U.S. patent application number 11/513126 was filed with the patent office on 2006-12-28 for compositions and methods involving direct write optical lithography.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Calvin F. Quate.
Application Number | 20060292628 11/513126 |
Document ID | / |
Family ID | 26776859 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292628 |
Kind Code |
A1 |
Quate; Calvin F. |
December 28, 2006 |
Compositions and methods involving direct write optical
lithography
Abstract
An improved optical photolithography system and method provides
predetermined light patterns generated by a direct write system
without the use of photomasks. The Direct Write System provides
predetermined light patterns projected on the surface of a
substrate (e.g., a wafer) by using a computer controlled means for
dynamically generating the predetermined light pattern, e.g., a
spatial light modulator. Image patterns are stored in a computer
and through electronic control of the spatial light modulator
directly illuminate the wafer to define a portion of the polymer
array, rather than being defined by a pattern on a photomask. Thus,
in the Direct Write System each pixel is illuminated with an
optical beam of suitable intensity and the imaging (printing) of an
individual feature is determined by computer control of the spatial
light modulator at each photolithographic step without the use of a
photomask. The Direct Write System including a spatial light
modulator is particularly useful in the synthesis of DNA arrays and
provides an efficient means for polymer array synthesis by using
spatial light modulators to generate a predetermined light pattern
that defines the image patterns of a polymer array to be
deprotected.
Inventors: |
Quate; Calvin F.; (Stanford,
CA) |
Correspondence
Address: |
AFFYMETRIX INC/ ATTN: Chief IP Counsel;LEGAL DEPT
3420 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
26776859 |
Appl. No.: |
11/513126 |
Filed: |
August 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11143476 |
Jun 3, 2005 |
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11513126 |
Aug 31, 2006 |
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10626627 |
Jul 25, 2003 |
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11513126 |
Aug 31, 2006 |
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10223719 |
Aug 20, 2002 |
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10626627 |
Jul 25, 2003 |
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09880058 |
Jun 14, 2001 |
6480324 |
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10223719 |
Aug 20, 2002 |
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09318775 |
May 26, 1999 |
6271957 |
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09880058 |
Jun 14, 2001 |
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60087333 |
May 29, 1998 |
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Current U.S.
Class: |
435/6.11 ;
356/322; 435/287.2 |
Current CPC
Class: |
B01J 2219/00596
20130101; B01J 2219/00605 20130101; B01J 2219/00659 20130101; B01J
2219/00689 20130101; G03F 7/704 20130101; B01J 2219/00439 20130101;
B01J 2219/00585 20130101; B01J 2219/0059 20130101; B01J 2219/00711
20130101; C40B 60/14 20130101; B01J 2219/00353 20130101; B01J
2219/00617 20130101; B01J 2219/00527 20130101; B01J 2219/00637
20130101; B01J 2219/00612 20130101; B82Y 30/00 20130101; C40B 40/06
20130101; G03F 7/70291 20130101; B01J 2219/00529 20130101; B01J
2219/00725 20130101; B01J 2219/00608 20130101; B01J 2219/00626
20130101; B01J 19/0046 20130101; B01J 2219/00722 20130101; G03F
7/70283 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 356/322 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; G01J 3/42 20060101
G01J003/42 |
Claims
1. A method for deprotecting reaction sites on a substrate
comprising the steps of: providing a substrate having protected
reaction sites; modulating light direction with a spatial light
modulator so as to generate a predetermined light pattern used for
deprotecting selected portions of said protected reaction
sites.
2. An apparatus for constructing DNA probes comprising: (a) a
reactor providing a reaction site at which nucleotide addition
reactions may be conducted; (b) a light source providing a light
capable of promoting nucleotide addition reactions; (c) a set of
electronically addressable micromirrors positioned along an optical
path between the light source and the reactor to receive and
reflect the light, the micromirrors separated by lanes having lane
widths; and (d) projection optics positioned along the optical path
between the reaction site and the image generator to focus an image
of the lanes on the reaction site; wherein the resolution of the
projection optics expressed as a separation distance between
resolvable line pairs is greater than half the lane width.
3. Apparatus for use in synthesis of arrays of DNA probes,
comprising: (a) a substrate with an active surface on which the
arrays may be formed; (b) a flow cell enclosing the active surface
of the substrate and having ports for applying DNA synthesis
reagents into the flow cell which can be flowed over the active
surface of the substrate; and (c) an image former providing a high
precision, two-dimensional light image projected onto the substrate
active surface, comprising: (1) a light source providing a light
beam; (2) a micromirror device receiving the light beam from the
source and formed of an array of electronically addressable
micromirrors, each of which can be selectively tilted between one
of at least two separate positions, wherein in one of the positions
of each micromirror the light from the source incident upon the
micromirror is deflected away from an optical axis and in a second
of the at least two positions of the micromirror the light is
reflected along the optical axis; and (3) projection optics
receiving the light reflected from the micromirrors along the
optical axis and imaging the pattern of the micromirrors onto the
active surface of the substrate, wherein the light is directed from
the micromirror onto the active surface by reflective optical
elements.
4. Apparatus for use in synthesis of arrays of DNA probes,
comprising: (a) a substrate with an active surface on which the
arrays may be formed; (b) a flow cell enclosing the active surface
of the substrate and having ports for applying DNA synthesis
reagents into the flow cell which can be flowed over the active
surface of the substrate; and (c) an image former providing a high
precision, two-dimensional light image projected onto the substrate
active surface, comprising: (1) a light source providing a light
beam; (2) a micromirror device receiving the light beam from the
source and formed of an array of electronically addressable
micromirrors, each of which can be selectively tilted between one
of at least two separate positions, wherein in one of the positions
of each micromirror the light from the source incident upon the
micromirror is deflected away from an optical axis and in a second
of the at least two positions of the micromirror the light is
reflected along the optical axis; and (3) projection optics
receiving the light reflected from the micromirrors along the
optical axis and imaging the pattern of the micromirrors onto the
active surface of the substrate, wherein the light is directed from
the micromirror onto the active surface by a reflective mirror.
5. Apparatus for use in synthesis of arrays of DNA probes,
comprising: (a) a substrate with an active surface on which the
arrays may be formed; (b) a flow cell enclosing the active surface
of the substrate and having ports for applying DNA synthesis
reagents into the flow cell which can be flowed over the active
surface of the substrate; and (c) an image former providing a high
precision, two-dimensional light image projected onto the substrate
active surface, comprising: (1) a light source providing a light
beam; (2) a micromirror device receiving the light beam from the
source and formed of an array of electronically addressable
micromirrors, each of which can be selectively tilted between one
of at least two separate positions, wherein in one of the positions
of each micromirror the light from the source incident upon the
micromirror is deflected away from an optical axis and in a second
of the at least two positions of the micromirror the light is
reflected along the optical axis; and (3) projection optics
receiving the light reflected from the micromirrors along the
optical axis and imaging the pattern of the micromirrors onto the
active surface of the substrate.
6. Apparatus for use in synthesis arrays of DNA probes, comprising:
(a) a substrate with an active surface on which the arrays may be
formed; (b) a flow cell enclosing the active surface of the
substrate and having ports for applying DNA synthesis reagents into
the flow cell which can be flowed over the active surface of the
substrate; and (c) an image former providing a high precision,
two-dimensional light image projected onto the substrate active
surface, comprising: (1) a light source providing a light beam; (2)
a micromirror device receiving the light beam from the source and
formed of an array of electronically addressable micromirrors,
wherein the micromirror device is formed of a two dimensional array
of micromirrors, cach of which can be selectively tilted between
one of at least two separate positions, wherein in one of the
positions of each micromirror the light from the source incident
upon the micromirror is deflected away from an optical axis and in
a second of the at least two positions of the micromirror the light
is reflected along the optical axis; and (3) projection optics
receiving the light reflected from the micromirrors along the
optical axis and imaging the pattern of the micromirrors onto the
active surface of the substrate, wherein the projection optics is
telecetric and wherein light is directed from the micromirrors to
the active surface is by reflective optical elements.
7. An apparatus for generating a time and spatially dependent light
spectrum comprising: a light source positioned to direct a light
output, wherein said light source comprises a light that is
redirected by a micromirror array under the control of a computer
to produce said light output; a variable spectrum filter placed in
the path of the light redirected by said light source, wherein said
variable spectrum filter passes one or more spatially separated
wavelengths of light; and said computer controls the relative
position of said variable spectrum filter and said light
output.
8. An apparatus for projecting one or more wavelengths of light
comprising: a light source positioned to redirect light from said
light source toward a target, wherein said light source is further
defined as comprising a light that produces one or more wavelengths
of light that are redirected by a micromirror array under the
control of a computer to produce said light; a variable spectrum
generator positioned to pass one or more spatially separated
wavelengths of light from said light source; and said computer
connected to, and controlling, said light source and said variable
spectrum generator to pass one or more wavelengths of light from
said light source toward said target.
9. An apparatus for generating a time and spatially dependent light
spectrum comprising: (a) a light source positioned to direct a
light output, wherein said light source comprises a light that is
redirected by a micromirror array under the control of a computer
to produce said light output; and (b) a filter placed in the path
of the light redirected by said light source, wherein said filter
passes one spatially separated wavelength of light.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of each of U.S. patent
application Ser. No. 11/143,476, filed Jun. 3, 2005, and U.S.
patent application Ser. No. 10/626,627, filed Jul. 25, 2003; and
each of U.S. patent application Ser. Nos. 11/143,476 and 10/626,627
are continuations of U.S. patent application Ser. No. 10/223,719,
filed Aug. 20, 2002, which is a continuation of U.S. patent
application Ser. No. 09/880,058, filed Jun. 14, 2001 (now U.S. Pat.
No. 6,480,324, issued Nov. 12, 2002), which is a divisional of U.S.
patent application Ser. No. 09/318,775, filed May 26, 1999 (now
U.S. Pat. No. 6,271,957, issued Aug. 7, 2001), and which claims
priority from U.S. Provisional Application No. 60/087,333, filed
May 29, 1998. The disclosures of the above-mentioned applications
are hereby incorporated by reference in their entireties in the
disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field of the Invention
[0003] This invention relates to optical lithography and more
particularly to direct write optical lithography.
[0004] 2. Description of the Related Art
[0005] Polymer arrays, such as the GeneChip.RTM. probe array
(Affymetrix, Inc., Santa Clara, Calif.), can be synthesized using
light-directed methods described, for example, in U.S. Pat. Nos.
5,143,854; 5,424,186; 5,510,270; 5,800,992; 5,445,934; 5,744,305;
5,384,261 and 5,677,195 and PCT published application No. WO
95/11995, which are hereby incorporated by reference in their
entireties. As an example, light-directed synthesis of
oligonucleotides employs 5'-protected nucleosidephosphoramidite
"building blocks." The 5'-protecting groups may be either
photolabile or acid-labile. A plurality of polymer sequences in
predefined regions are synthesized by repeated cycles of
deprotection (selective removal of the protective group) and
coupling. Coupling (i.e., nucleotide or monomer addition) occurs
only at sites that have been deprotected. Three methods of
light-directed synthesis are: use of photolabile protecting groups
and direct photodeprotection (DPD); use of acid-labile
4,4'-dimethoxytrityl (DMT) protecting groups and a photoresist; use
of DMT protecting groups and a polymer film that contains a
photoacid generator (PAG).
[0006] These methods have many process steps similar to those used
in semiconductor integrated circuit manufacturing. These methods
also often involve the use of photomasks (masks) that have a
predefined image pattern which permits the light used for synthesis
of the polymer arrays to reach certain regions of a substrate but
not others. The substrate can be non-porous, rigid, semi-rigid,
etc. It can be formed into a well, a trench, a flat surface, etc.
The substrate can include solids, such as siliceous substances such
as silicon, glass, fused silica, quart and other solids such as
plastics and polymers, such as polyacrylamide, polystyrene,
polycarbonate, etc. Typically, the solid substrate is called a
wafer from which individual chips are made (See the U.S. patents
above which are incorporated herein by reference). As such, the
pattern formed on the mask is projected onto the wafer to define
which portions of the wafer are to be deprotected and which regions
remain protected. See, for example, U.S. Pat. Nos. 5,593,839 and
5,571,639 which are hereby incorporated by reference in their
entireties.
[0007] The lithographic or photochemical steps in the synthesis of
nucleic acid arrays may be performed by contact printing or
proximity printing using photomasks. For example, an emulsion or
chrome-on-glass mask is placed in contact with the wafer, or nearly
in contact with the wafer, and the wafer is illuminated through the
mask by light having an appropriate wavelength. However, masks can
be costly to make and use and are capable of being damaged or
lost.
[0008] In many cases a different mask having a particular
predetermined image pattern is used for each separate photomasking
step, and synthesis of a wafer containing many chips requires a
plurality of photomasking steps with different image patterns. For
example, synthesis of an array of 20mers typically requires
approximately seventy photolithographic steps and related unique
photomasks So, using present photolithographic systems and methods,
a plurality of different image pattern masks must be pre-generated
and changed in the photolithographic system at each photomasking
step. This plurality of different pattern masks adds lead time to
the process and complexity and inefficiency to the
photolithographic system and method. Further, contact printing
using a mask can cause defects on the wafer so that some of the
reaction sites are rendered defective. Thus, a photolithographic
system and method that does not require such masks and obviates
such difficulties would be generally useful in providing a more
efficient and simplified lithographic process.
SUMMARY OF THE INVENTION
[0009] In view of the above, one advantage of the invention is
providing an improved and simplified system and method for optical
lithography.
[0010] Another advantage of the present invention is providing an
optical lithography system and method that dynamically generates an
image using a computer and reconfigurable light modulator.
[0011] A further advantage of the present invention is providing an
optical lithography system and method that does not use
photomasks.
[0012] A still further advantage of the present invention is
providing an optical lithography system and method that uses
computer generated electronic control signals and a spatial light
modulator, without any photomask, to project a predetermined light
pattern onto a surface of a substrate for the purposes of
deprotecting various areas of a polymer array.
[0013] According to one aspect of the invention, polymer array
synthesis is performed using a system without photomasks.
[0014] According to a second aspect of the invention, polymer array
synthesis is performed using a system with a transmissive spatial
light modulator and without a lens and photomask.
[0015] According to another aspect of the invention, a Direct Write
System transmits image patterns to be formed on the surface of a
substrate (e.g., a wafer). The image patterns are stored in a
computer. The Direct Write System projects light patterns generated
from the image patterns onto a surface of the substrate for
light-directed polymer synthesis (e.g., oligonucleotide). The light
patterns are generated by a spatial light modulator controlled by a
computer, rather than being defined by a pattern on a photomask.
Thus, in the Direct Write System each pixel is illuminated with an
optical beam of suitable intensity and the imaging (printing) of an
individual feature on a substrate is determined dynamically by
computer control.
[0016] According to a further aspect of the invention, polymer
array synthesis is accomplished using a class of devices known as
spatial light modulators to define the image pattern of the polymer
array to be deprotected.
[0017] An even further aspect of the present invention provides
methods For synthesizing polymer arrays using spatial light
modulators and the polymer arrays synthesized using the methods
taught herein.
[0018] As can be appreciated by one skilled in the art, the
invention is relevant to optical lithography in general, and more
specifically to optical lithography for polymer array synthesis
using photolithograpic processes. However, it is inherent that the
invention is generally applicable to eliminating the need for a
photomask in optical lithography.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The above objects, features, and advantages of the present
invention will become more apparent from the following detailed
description taken with the accompanying drawings in which:
[0020] FIG. 1 shows a first embodiment of the invention having a
light source, a reflective spatial light modulator, such as a
micro-mirror array, and a lens.
[0021] FIG. 2 is a diagrammatic representation of a second
embodiment of the invention employing an array of, for example,
micro-lenses.
[0022] FIG. 3 illustrates a micro-lens array in the form of Fresnel
Zone Plates, which may be used in the invention.
[0023] FIG. 4 shows a third embodiment of the invention having a
transmissive spatial light modulator.
[0024] FIG. 5 illustrates a reactor system for forming a plurality
of polymers on a substrate.
[0025] FIG. 6A is a top view and FIG. 6B is a cross-sectional view
of a device used to synthesize arrays of polymer sequences.
[0026] FIG. 7 is a cross-sectional view of a channel block and
associated flow ports.
[0027] FIG. 8 is a diagram of a flow system used to deliver
coupling compounds and reagents to a flow cell.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention refers to articles and patents that
contain useful supplementary information. These references are
hereby incorporated by reference in their entireties.
[0029] The presently preferred invention is based on the principle
that a Direct Write Optical Lithography System will significantly
improve the cost, quality, and efficiency of polymer array
synthesis by providing a maskless optical lithography system and
method where predetermined image patterns can be dynamically
changed during photolithographic processing. As such, an optical
lithography system is provided to include a means for dynamically
changing an intended image pattern without using a photomask. One
such means includes a spatial light modulator that is
electronically controlled by a computer to generate unique
predetermined image patterns at each photolithograpic step in
polymer array synthesis. The spatial light modulators can be, for
example, micromachined mechanical modulators or microelectronic
devices (e.g. liquid crystal display (LCD)). The Direct Write
System of the present invention using such spatial light modulators
is particularly useful in the synthesis of polymer arrays, such as
polypeptide, carbohydrate, and nucleic acid arrays. Nucleic acid
arrays typically include polynucleotides or oligonucleotides
attached to glass, for example, Deoxyribonucleic Acid (DNA)
arrays.
[0030] Certain preferred embodiments of the invention involve use
of the micromachined mechanical modulators to direct the light to
predetermined regions (i.e., known areas on a substrate predefined
prior to photolithography processing) of the substrate on which the
of polymers are being synthesized. The predetermined regions of the
substrate associated with, for example, one segment (referred to
herein as a pixel) of a micromachined mechanical modulator (e.g., a
micro-mirror array) are referred to herein as features. In each
predetermined region or feature a particular oligonucleotide
sequence, for example, is synthesized. The mechanical modulators
come in a variety of types, two of which will be discussed in some
detail below.
[0031] One type of mechanical modulator is a micro-mirror array
which uses small metal mirrors to selectively reflect a light beam
to particular individual features; thus causing the individual
features to selectively receive light from a light source (i.e.,
turning light on and off of the individual features). An example is
the programmable micro-mirror array Digital Micromirror Device
(DMD.TM.) manufactured by Texas Instruments, Inc., Dallas, Tex.,
USA. Texas Instruments markets the arrays primarily for projection
display applications (e.g., big-screen video) in which a highly
magnified image of the array is projected onto a wall or screen.
The present invention shows, however, that with appropriate optics
and an appropriate light source, a programmable micro-mirror array
can be used for photolithographic synthesis, and in particular for
polymer array synthesis.
[0032] The Texas Instruments DMD.TM. array consists of
640.times.480 mirrors (the VGA version) or 800.times.600 mirrors
(the super VGA (SVGA) version). Devices with more mirrors are under
development. Each mirror is 16 .mu.m.times.16 .mu.m and there are
1-.mu.m gaps between mirrors. The array is designed to be
illuminated 20 degrees off axis. Each mirror can be turned on
(tilted 10 degrees in one direction) or off (tilted 10 degrees in
the other direction). A lens (on axis) images the array onto a
target. When a micro-mirror is turned on, light reflected by the
micro-mirror passes through the lens and the image of the
micro-mirror appears bright. When a micro-mirror is turned off,
light reflected by the micro-mirror misses the lens and the image
of the micro-mirror appears dark. The array can be reconfigured by
software (i.e., every micro-mirror in the array can be turned on or
off as desired) in a fraction of a second.
[0033] An optical lithography system including a micro-mirror array
1 based spatial light modulator according to one embodiment of the
invention is shown in FIG. 1. This embodiment includes a spatial
light modulator made of a micro-mirror array 1, and arc lamp 3, and
a lens 2 to project a predetermined image pattern on a chip or
wafer (containing many chips) 4. In operation, collimated, filtered
and homogenized light 5 from the arc lamp 3 is selectively
reflected as a light beam 6 according to dynamically turned on
micro mirrors in the micro-mirror array 1 and transmitted through
lens 2 on to chip or wafer 4 as reflected light beam 8. Reflected
light from micro-mirrors that are turned off 7 is reflected in a
direction away from the lens 2 so that these areas appear dark to
the lens 2 and chip or wafer 4. Thus, the spatial light modulator,
micro-mirror array 1, modulates the direction of reflected light (6
and 7) so as to define a predetermined light image 8 projected onto
the chip or wafer 4. The direction of the reflected light alters
the light intensity transmitted from each pixel to each feature. In
essence, the spatial light modulator operates as a directional and
intensity modulator. Further, additional reflective optical
elements may be provided between the micro-mirror array and the
chip or wafer, such as the reflective mirror 122, for directing
light from a light source onto the chip or wafer, as described in
U.S. Pat. No. 5,143,854, which is incorporated by reference herein
in its entirety.
[0034] The micro-mirror array 1 can be provided by, for example,
the micro-mirror array of the Texas Instruments (TI) DMD, in
particular, the TI "SVGA DLP.TM." subsystem. The Texas Instruments
"SVGA DLP.TM." subsystem with optics may be modified for use in the
present invention. The Texas Instruments "SVGA DLP.TM." subsystem
includes a micro-mirror array (shown as micro-mirror array 1 in
FIG. 1), a light source, a color filter wheel, a projection lens,
and electronics for driving the array and interfacing to a
computer. The color filter wheel is replaced with a bandpass filter
having, for example, a bandpass wavelength of 365-410 nm
(wavelength dependent upon the type of photochemicals selected for
used in the process). For additional brightness at wavelengths of,
for example, 400-410 nm, the light source can be replaced with arc
lamp 3 and appropriate homogenizing and collimating optics. The
lens included with the device is intended for use at very large
conjugate ratios and is replaced with lens 2 or set of lenses
appropriate for imaging the micro-mirror array 1 onto chip or wafer
4 with the desired magnification. Selection of the appropriate lens
and bandpass filter is dependent on, among other things, the
requisite image size to be formed on the chip, the type of spatial
light modulator, the type of light source, and the type of
photoresist and photochemicals being used in the system and
process.
[0035] A symmetric lens system (e.g., lenses arranged by type
A-B-C-C-B-A) used at 1:1 magnification (object size is the same as
the image size) is desirable because certain aberrations
(distortion, lateral color, coma) are minimized by symmetry.
Further, a symmetric lens system results in a relatively simple
lens design because there are only half as many variables as in an
asymmetric system having the same number of surfaces. However, at
1:1 magnification the likely maximum possible chip size is 10.88
mm.times.8.16 mm with a VGA device, or 10.2 mm.times.13.6 mm with
an SVGA device. Synthesis of, for example, a standard GeneChip.RTM.
12.8 mm.times.12.8 mm chip uses an asymmetric optical system (e.g.,
a magnification of about 1.25:1 with SVGA device) or a larger
micro-mirror array (e.g. 1028.times.768 mirrors) if the mirror size
is constant. In essence, the lens magnification can be greater than
or less than 1 depending on the desired size of the chip.
[0036] In certain applications of the invention, a relatively
simple lens system, such as a back-to-back pair of achromats or
camera lens, is adequate. A particularly useful lens for some
applications of the invention is the Rodenstock (Rockford, Ill.)
Apo-Rodagon D. This lens is optimized for 1:1 imaging and gives
good performance at magnifications up to about 1.3:1. Similar
lenses may be available from other manufacturers. With such lenses,
either the Airy disk diameter or the blur circle diameter will be
rather large (maybe 10 um or larger). See Modern Optical
Engineering, 2d Edition, Smith, W. J., ed., McGraw-Hill, Inc., New
York (1990). For higher-quality synthesis, the feature size is
several times larger than the Airy disk or blur circle. Therefore,
a custom-made lens with resolution of about 1-2 um over a 12.8
mm.times.12.8 mm field is particularly desirable.
[0037] FIG. 5 schematically illustrates a preferred embodiment of a
reactor system 100 for synthesizing polymers on the prepared
substrate in accordance with one aspect of the invention. The
reactor system includes a body 102 with a cavity 104 on a surface
thereof. In preferred embodiments the cavity 104 is between about
50 and 1000 .mu.m deep with a depth of about 500 .mu.m
preferred.
[0038] The bottom of the cavity is preferably provided with an
array of ridges 106 which extend both into the plane of the Figure
and parallel to the plane of the Figure. The ridges are preferably
about 50 to 200 .mu.m deep and spaced at about 2 to 3 mm. The
purpose of the ridges is to generate turbulent flow for better
mixing. The bottom surface of the cavity is preferably light
absorbing so as to prevent reflection of impinging light.
[0039] A substrate 112 is mounted above the cavity 104. The
substrate is provided along its bottom surface 114 with a
photoremovable protective group such as NVOC with or without an
intervening linker molecule. The substrate is preferably
transparent to a wide spectrum of light, but in some embodiments is
transparent only at a wavelength at which the protective group may
be removed (such as UV in the case of NVOC). The substrate in some
embodiments is a conventional microscope glass slide or cover slip.
The substrate is preferably as thin as possible, while still
providing adequate physical support. Preferably, the substrate is
less than about 1 mm thick, more preferably less than 0.5 mm thick,
more preferably less than 0.1 mm thick, and most preferably less
than 0.05 mm thick. In alternative preferred embodiments, the
substrate is quartz or silicon.
[0040] The substrate and the body serve to seal the cavity except
for an inlet port 108 and an outlet port 110. The body and the
substrate may be mated for sealing in some embodiments with one or
more gaskets. According to a preferred embodiment, the body is
provided with two concentric gaskets and the intervening space is
held at vacuum to ensure mating of the substrate to the
gaskets.
[0041] Fluid is pumped through the inlet port into the cavity by
way of a pump 116 which may be, for example, a model No. B-120-S
made by Eldex Laboratories. Selected fluids are circulated into the
cavity by the pump, through the cavity, and out the outlet for
recirculation or disposal. The reactor may be subjected to
ultrasonic radiation and/or heated to aid in agitation in some
embodiments.
[0042] Above the substrate 112, a lens 120 is provided which may
be, for example, a 2'' 100 mm focal length fused silica lens. For
the sake of a compact system, a reflective mirror 122 may be
provided for directing light from a light source 124 onto the
substrate. Light source 124 may be, for example, a Xe(Hg) light
source manufactured by Oriel and having model No. 66024. A second
lens 126 may be provided for the purpose of projecting a mask image
onto the substrate in combination with lens 120.
[0043] Light from the light source is permitted to reach only
selected locations on the substrate as a result of mask 128. Mask
128 may be, for example, a glass slide having etched chrome
thereon. The mask 128 in one embodiment is provided with a grid of
transparent locations and opaque locations. Such masks may be
manufactured by, for example, Photo Sciences, Inc. Light passes
freely through the transparent regions of the mask, but is
reflected from or absorbed by other regions. Therefore, only
selected regions of the substrate are exposed to light.
[0044] Light valves (LCD's) may be used as an alternative to
conventional masks to selectively expose regions of the
substrate.
[0045] A preferred embodiment of synthesizing polymer arrays with a
programmable micro-mirror array using the DMT process with
photoresist takes place as follows. First, a computer file is
generated and specifies, for each photolithography step, which
mirrors in the micro-mirror array 1 need to be on and which need to
be off to generate a particular predetermined image pattern. Next,
the individual chip or the wafer from which it is made 4 is coated
with photoresist on the synthesis surface and is mounted in a
holder or flow cell (not shown) on the photolithography apparatus
so that the synthesis surface is in the plane where the image of
the micro-mirror array 1 will be formed. The photoresist may be
either positive or negative thus allowing deprotection at locations
exposed to the light or deprotection at locations not exposed to
the light, respectively (example photoresists include: negative
tone SU-8 epoxy resin (Shell Chemical) and those shown in the above
cited patents and U.S. patent application Ser. No. 08/634,053,
filed Apr. 17, 1996, now U.S. Pat. No. 5,959,098, issued Sep. 28,
1999). A mechanism for aligning and focusing the chip or wafer is
provided, such as a x-y translation stage. Then, the micro-mirror
array 1 is programmed for the appropriate configuration according
to the desired predetermined image pattern, a shutter in the arc
lamp 3 is opened, the chip or wafer 4 is illuminated for the
desired amount of time, and the shutter is closed. If a wafer
(rather than a chip) is being synthesized; a stepping-motor-driven
translation stage moves the wafer by a distance equal to the
desired center-to-center distance between chips and the shutter of
the arc lamp 3 is opened and closed again, these two steps being
repeated until each chip of the wafer has been exposed.
[0046] Next, the photoresist is developed and etched. Exposure of
the wafer 4 to acid then cleaves the DMT protecting groups from
regions of the wafer where the photoresist has been removed. The
remaining photoresist is then stripped. Then DMT-protected
nucleotides containing the desired base (adenine (A), cytosine (C),
guanine (G), or thymine (T)) are coupled to the deprotected
oligonucleotides.
[0047] Subsequently, the chip or wafer 4 is re-coated with
photoresist. The steps from mounting the photoresist coated chip or
wafer 4 in a holder through re-coating the chip or wafer 4 with
photoresist are repeated until the polymer array synthesis is
complete.
[0048] The flow cell may include ports through which reagents can
be supplied from a synthesizer, such as those described in U.S.
Pat. No. 6,136,269, which is incorporated by reference herein in
its entirety.
[0049] Methods for Mechanical Delivery of Reagents
[0050] In preferred embodiments of the present invention, reagents
are delivered to the substrate by either (1) flowing within a
channel defined on predefined regions or (2) "spotting" on
predefined regions. However, other approaches, as well as
combinations of spotting and flowing, may be employed. In each
instance, certain activated regions of the substrate are
mechanically separated from other regions when the monomer
solutions are delivered to the various reaction sites.
[0051] A typical "flow channel" method of the present invention can
generally be described as follows. Diverse polymer sequences are
synthesized at selected regions of a substrate by forming flow
channels on a surface of the substrate through which appropriate
reagents flow or in which appropriate reagents are placed. For
example, assume a monomer "A" is to be bound to the substrate in a
first group of selected regions. If necessary, all or part of the
surface of the substrate in all or a part of the selected regions
is activated for binding by, for example, flowing appropriate
reagents through all or some of the channels, or by washing the
entire substrate with appropriate reagents. After placement of a
channel block on the surface of the substrate, a reagent having the
monomer A flows through or is placed in all or some of the
channel(s). The channels provide fluid contact to the first
selected regions, thereby binding the monomer A on the substrate
directly or indirectly (via a linker) in the first selected
regions.
[0052] Thereafter, a monomer B is coupled to second selected
regions, some of which may be included among the first selected
regions. The second selected regions will be in fluid contact with
a second flow channel(s) through translation, rotation, or
replacement of the channel block on the surface of the substrate;
through opening or closing a selected valve; or through deposition
of a layer of photoresist. If necessary, a step is performed for
activating at least the second regions. Thereafter, the monomer B
is flowed through or placed in the second flow channel(s), binding
monomer B at the second selected locations. In this particular
example, the resulting sequences bound to the substrate at this
stage of processing will be, for example, A, B, and AB. The process
is repeated to form a vast array of sequences of desired length at
known locations on the substrate.
[0053] After the substrate is activated, monomer A can be flowed
through some of the channels, monomer B can be flowed through other
channels, a monomer C can be flowed through still other channels,
etc. In this manner, many or all of the reaction regions are
reacted with a monomer before the channel block must be moved or
the substrate must be washed and/or reactivated. By making use of
many or all of the available reaction regions simultaneously, the
number of washing and activation steps can be minimized.
[0054] Flow Channel Embodiments
[0055] FIGS. 6A and 6B illustrate details of a first embodiment of
a device used for performing the synthesis steps described above.
In particular, FIG. 6A illustrates the device in top view, while
FIG. 6B illustrates the device in cross-sectional side view. In the
particular embodiment shown in FIGS. 6A and 6B, the device is used
to synthesize polymer sequences on substrate 401. Substrate 401 is
coupled to a rotating stage 403 and removably held by clamp 405 to
channel block 407. Channel block 407 has etched therein a plurality
of channels 409 in the form of stripes therein. Each channel is
provided with a flow inlet 411 and an outlet 413. A vacuum source
415 is applied to one or more of the outlets 413, while a pipettor
417 is slidably mounted on arm 419 to deliver selected reagents
from reservoir(s) 421 to selected flow inlets 411.
[0056] As shown in FIG. 7, the fluid delivery ports are accessed
from holes in the back surface of a stabilizing plate 108 on the
channel block. The stabilizing plate, which is preferably made from
fused pyrex, provides structural integrity to the channel block
during clamping in the pressure chamber. It may also provide a
means to access the channel block ports and reduce leakage between
ports or channels. In preferred embodiments, the channels 123 of
the channel block are formed on a wafer 106 which generally may be
any machinable or cast material, and preferably may be etched
silicon or a micromachined ceramic. In other embodiments, the
channel block is pressure-formed or injection-molded from a
suitable polymer material. The entire channel block arrangement is
mounted on a rigid channel block sub-plate 110 including a vacuum
line 112, ports for fluid delivery lines 115, ports for fluid
outlet lines 117, and recessed regions for plug ends 151 and 153.
With this arrangement, the substrate can be clamped against the top
surface of the channel block while fluid enters and exits from
below.
[0057] FIG. 8 shows a fluid flow diagram of a preferred system of
the present invention. The pressure is controlled at point 25 (P1)
and point 21 (P2) so that a pressure drop (P1-P2) is maintained
across the system. Coupling compounds such as activated monomers
are supplied from reservoirs 31, 32, and 33. Additional reagents
are supplied from reservoirs 15, 17, and 19. Of course, the monomer
and coupling reagent reservoirs shown in FIG. 8 are representative
of a potentially much larger series of reservoirs. The reagents and
coupling compounds are combined at nodes 27, 28, and 29 before
being directed to channel block 139. Mixing of the appropriate
reagents and coupling compounds is controlled by valves at the
nodes which are in turn controlled by electronic control 23. Waste
fluids that have been directed across the substrate are removed
through line 35.
[0058] The system displayed in FIG. 8 allows control of all
channels in parallel by regulating only a few variables. For
example, a constant pressure gradient is maintained across all
channels simultaneously by fixing P1 and P2. Thus, the flow rate in
each channel is dependent upon the cross-sectional area of the flow
channel and the rheological properties of the fluids. Because the
channels have a uniform cross-section and because the coupling
compounds are typically provided as dilute solutions of a single
solvent, a uniform flow rate is created across all channels. With
this system the coupling time in all channels can be varied
simultaneously by simply adjusting the pressure gradient across the
system. The valves of the system are preferably controlled by a
single electronic output from control 23.
[0059] Spotting Embodiments
[0060] According to some embodiments, monomers (or other reactants)
are deposited from a dispenser in droplets that fill predefined
regions. For example, in a single coupling step, the dispenser
deposits a first monomer in a series of predefined regions by
moving over a first region, dispensing a droplet, moving to a
second region, dispensing a droplet, and so on until the each of
the selected regions has received the monomer. Next the dispenser
deposits a second monomer in a second series of predefined regions
in much the same manner. In some embodiments, more than one
dispenser may be used so that more than one monomer are
simultaneously deposited. The monomers may react immediately on
contact with the reaction regions or may require a further
activation step, such as the addition of catalyst. After some
number of monomers have been deposited and reacted in predefined
regions throughout the substrate, the unreacted monomer solution is
removed from the substrate.
[0061] It is worth noting that if a DPD method, using for example
1-(6-nitro-1,3-benzodioxol-5-yl)ethyloxycarbonyl (MeNPOC)
chemistry, or a PAG method, using a polymer film containing a
photoacid generator (PAG), are used for polymer array synthesis
then photoresist would not be used and the process is somewhat
simplified. However, the use of a direct write optical Lithography
system with a spatial light modulator is also applicable to
performing a process of deprotection of reaction sites using the
DPD and PAG methods without photoresist.
[0062] As is clear from the above described method for polymer
array synthesis, no photomasks are needed. This simplifies the
process by eliminating processing time associated with changing
masks in the optical lithography system and reduces the
manufacturing cost for polymer array synthesis by eliminating the
cost of the masks as well as processing defects associated with
using masks. In addition, the process has improved flexibility
because reprogramming the optical lithography system to produce a
different generate and verify new photomasks, thus making it
possible to transfer an image pattern computer file directly from a
CAD or similar system to the optical lithography system or
providing electronic signals directly from the CAD system to drive
the optical lithography system's means for dynamically producing
the desired light pattern (e.g., spatial light modulator).
Therefore, the optical lithography system is simplified and more
efficient than conventional photomask based optical lithography
systems. This is particularly valuable in complex multiple step
photolithography processing; for example polymer array synthesis of
GeneChip.RTM. probe arrays having upwards of seventy or more
cycles, especially when many different products are made and
revised
[0063] As indicated above, substrates coated with photoresist are
employed in preferred embodiments of the invention, e.g., using the
DMT process with photoresist. The use of photoresist with
photolithographic methods for fabricating polymer arrays is
discussed in McGall et al., Chemtech, pp. 22-32 (February 1997);
McGall et al., Proc. Natl. Acad. Sci., U.S.A., Vol. 93, pp.
13555-13560 (November 1996) and various patents cited above, all of
which are incorporated by reference in their entireties.
Alternatively, polymer array synthesis processing can be performed
using photoacid generators without using a conventional
photoresist, e.g. using the PAG process, or using direct
photodeprotection without using any photoresist, e.g., using the
DPD process. The use of photoacid generators is taught in U.S.
application Ser. No. 08/969,227, filed Nov. 13, 1997, now U.S. Pat.
No. 6,083,697, issued Jul. 4, 2000. However, the present invention
is particularly useful when using the DMT and PAG processes for
polymer array synthesis.
[0064] When synthesizing nucleic acid arrays, the photochemical
processes used to fabricate the arrays is preferably activated with
light having a wavelength greater than 365 nm to avoid
photochemical degradation of the polynucleotides used to create the
polymer arrays. Other wavelengths may be desirable for other
probes. Many photoacid generators (PAGs) based on o-nitrobenzyl
chemistry are useful at 365 nm. Further, when using the mirror
array from Texas Instruments discussed above, the PAG is preferably
sensitive above 400 nm to avoid damage to the mirror array. To
achieve this, p-nitrobenzyl esters can be used in conjunction with
a photosensitizer. For example, p-nitrobenzyltosylate and
2-ethyl-9,10-dimethoxy-anthracene can be used to photochemically
generate toluenesulfonic acid at 405 nm. See S. C. Busman and J. E.
Trend, J. Imag. Technol., 1985, 11, 191; A. Nishida, T. Hamada, and
O. Yonemitsu, J. Org. Chem., 1988, 53, 3386. In this system, the
sensitizer absorbs the light and then transfers the energy to the
p-nitrobenzyltosylate, causing dissociation and the subsequent
release of toluensulfonic acid. Alternate sensitizers, such as
pyrene, N,N-dimethylnapthylamine, perylene, phenothiazine,
canthone, thiocanthone, actophenone, and benzophenone that absorb
light at other wavelengths are also useful.
[0065] A variety of photoresists sensitive to 436-nm light are
available for use in polymer array synthesis and will avoid
photochemical degradation of the polynucleotides.
[0066] A second preferred mechanical modulator that may be used in
the invention is the Grating Light Valve.TM. (GLV.TM.) available
from Silicon LightMachines, Sunnyvale, Calif., USA. The GLV.TM.
relies on micromachined pixels that can be programmed to be either
reflective or diffractive (Grating Light Valve.TM. technology).
Information regarding certain of the mechanical modulators
discussed herein is obtained at http://www.ti.com (Texas
instruments) and http://siliconlight.com. (Silicon
LightMachines).
[0067] Although preferred spatial light modulators include the
mechanical modulators DMD.TM. available from Texas Instruments and
the GLV.TM. available from Silicon LightMachines, various types of
spatial light modulators exist and may be used in the practice of
the present invention. See Electronic Engineers' Handbook, 3.sup.rd
Ed., Fink, D. G. and Christiansen, D. Eds., McGraw-Hill Book Co.,
New York (1989). Deformable membrane mirror-arrays are available
from Optron Systems Inc. (Bedford, Mass.). Liquid-crystal spatial
light modulators are available from Hamamatsu (Bridgewater, N.J.),
Spatialight (Novato, Calif.), and other companies. However, one
skilled in the art must be careful to select the proper light
source and processing chemistries to ensure that the liquid-crystal
spatial light modulator is not damaged since these devices may be
susceptible to damage by various ultraviolet (UV) light.
Liquid-crystal displays (LCD; e.g., in calculators and notebook
computers) are also spatial light modulators useful for
photolithography particularly to synthesize large features.
However, reduction optics would be required to synthesize smaller
features using LCDs.
[0068] Some spatial light modulators may be better suited than the
Texas Instruments device for use with UV light and would therefore
be compatible with a wider range of photoresist chemistries. One
skilled in the art will choose the spatial modulator that is
compatible with the chosen wavelength of illumination and synthesis
chemistries employed. For example, the device from Texas
Instruments DMD.TM. should not be used with UV illumination because
its micro-mirror array may be damaged by UV light. However, if the
passivation layer of the micro-mirror array is modified or removed,
the Texas Instruments DMD.TM. could be used in the invention with
UV light.
[0069] One embodiment that is particularly useful when extremely
high resolution is required involves imaging the micro-mirror array
using a system of the type shown in FIG. 2. In this system, a lens
12 images the micro-mirror array 11 (e.g., DMD.TM. or GLV.TM.) onto
an array 10 having an array of micro-lenses 15 or non-imaging light
concentrators. Each element of the array 10 focuses light onto the
chip or wafer, e.g., Gene Chip array 14. Each micro-lens 15
produces an image of one pixel of the micro-mirror array 11. Optics
16, including a shaping lens 17 may be included to translate light
from a light source 13 onto the micro-mirror array 11.
[0070] For example, if an SVGA DLP.TM. device is imaged with 1:1
magnification onto a micro-lens array 10, an appropriate micro-lens
array 10 can consist of 800.times.600 lenses (micro-lenses 15) with
17 .mu.m center-to-center spacing. Alternatively, the micro-lens
array can consist of 400.times.300 17 .mu.m diameter lenses with 34
.mu.m center-to-center spacing, and with opaque material (e.g.,
chrome) between micro-lenses 15. One advantage of this alternative
is that cross-talk between pixels is reduced. The light incident
upon each micro lens 15 can be focused to a spot size of 1-2 .mu.m.
Because the spot size is much less than the spacing between
micro-lenses, a 2-axis translation stage (having, in these
examples, a range of travel of at least either 17 .mu.m.times.17
.mu.m or 34 .mu.m.times.34 .mu.m) is necessary if complete coverage
of the chip or wafer 14 is desired.
[0071] Micro-lenses 15 can be diffractive, refractive, or hybrid
(diffractive and refractive). Refractive micro-lenses can be
conventional or gradient-index. A portion of a diffractive
micro-lens array 10 is shown in FIG. 3 and has individual
micro-lenses formed as circles commonly known as Fresnel Zone
Plates 20. Alternatively an array of non-imaging light
concentrators can be employed. An example of such an approach would
include a short piece of optical fiber which may be tapered to a
small tip.
[0072] Furthermore, some spatial light modulators are designed to
modulate transmitted rather than reflected light. An example of a
transmissive spatial light modulator is a liquid crystal display
(LCD) and is illustrated in another embodiment, shown in FIG. 4.
This embodiment includes a light source 33 providing light 35,
transmissive spatial light modulator 31 and a computer 39 providing
electronic control signals to the transmissive spatial light
modulator 31 through cables 40 so as to transmit a desired light
image 38 on the chip or wafer 34. The computer 39 may be, for
example, a unique programmable controller, a personal computer
(PC), or a CAD system used to design the desired image pattern.
[0073] Using a transmissive spatial light modulator has even
additional advantages over the conventional optical lithography
system. Reflective spatial light modulators require a large working
distance between the modulator and the lens so that the lens does
not block the incident light. Designing a high performance lens
with a large working distance is more difficult than designing a
lens of equivalent performance with no constraints on the working
distance. With a transmissive spatial light modulator the working
distance does not have to be long and lens design is therefore
easier. In fact, as show in FIG. 4, some transmissive spatial light
modulators 31 might be useful for proximity or contact printing
with no lens at all, by locating the modulator very close to the
chip or wafer 34.
[0074] In fact, the transmissive spatial light modulator in the
embodiment of FIG. 4 could be replaced by an LED array or a
semiconductor laser arrays emitting light of the appropriate
wavelength, each of which not only may be operated to dynamically
define a desired image but also act as the light source. Thus, as
modified, this embodiment would be simplified so as to not require
a separate light source.
[0075] Although discussed herein in reference to polymer array
synthesis, one skilled in the art will appreciate that the present
invention has a variety of applications including, among others,
silicon micromachining and custom semiconductor chip manufacturing.
However, use of some types of spatial light modulators with the
invention may result in limiting the types of geometries available
in silicon micromachining and custom semiconductor chip
manufacturing applications. It is understood that the examples and
embodiments described herein are for illustrative purposes only and
that various modifications or changes in light thereof will be
suggested to persons skilled in the art and are to be included
within the spirit and purview of this application and scope of the
appended claims.
[0076] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes application Ser. No. 08/426,202 (filed Apr. 21, 1995),
now U.S. Pat. No. 6,136,269, issued Oct. 24, 2000, relates to the
present invention and is hereby incorporated by reference for all
purposes.
* * * * *
References