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Publication numberUS20060292628 A1
Publication typeApplication
Application numberUS 11/513,126
Publication dateDec 28, 2006
Filing dateAug 31, 2006
Priority dateMay 29, 1998
Also published asCA2273387A1, DE69934631D1, DE69934631T2, EP0961174A2, EP0961174A3, EP0961174B1, US6271957, US6480324, US20010028495, US20030002130, US20050088722, US20050233256
Publication number11513126, 513126, US 2006/0292628 A1, US 2006/292628 A1, US 20060292628 A1, US 20060292628A1, US 2006292628 A1, US 2006292628A1, US-A1-20060292628, US-A1-2006292628, US2006/0292628A1, US2006/292628A1, US20060292628 A1, US20060292628A1, US2006292628 A1, US2006292628A1
InventorsCalvin Quate
Original AssigneeAffymetrix, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Compositions and methods involving direct write optical lithography
US 20060292628 A1
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.
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Claims(9)
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

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

1. Technical Field of the Invention

This invention relates to optical lithography and more particularly to direct write optical lithography.

2. Description of the Related Art

Polymer arrays, such as the GeneChip® 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).

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.

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.

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

In view of the above, one advantage of the invention is providing an improved and simplified system and method for optical lithography.

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.

A further advantage of the present invention is providing an optical lithography system and method that does not use photomasks.

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.

According to one aspect of the invention, polymer array synthesis is performed using a system without photomasks.

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.

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.

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.

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.

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

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:

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.

FIG. 2 is a diagrammatic representation of a second embodiment of the invention employing an array of, for example, micro-lenses.

FIG. 3 illustrates a micro-lens array in the form of Fresnel Zone Plates, which may be used in the invention.

FIG. 4 shows a third embodiment of the invention having a transmissive spatial light modulator.

FIG. 5 illustrates a reactor system for forming a plurality of polymers on a substrate.

FIG. 6A is a top view and FIG. 6B is a cross-sectional view of a device used to synthesize arrays of polymer sequences.

FIG. 7 is a cross-sectional view of a channel block and associated flow ports.

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

The present invention refers to articles and patents that contain useful supplementary information. These references are hereby incorporated by reference in their entireties.

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.

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.

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™) 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.

The Texas Instruments DMD™ array consists of 640×480 mirrors (the VGA version) or 800×600 mirrors (the super VGA (SVGA) version). Devices with more mirrors are under development. Each mirror is 16 μm×16 μm and there are 1-μm 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.

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.

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™” subsystem. The Texas Instruments “SVGA DLP™” subsystem with optics may be modified for use in the present invention. The Texas Instruments “SVGA DLP™” 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.

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×8.16 mm with a VGA device, or 10.2 mm×13.6 mm with an SVGA device. Synthesis of, for example, a standard GeneChip® 12.8 mm×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. 1028×768 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.

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×12.8 mm field is particularly desirable.

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 μm deep with a depth of about 500 μm preferred.

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 μm 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.

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.

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.

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.

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.

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.

Light valves (LCD's) may be used as an alternative to conventional masks to selectively expose regions of the substrate.

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.

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.

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.

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.

Methods for Mechanical Delivery of Reagents

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.

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.

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.

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.

Flow Channel Embodiments

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.

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.

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.

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.

Spotting Embodiments

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.

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.

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® probe arrays having upwards of seventy or more cycles, especially when many different products are made and revised

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.

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.

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.

A second preferred mechanical modulator that may be used in the invention is the Grating Light Valve™ (GLV™) available from Silicon LightMachines, Sunnyvale, Calif., USA. The GLV™ relies on micromachined pixels that can be programmed to be either reflective or diffractive (Grating Light Valve™ 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).

Although preferred spatial light modulators include the mechanical modulators DMD™ available from Texas Instruments and the GLV™ 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, 3rd 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.

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™ 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™ could be used in the invention with UV light.

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™ or GLV™) 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.

For example, if an SVGA DLP™ device is imaged with 1:1 magnification onto a micro-lens array 10, an appropriate micro-lens array 10 can consist of 800×600 lenses (micro-lenses 15) with 17 μm center-to-center spacing. Alternatively, the micro-lens array can consist of 400×300 17 μm diameter lenses with 34 μm 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 μm. 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 μm×17 μm or 34 μm×34 μm) is necessary if complete coverage of the chip or wafer 14 is desired.

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.

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.

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.

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.

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.

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.

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US7910312Jul 16, 2010Mar 22, 2011Affymetrix, Inc.Use of acid scavengers for the synthesis of standard length and long-mer polymer arrays
US8062844Jan 21, 2011Nov 22, 2011Affymetrix, Inc.Use of acid scavengers for the synthesis of standard length and long-mer nucleic acid arrays
US8318427Sep 21, 2011Nov 27, 2012Affymetrix, Inc.Use of acid scavengers for the synthesis of standard length and long-mer nucleic acid arrays