materials, as described in the literature, and then spun (step 66c)
at high speed to remove the washing fluids. After the washing and
spin steps 64b, 66c, an oxidizing reagent is dispensed (step 67)
from dispenser 29 and flooded over the array substrate surface 12.
The oxidizing reagent stabilizes the oxidation state of the
phosphorous group of the newly attached base. The oxidizing reagent
is spun off (step 66d) the substrate 11 at high speed. The
substrate 12 is then washed (step 64c) in with conventional
solutions, as described in the literature, and then spun (step 66e)
at high speed to remove the wash fluids. Lastly, the
dimethoxytrityl is removed according to conventional methods with
deblocking reagents in a deblocking step (step 68) so that the base
addition cycle can be repeated. The substrate is then spun (step
66f) at high speed to remove the deblocking reagent. The substrate
11 is then washed (step 64d) in with conventional solutions, as
described in the literature, and then spun (step 66g) at high speed
before the synthesis cycle (steps 61, 62, 63, 64a-64d and 65,
66a-66g, 67 and 68) is repeated and the next base is added.
[0090] The array 10 and array writer 20 of the present invention
provide efficient deposition of the phosphoramidites. Each of the
ink jets 27 can rapidly address all locations on the substrate 11
and avoids the complex x, y movement found in the conventional
systems. Ink jet assemblies 27 with multiple nozzles eliminate any
ink jet pen movement. The alignment of the nozzles in the pen
assemblies 27 is permanent. The deposition of nucleotide bases 13
is continuous and without changes in direction, as in the
conventional x, y format.
[0091] The spin steps (66a-66g) provide efficient washing between
nucleotide base additions. In the washing steps 64a-64d, 66a-66g,
the reagents are rapidly and cleanly removed without loss of
substrate alignment. Advantageously, "carryover" typically found in
the conventional methods of in situ synthesis, which could block
the action of a chemical during synthesis, is negligible in the
present invention. Moreover, the surface 12 of the substrate 11 is
essentially dry before the next reagent is added.
[0092] The array 10 and array writer 20 of the present invention
advantageously facilitates rapid manufacturing of custom arrays.
The array writer 20 supports customer's needs for "experiment on
demand" research. The specific information needed to synthesize a
custom array is captured in an electronic file. The array writer 20
of the present invention reads the electronic file. A customer can
order a single array and pay only for the costs of running the
array writer 20 for the time needed to synthesize the single array.
There are no tooling requirements or minimum lot size requirements,
as is found in the conventional synthesis equipment using the x, y
format.
[0093] In the conventional photolithographic methods of
synthesizing arrays, the steps of deblocking, for example, require
very expensive masks for each step. Approximately 80 masks are
needed to make an array of oligonucleotides having 20 bases in
length in the conventional photolithographic synthesis method.
Therefore, the cost of making a custom array by photolithographic
methods for a customer is typically prohibitive.
[0094] In the conventional pre-synthesized oligonucleotide method
of forming arrays, the oligonucleotides, typically 1000 of them,
are deposited onto the surface of the array substrate sequentially.
This deposition process requires a potentially large production
line and the arrays are typically manufactured in large quantities
to minimize cost. Therefore, this method is not conducive to
providing custom arrays in small quantities. However, the present
invention not only can provide economically feasible custom arrays,
but also can provide the custom arrays in an economically
affordable lot size as small as one array.
[0095] Printing pre-synthesized (cDNAs) or oligonucleotide probes
13a in an annular region on the substrate 11 may be performed using
a thermal inkjet printing system, as mentioned above. However, an
oligonucleotide probe 13a at a particular location must be printed
on each substrate in the manufacturing run independently of another
probe 13a being printed. This may be more time consuming.
Therefore, it is preferred that several substrates 11 are
manufactured with pre-synthesized (cDNAs) or oligonucleotide probes
13a in a single production run in accordance with the present
invention.
[0096] The array 10 may or may not be packaged for handling and
processing in accordance with the invention after the synthesis of
the oligonucleotide probes 13a on the surface 12. In the preferred
embodiment, the array 10 is packaged in a custom tooled carbon
black loaded polypropylene or ABS black plastic housing 17, which
can be injection molded by most injection molding companies. The
packaged array of the preferred embodiment is designated
hereinafter as 17a. FIG. 3A illustrates a perspective top view of
the array 10 in a housing 17, 17b according to the preferred
embodiment. FIG. 3B illustrates a cross sectional view of the array
10 in one embodiment of the housing 17 assembly of FIG. 3A. FIG. 3C
illustrates an exploded view of the array 10 and a preferred
embodiment of the housing 17b assembly, which is further
illustrated in cross-sectional view in FIG. 3D.
[0097] Referring to FIGS. 3A-3D, the housing 17, 17b comprises a
base portion 18. The base portion 18 has a first annular recess
18a-1 on a first side 18g where the array 10 is inserted. The base
portion 18 has a second annular recess 18a-2, positioned coaxially
within the first annular recess 18a-1. The second annular recess
18a-2 is shallower than the first annular recess 18a-1. The base
portion has third recess 18a, positioned coaxially and preferably
centered within the second annular recess 18a-2 and the first
recess 18a-1. The third recess 18a is preferably circular and
deeper than each of the first and second annular recesses 18a-1 and
18a-2. The third recess 18a is in at least fluid communication with
at least the second annular recess 18a-2.
[0098] The base portion 18 further includes a first radially
extending recess 18f-1. The first radial recess 18f-1 is in at
least fluid communication with second annular recess 18a-2 and
therefore, third recess 18a via pathway 18a-4. The first radial
recess 18f-1 extends radially inward from the outer periphery 18e
of the base portion 18.
[0099] The base portion 18 further comprises a valve mechanism 18d
within first radial recesses 18f-1, or valve chamber 18f-1. The
valve mechanism 18d comprises an actuator means 18d-1, a valve
chamber cap 18d-2 and a biasing means 18d-3 located between
actuator 18d-1 and cap 18d-2. The actuator means 18d-1 and biasing
means 18d-3 are assembled into the first valve chamber 18f-1. The
valve chamber cap 18d-2 is then attached to the housing 17,
preferably by ultrasonic welding. In the embodiment of housing 17
illustrated in FIG. 3B, the valve chamber cap 18d-2 has a through
hole to allow the fluids to exit the package via the valve chamber
18f-1 when the valve is open. The valve mechanism 18d provides a
controlled means for at least fluids to exit the housing 17 via
pathway 18a-4. The valve mechanism 18d is controlled by centrifugal
force and is further described in pending U.S. patent application
Ser. No. 09/133,102, filed Aug. 12, 1998, assigned to the assignee
of the present invention, and incorporated herein in its entirety
by reference.
[0100] In the preferred embodiment of the housing 17b illustrated
in FIGS. 3C-3D, the base portion further comprises a second
radially extending recess or valve chamber 18f-2 which is located
preferably diametrically opposite from the first valve chamber
18f-1. The second valve chamber 18f-2 holds a second valve
mechanism 18d similar to the first mechanism. The second valve
mechanism is assembled in the second valve chamber 18f-2 in the
same manner as described above for housing 17 in FIG. 3B. The
housing 17b further comprises an annular frame 19 and a fourth
annular recess 18a-3 on a second side 18h of the base portion 18
opposite to the first side 18g. The fourth annular recess 18a-3 is
preferably deeper and narrower than the first annular recess and
the second annular recess 18a-1 and 18a-2 and is located near the
periphery 18e of the base portion 18. The frame 19 is attached to
the second side 18h of the base portion 18, preferably by
ultrasonic welding using conventional methods, thereby enclosing
the fourth annular recess 18a-3 as a waste cavity 18a-3. The waste
cavity 18a-3 intersects with the first valve chamber 18f-1 and the
second valve chamber 18f-2, thereby providing at least fluid
communication between first and second valve chambers 18f-1 and
18f-2. In this embodiment of the housing 17b, the valve caps 18d-2
do not have through holes for fluid to escape. Instead, at least
one valve mechanism 18d provides a controlled means for venting the
annular waste cavity 18a-3 of air via a pathway 18f-3 to the
exterior of the housing 17b adjacent to the second side 18h of base
portion 18. The air is vented to allow fluids to enter the waste
cavity 18a-3. The valve mechanisms 18d are controlled by
centrifugal force.
[0101] In both embodiments of the housing 17, 17b illustrated in
FIGS. 3A-3D, the base portion 18 further comprises a fluid
introduction port 18a-5 and a septum or diaphragm 18c located
substantially in the center of the circular recess 18a of base
portion 18. The septum 18c is captured within the fluid
introduction port 18a-5 with a cover (not shown) that is
ultrasonically welded into place, preferably after the base portion
18 (and frame 19, in the preferred embodiment) are attached. The
fluid introduction port 18a-5 provides a passageway for the target
sample 13b and other ancillary materials to be introduced into
housing 17, 17b after the array 10 is attached. The septum 18c
functions to allow fluids into the housing 17, 17b but prevents the
fluids from escaping. The septum 18c can be made of any type of
rubber.
[0102] After the housing 17, 17b is assembled, the array 10 is
inserted into first annular recess 18a-1 of housing 17, 17b with
the array surface 12 adjacent to second annular recess 18a-2 of
base portion 18 in accordance with the preferred embodiment of the
invention. The array 10 is bonded into place using conventional
adhesives and the adhesive manufacturer's recommended bonding
procedures. The bonded array 10 and the base portion 18 form an
assay chamber 18b within the third recess 18a and second annular
recess 18a-2 regions. The array surface 12 of the array 10 faces
into the assay chamber 18b adjacent the second annular region
18a-2. The second annular cavity region 18a-2 is preferably from
approximately 25 to 50 microns in depth.
[0103] In order to perform gene analyses, the array 10, containing
oligonucleotide probes 13a, is placed in a hybridization chamber
where the probes 13a are exposed to a solution of the target
nucleotide sequences 13b under controlled conditions for
hybridization to occur. The target sample 13b is expected to have
complementary nucleotide sequences to the probe nucleotides 13a. In
accordance with the invention, the conventional hybridization
solutions and processes for hybridization can be used, such as
those referenced above.
[0104] However, in accordance with the invention, the hybridization
chamber is adapted with a pedestal shaped stage similar to the
stage 22 in the array writer 20 and a spinner assembly, similar to
the assembly 23 in the array writer 20 (see FIG. 2). In the
preferred embodiment, the pedestal shaped stage holds the housing
17, 17b, which houses the array 10, as described above (the
packaged array 17a) and therefore, the hybridization chamber or
enclosure is not needed.
[0105] Referring to FIG. 8C, the method 70 of hybridization in
accordance with the invention includes introducing the target
sample 13b (step 71) into the center 11b of the array substrate
surface 12. The array 10 is spun (step 73) by the spinner assembly
to move the target sample 13b centrifugically toward the periphery
11a of the substrate 11 where the annular array of probes 13a (15a,
15b, 15c, etc.) is located. In the preferred packaged array 17a
embodiment, the target sample 13b is introduced (step 71) into the
assay chamber 18b through septum 18c of housing 17, 17b. The sample
13b travels from the chamber 18b directly to the annular cavity
region 18a-2 where the probes 13a are located during the spinning
step 73.
[0106] The amount of target sample 13b available for analysis at
any one time is typically very small. Therefore, the target sample
13b should not be wasted during its introduction to the probes 13a
for analysis. The present invention advantageously conserves the
target sample 13b during the hybridization process. The amount of
target sample 13b needed is minimized by the present invention,
because the target sample 13b is effectively spread by the
centrifugal force of the spinner assembly in a film over the probes
13a in a thickness from approximately 25 to 50 microns. According
to the preferred packaged array 17a embodiment, the centrifugal
force from spinning (step 73) the packaged array 17a after the
target sample 13b is introduced (step 71) into the assay chamber
18b, leaves little or no target sample 13b wasted in the assay
chambers 18b, 18a-2 of the housing 17, 17b. Therefore, the use of
the valuable target sample 13b, which is typically in very short
supply, is optimized in accordance with the present invention.
[0107] A conventional hybridization environment is provided (step
75). After the hybridization is completed the hybridized array 10a
is spun again (step 77) at a speed high enough so that the excess
unhybridized target sample 13b is forced off of the substrate
surface 12 by centrifugal force prior to washing the surface. The
spin speed used at any one step in the invention is dependent upon
the fluids that need to be moved. Basically, the higher the spin
speed, the greater the centrifugal force obtained.
[0108] In the preferred packaged array 17a embodiment, the
unhybridized target sample 13b is moved from the assay chamber 18b
to the waste cavity 18a-3 via pathway 18a-4 and first valve chamber
18f-1 by activating the valve assembly 18d therein during the spin
step 77. The valve assembly 18d is activated by the centrifugal
force of spinning the packaged array 17a at a sufficient speed.
[0109] The hybridized array is designated hereinafter as 10a.
Ancillary materials, e.g., conventional wash fluids, are then
introduced (step 78) to the center of the hybridized array
substrate 11 and spun (step 79) across the hybridized array 10a
many times until the non-specifically bound (unhybridized) material
is removed. The array surface 12 is flooded with wash fluids in a
volume of 3 to 10 times the volume of the original target sample
13b introduced. Depending on whether the hybridized array 10a will
be optically evaluated in a dry state or wet state, the final wash
fluids are either spun out or left in place, respectively.
[0110] In the packaged array 17a of the preferred embodiment, the
wash fluids are introduced (step 78) into the assay chamber 18b via
septum 18c and spun (step 79) at a speed high enough to spread the
wash fluids over the hybridized array area 18a-2. The spin step
also functions to discharge the wash fluids into at least the first
valve chamber 18f-1 via pathway 18a-4. The preferred housing 17b
collects and holds the waste of the hybridization procedure in its
waste cavity 18a-3. In the preferred packaged array 17a, either the
housing 17 or 17b advantageously holds the fluid waste in a
controlled fashion. Without the housing 17b in accordance with the
preferred embodiment, the fluid waste is collected in the
hybridization chamber.
[0111] The present invention facilitates automation of the
hybridization 71, 75 and washing 78 steps. The transport of
ancillary fluid materials is accomplished by centrifugal force
inherent in the spinning steps 73, 77, 79. The amount of force
applied is controlled by the spinner motor speed. Advantageously,
all of the target sample 13b is moved into the probe 13a array
area. The target sample 13b is evenly distributed by the spinning
motion. No bubbles are formed during the hybridization process, or
if they are formed, they are moved to the center of the array 10
during the spinning steps, because they are more buoyant than the
fluids. Spreading of reagents, target sample 13b and wash solutions
over the surface 12 of the substrate 11 are easily implemented with
the spinning steps 73, 77, 79. When the hybridization process is
complete, the unhybridized target sample 13b is quickly and cleanly
moved out of the probe 13a array area (steps 77-79). The washing
steps 77, 78 are completed faster with the invention. The housing
17, 17b of the preferred packaged array 17a embodiment
advantageously functions to contain the fluid waste until disposal
is appropriate. The valve assemblies 18d in the housing 17, 17b of
the preferred packaged array 17a embodiment facilitates the removal
of the unhybridized sample and ancillary materials, such as the
reagents and washing solutions, and is controlled by centrifugal
force.
[0112] After the hybridization step of the array 10, the hybridized
array 10a is optically interrogated to determine the extent and
location of hybridized features 16 among the discrete features 14.
In accordance with the invention, the hybridized array 10a is
interrogated with an optical scanning system 30 illustrated in
FIGS. 4 and 5. The optical scanning system 30 uses either an
off-axis method of optical scanning, as described in U.S. Pat. No.
5,585,639, or a confocal method of optical scanning, as described
in U.S. Pat. No. 5,760,951 or U.S. Pat. No. 5,763,870 (also
describes an autofocus capability), all incorporated herein by
reference.
[0113] Referring to FIG. 4, the optical scanning system 30 of the
present invention includes a light source 31, typically a laser,
which emits light of a wavelength and with sufficient intensity to
cause fluorescence from a fluorophore label. The light beam 31a is
focussed and directed by optics subsystem 32 to the hybridized
array 10a. A scanning subsystem 36 provides annular movement of the
hybridized array 10a in the r, .theta. format and linear movement
to either the array 10a or the optics subsystem 32. A portion of
optics subsystem 32 directs a fluorescence signal 34a from the
surface of the hybridized array 10a into a detector subsystem 35.
The detector subsystem 35 detects the fluorescence signal 34a. The
data from the detector subsystem 35 is analyzed by an analysis
subsystem 39 and information about the target samples 13b is
determined.
[0114] The light source 31 may be an Argon ion type laser (off-axis
scanner) or an internally doubled Nd-YAG (532 nm) laser and a HeNe
(633 nm) laser (confocal scanner). In the preferred embodiment, a
confocal scanner with both the internally doubled Nd-YAG (532 nm)
and a HeNe (633 nm) laser light source 31 was used.
[0115] FIG. 5 illustrates a schematic view of the subsystems 32,
35, 36, and 39 of a preferred embodiment of the present scanning
system 30. FIG. 8D illustrates the method 80 of optical
interrogation in accordance with the invention. The hybridized
array 10a is mounted into the scanning subsystem 36 (step 81). The
scanning subsystem 36 comprises an annular movement subassembly 37
and a linear movement subassembly 38. The annular movement
subassembly 37 comprises a holder 37a for holding the hybridized
array 10a in place. An exploded view of the holder 37a is
illustrated in FIG. 6. The holder 37a comprises a ring plate 37a-1
having a recess 37a-2 into which the hybridized array 10a is
inserted. The holder 37a further comprises a solid plate 37a-3 and
an O-ring 37a-4 to seat against the array 10a. The ring plate
37a-1, solid plate 37a-3 and O-ring 37a-4 hold the array 10a flat
inside the holder 37a such that the annular array of features 14,
16 are unobstructed and light beam 31a can impinge upon the
features 14, 16. The ring plate 37a-1 and solid plate 37a-3 of
holder 37a are preferably made of stainless steel and are held
together with screws or other appropriate fasteners. In the
preferred embodiment, the packaged array 17a is mounted directly
into the annular movement subassembly 37 without the need for a
holder 37a, as described above.
[0116] The annular movement subassembly 37 further comprises a
spinner subassembly 37b that provides rotational movement to the
holder 37a or the preferred packaged array 17a. The spinner
subassembly 37b comprises a spinner motor 37b-1 and a spindle 37b2,
which may be similar to the spinner subassembly 23 in FIG. 2. The
spindle 37b-2 has an end 37b-3 for receiving holder 37a or packaged
array 17a. The holder 37a or packaged array 17a is removably
connected to end 37b-3 of the spindle 37b-2, preferably by way of
detents (not shown) so that the holder 37a or the packaged array
17a snaps in and out of the spindle end 37b-3. The spindle 37b-2 is
connected for rotational movement at its other end to the spinner
motor 37b-1. The spinner subassembly 37b rotates the hybridized
array 10a in the holder 37a, or rotates the housing 17, 17b that
encloses the array 10a in the preferred packaged array 17a
embodiment, while the laser 31, the light source 31a and the optics
subsystem 32 remain stationary.
[0117] The scanning subsystem 36 further comprises a linear
subassembly 38. The linear subassembly 38 comprises a linear
actuator (not shown) and a linear stage 38a. The linear subassembly
can be used to move the optic subassembly 32 or the annular
subassembly 37 in a radial direction. In the preferred embodiment,
the spinner subassembly 37b is removably mounted to the linear
stage 38a with screws or other appropriate fasteners. The linear
subassembly 38 moves the spinner subassembly 37b, along with the
hybridized array 10a in holder 37a, or as package array 17a,
radially (in FIG. 5, in a direction in or out of the page or along
the arrows shown). The movement of the spinner subassembly 37b by
the linear subassembly 38 is incremental, in microns, in a radial
direction, such that the light beam 31a can scan all of the
features 14, 16 in the annular region of the array substrate 11.
The linear subassembly 38 does not need to be particularly precise
for the present invention, because it provides the radial movement
to the spinner subassembly 37b. The linear subassembly 38 can be
any commercially available controllable linear system, such as
those manufactured by Newport of Irvine, Calif. The present
invention was demonstrated with several linear subassemblies from
the UTM series of Mid Range Travel Translation Stages.
[0118] FIG. 5 further illustrates the light beam 31a from light
source 31 entering first optics subsystem 32 where it is reflected
by a mirror 32a to a dichroic reflector 32c, where it is directed
through an objective lens 32d to impinge (step 82) on the
hybridized array 10a. The annular subassembly 37 rotates (step 84)
the hybridized array 10a 360 degrees so that the light beam 31a
impinges on all of the features 14, 16 in the annular region. The
linear subassembly 38 preferably moves the array 10a radially (step
86), so that the light beam 31a impinges on all of the discrete
features 14, 16 in the annular region. When the light beam 31a
impinges on a fluorophore label on the hybridized features 16, a
fluorescence signal 34a is emitted. The fluorescence signal 34a is
directed through the objective lens 32d to the dichroic reflector
32c of optics subsystem 32 and the dichroic reflector 32c directs
the fluorescence signal 34a into the detector subsystem 35, where
the signals 34a are detected (step 88). The detector subsystem 35
comprises a lens 35a, a pinhole or slit 35b to direct the signals
34a into a detector 35c, preferably a photomultiplier tube.
[0119] Back reflected light or "signal noise" may also be generated
from the surface 12 of the hybridized array 10a substrate 11 in
response to the light beam 31a. If generated, the back reflected
light also passes through the objective lens 32d, but is filtered
out by the dichroic mirror 32c or other filtering means, such as an
interference filter, so that the signal noise does not interfere
with the detection system 35 and analysis 39.
[0120] The detected signals 34a are gathered and analyzed (step 89)
by the analysis subsystem 39 comprising a computer. Information
about the hybridized targets samples 13b is determined from what is
known about the probes 13a at the hybridized locations. Analysis
subsystem 39 can use any of the commercially available analysis
software programs mentioned previously to analyze the hybridization
signals 34a.
[0121] In the preferred embodiment, the scanner 30, 30a scans an
annulus or ring of about 5 to 10 microns wide then steps over to an
adjacent 5 to 10 micron wide annulus to scan until the hybridized
array 10a surface 12 is completely scanned in a continuous fashion.
Therefore, the scanner 30, 30a scans between discrete features 14,
16, as well as the features 14, 16 themselves. The features 14, 16
are nominally 100 microns in diameter. Therefore, it takes 10-20
scanning rings to scan an annular ring, for example, of features
14, 16. An increased scan speed for rescanning, as described in
U.S. Pat. No. 5,837,475, assigned to the assignee of the present
invention and incorporated herein by reference, is simply an
increase in rotation rate of the spinner subsystem 36 in the
preferred embodiment without the acceleration problems of a
reciprocating motion scan, as is found in the conventional x, y
format scanners described in the U.S. Pat. No. 5,837,475. In this
case, subpixel steps in the radial direction are conceivable.
Subpixel scanning is achieved by stepping the scan less than the
spot size of the light beam 31a.
[0122] The optical scanning system 30 of the present invention
provides for faster scan processing. There is a 100 percent duty
cycle during the scan because overscanning is limited to
compensating for run-out, which may occur during the manufacturing
process. Run out is a term that defines possible alignment errors
during manufacturing of the array 10 rendering the array pattern
slightly off center. The present invention looks at the entire
range of possible manufacturing errors to be sure that the actual
array of features 14, 16 is scanned completely.
[0123] The present optical scanning system 30 and method 80 have no
direction changes between scan lines, as found in the conventional
x, y format scanners mentioned above. There is no risk of the laser
beam 31a impinging on adhesive glue lines, since the scan is
circular. The instrumentation for the optical scanning system 30 is
less expensive than the conventional systems for comparable
performance, because neither an x, y stage nor a galvanometer is
required. Moreover, since the centrifugal force from spinning
during the hybridization method 70 eliminates bubbles or spins the
bubbles out of the scanned region, the present invention
interrogation system 30 and method 80 provide more reliable
results. The movement of the hybridized array 10a rather than the
optics 32 or the laser assembly 31 allows for a very small, high
depth discrimination lens 32d at a reasonable cost. In addition,
the present optical scanning system 30 and method 80 facilitate
dynamic autofocussing, as described in U.S. Pat. No. 5,763,870.
[0124] FIG. 7 illustrates an example of the analysis obtained from
analysis subsystem 39 of a hybridized array 10a. The hybridization
results in FIG. 7 are merely illustrative of the many hybridization
scenarios possible in a biological assay. The results illustrated
in FIG. 7 are not intended to limit the scope of the invention or
to represent a preferred embodiment of the invention. It should be
understood by those skilled in the art that the hybridization
efficiency between the probes 13a and the target nucleotide
sequences 13b is dependent upon their complementarity and the
hybridization processes used, among other things.
[0125] Moreover, the hybridization results in FIG. 7 are presented
in a linear mode. Each circular sweep made by the substrate 11
while the light beam is impinging on the surface is translated to a
straight line at the output of the analysis subsystem 39. It is not
the intent of the inventors to be limited to a linear
representation of the hybridization results and the results can be
presented in any format and still be within the scope of the
invention.
[0126] FIG. 8A illustrates the method 50 of assaying biological
materials in accordance with the present invention. Referring to
FIG. 8A, a first biological material, such as the oligonucleotide
probes 13a, are provided (step 51) on the substrate 11 in an
annular array pattern of discrete features 14 with the synthesis
system 20 and synthesis method 60 of the present invention. A
second biological material, such as the target nucleotide sequences
or sample 13b, is hybridized (step 53) with the probes 13a with the
hybridization system and method 70 of the present invention. The
hybridized discrete features 16 have a signal producing system for
producing a signal 34a when illuminated with a light beam 31a. As
mentioned above, the signal producing system can be a conventional
label, and preferably a fluorophore label. The hybridized array 10a
is optically interrogated (step 55) with the optical interrogation
system 30 and method 80 of the present invention. The information
obtained from the fluorescence signals 34a is used to determine
(step 57) the characteristics about the first biological material
13a and the second biological material 13b.
[0127] The method 50 of assaying biological materials 13 is
particularly conducive to automation and therefore, has inherent
advantages. Each step 51-57 of the assay process mentioned above
can be automated to provide flexibility and accuracy to the
assaying process 50.
[0128] Thus there has been described a new apparatus, systems and
methods for assaying biological materials that uses an r, .theta.
format. The biological array apparatus is manufactured on a
substrate and the annular array pattern is interrogated optically
in the r, .theta. format using an optical scanning system that
rotates or spins the array 360 degrees and moves the array or the
optics in a radially fashion. It should be understood that the
above-described embodiments are merely illustrative of the some of
the many specific embodiments that represent the principles of the
present invention. Clearly, numerous other arrangements can be
readily devised by those skilled in the art without departing from
the scope of the present invention.
[0129] For example, the array 10 of the invention is not limited to
a particular size or array substrate thickness. The array substrate
10 need not be circular, as long as the array pattern of surface
bound features is within an annular region of the substrate and the
synthesis, hybridization or optical interrogation of the array can
take advantage of the rotational motion in the r, .theta. format.
It is within the scope of the present invention to use a variety of
substrate shapes, diameters and thicknesses. Moreover, the array
substrate does not have to be clear. Substrates that are opaque are
within the scope of the invention. Opaque substrates can be scanned
from the array side as opposed to scanning through the clear
substrate material to the array. As mentioned above, packaging the
array is a preferred embodiment and the array does not have to be
packaged in accordance with the invention. Moreover, the array
pattern can be arranged in any annular pattern, including but not
limited to a spiral or concentric and/or segmented annular rings
and still be within the scope of the invention. It is also within
the scope of the invention to manufacture the array by printing the
monomers with an x, y format system during in situ synthesis, but
optically scan the array in a annular fashion. Moreover, the
features could be printed in the x, y format in a closely packed
pattern that approximates a circle, where the center of the pattern
is missing. This pattern would allow for x, y deposition of
pre-synthesized DNA and annular scanning using the present
invention.
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