CA2391491A1 - Very large scale immobilized peptide synthesis - Google Patents

Abstract

A method and device for preparing desired sequences on a substrate at known locations. Known locations (10) of a substrate (2) are irradiated by way of a mask (8) so as to activate a material (4) for binding. The substrate is then exposed to a first material (12) for binding thereto. Second locations (14) are then irradiated through a mask and exposed to a second material (16). A variety of sequences may be formed through selective irradiation of the substrate followed by application of selected materials. A reactor system and fluorescence detection system are also dis-closed.

Description

1' VERY LARGE SCALE IMMOBILIZED PEPTIDE SYNTHESIS
COPYRIGHT NOTICE
A portion of the disclosure of this patent document contains material which is subject to copyright protection.
The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in'the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
This application is a divisional application of Canadian Application No: 2,054;70 filed on June 7, 1990. A second divisional application, Canadian Application No. 2;278,883 was filed on August 17, 1999.
BACKGROUND OF THE INVENTION
The present invention relates to the synthesis and replacement materials at known locations. In particular, one embodiment of the invention provides a method and associated apparatus for preparing diverse chemical sequences at known locations on a single substrate surface. The inventions may be applied, for example, in the field of preparation of oligomer, peptide, nucleic acid, oligosaechari.de, phospholipid, polymer, or drug congener preparation; especially to create sources of chemical diversity for use in screening for biological activity.

'la The relationship betwEen structure and activity of molecules is a fundamental issue in the study of biological systems. Structure-activity relationships are important in understanding, for example; the function of enzymes, the ways in which cells communicate with each other, as well as cellular control and feedback systems.
Certain macromolecules are known to interact and bind to other molecules having a very specific three-dimensional spatial and electronic distribution. Any large molecule having such specificity can be considered a receptor, whether it is an enzyme catalyzing hydrolysis of a metabolic intermediate, a cell-surface protein mediating membrane transport of ions, a glycoprotein serving to identify a particular cell to its neighbors, an IgG-class antibody circslat:ng in the plasma, an alidcnucleotide saque.~.ce of DNA in the nucleus, o~ t:ne like. The various molecules whicz receptors.seect.ivelv bind are known as ligands.
Many assays are available for measuring the binding affinity of known receators and ligands, but the information which can be gained from such exper~.men~s is often limited by the number and type of ligancs which ale available. Novel ligands are sometimes discovered by c=Dance or by application of new technirues for t:~e elucidation o' molecular structure, incjuding x-ray crystallographic analysis and recombinant gene~.ic tec hricues f or pr o rains Small peptides are an exemplary syste:.~. ~cr explcr ing the relationship between s tructure and f~~.~.c ~:cn in bio.icgy: A peptide is a sza_uence of amino acids.
When the twenty naturally occurring amino acids are condensed into polymeric molecules they form a wide variety of three-dimensional configurations, each resulting from a particular amino acid sequence and solvent condition. The number of possible pentapeptides of the 20 naturally occurring amino acids, for example, is 20' or 3.2_million;different peptides. The likelihood than molecules of this size might be useful in recep'or-binding studies is supported by epitope analysis s.udies showing that some antibodies recognize sequences as shcr~
as a few amino acids with high speci~ici y. Furthe:.-.~,iore, the average molecular'weight of amino acids puts s:aall peptides in the size range of many currently useful pharmaceutical products.
Phar:uaceutical drug discavery is one type of research which relies on such a study of structure-acti~rity relationships. In most cases, contemporary pharmaceutical research can be desc:ibed as the process of discovering novel ligands with desirable patterns c~
specificity for biologica2ly important receptors.

Another example is research to niscover new compouncs for use in agriculture, such :as pesticides and herbicides.
Sometimes; the solution to a rational process of designing ligands is difficult or unyielding. .Prior methods of preparing large numbers of different polymers have been painstaking3y slow when used 'at a scale sufficient to permit effective rational or random screening. For example, the "Merrifi~ld" method (J. Ar,.
them. Soc. (2963) 85:2149-2154 has been used to synthesize peptides on a solid support. In the Merrifield method, an amino acid is covalently bonded to a support made of ~n insoluble polymer. Another amino acid with an alpha protected group is reacted wzth to covalently banded amino acid to for: a dipeptide. A°tew washing, the protective group '1s removed and a thin;
amino acid with an alpha protective group is added to t::e dipeptide. This process is continued until a peptide c~
a desired length and sequence is obtained. Using the Merrifield method, it is not economically practical ~o synthesize more than a handful of peptide seguences in a day.
To synthesize larger numbers of polymer sequences, it has also been prayosed to use a series o~
reaction vessels for polymer synthesis. For example, a tubular reactor syste~a'may be used to synthesize a linear polymer on a solid phase support by automated sec_uer.tia' addition of reagents. This method still does not enable the synthesis of a sufficiently large number of polymer sequences for effective economical screening.
Methods of preparing a plurality of polyner sequences are also known 2n which a foraminous container encloses a known quantity of reactive particles, the particles being larger in size than foramina of the 3c container. The contyiners may be selectively reacted with desired materials to synthesize desired sequences c~
product molecules. As with other'methods known in the art, this method cannot practically be used to synthesize a sufficient variety of polypeptides for effective screening.
Other techniques have also been described.
These methods include the synthesis of peptides on 96 plastic pins which fit the Format of standard microtiter plates. Unfortunately, while these tzchnicues have been somewhat useful, substantia3 problems remain. ror example, these methods continue to be limited in the I0 diversity of sequences which can be economically synthesized and screened:
From the above; it is seen that an improved method and apparatus for synthesizing a variety of chemical sequences at known locations is desired.
SUI~B~iA,RY OF THE INVENTION
An improved method and apparatus for the preparation of a variety of polymers is disclosed.
In one preferred embodiment, linker molecules are provided on a substrate. A fierminal end of the linker molecules is provided with a reactive functional group protected with a photoremovable protective grcup.
Using lithographic methbds, the photoremovable protective group is exposed to light and removed from the linker molecules in first selected regions. The substrate is then washed or otherwise contacted with a first monomer that reacts with exposed functional groups on the linker molecules. In a preferred embodiment, the monomer is an amiwo acid containing a photoremovable protective group at its amino or carboxy terminus and the linker molecule terminates in an amino or carboxy acid group bearing a photoremovable protective group.
A second set of selected regions is, thereafter, exposed to light and the photoremovable ;5 protective group on the linker molecule/protected aui~to acid is removed at the second set of regions. The substrate is then contacted with a second monomer containing a photpremovable protective group for raacticn with exposed functional groups: This process is rape3ted to selective)y app3y menomers until polymers of a desired length and desired chemical sequence are obtained..
Photolabile groups are then optionally removed and the sequence is, thereafter, optionally capped. Side chain protective graups, if present; are also removed.
By using the!lithographic technigues disclosed herein, it is possible to direct light to relatively small and precisely known locations on the substrate.
2t is, therefore, possible to synthesize polymers of a known chemical sequence at known.locations on the substrate.
The resu2ting substrate w'xll have a variety of uses incyuding; for example, screening large numi~ers of polymers for biological activity, To screen for biological activity, the substrate is exposed to one or more receptors such; as antibody whole cells, receptors on vesicles, lipids, or any one of a variety of other receptors. The receptors are preferably labeled with, for example, a fluorescent marker; radioactive marker, or a labeled antibody reactive with the receptor. The location of the marker on the substrate is.detected with, for example, photon detection or autoradiogranric techniques. Through knowledge of the sequence of the material at the location where binding is detected, it is possible to quickly determine which sequence binds with the receptor and, therefore; the technique can be used to screen large numbers of peptides. Other possible applications of the inventions herein include-diagnos;.ics in which various antibodies for particular receptors would be placed on a substrate and, for example, blood sera would be screened fo= immune deficiencies. Still further applications include, for example, selective "doping" of organic materia3s in semiconductor devices, and the like:

In connection with one aspect of the invention an improved reactor system,for synthesizing polymers is also dis-closed. The reactor system ine3udes a substrata mount which engages a sub trate around a periphery thereof. The substrate mount provides for a reactor space between the substrate and the mount through or into which reaction fluids are ptuaped or flowed. A mask is placed on or focused on~the substrate and illuminated so as to deprotect selected regions of the substrate in the reactor space. A monomer is pumped through the reactor space or otherwise contacted with the substrate and reacts with the deprotected regions., Hy se3.ectivel.y deprotecting regions on the substrate and flowing predeter~ained monomers through the reactor space, desired polymers at known locations may be synthesized.
Imprcved detection apparatus and methods are also disclosed. The detection method and apparatus utilize a substrate having a large variety of potymer sequences at known locations on a surface thereof. The substrate is exposed to a fluorescently labeled receptor which binds o one or more of the polymer sequences. The substrate is placed in a microscope detection apparatus for identification of locations where binding takes place. The mxcroscope;detection apparatus includes a monochromatic or polychromatic light source for directing light at the substrate, means for detecting fluoresced light from the substrate, and means for determining a location of the fluoresced light. The means for detecting light f3uoresced on the substrate may in some embodiments include a photon counter. The means for determining a .location of thefluoresced light may include an x/y translat~:on table for the substrate.
Translation of the slide and data coT3ection are recorded and managed by an appropriately programmed digital computer.

Therefore, in accordance with the invention of the present divisional application there is provided apparatus for detection of fluoreseently marked regions on a substrate comprising: a) a substrate bearing a plurality of different polymer sequences coupled to a surface of said substrate, each of said different polymer sequences being coupled in a different known location of said surface, each of said known location having an area of 102 em2 or less; b) a light source for directing light at said surface of said substrate; c) a means for deteetW g light fluoresced from a fluorescent label bound to the polymer sequences on said surface in response to said light source; d) means for translating said substrate from a first position to a second position relative to said'light source and/or said means for detecting light; and e) means for storing fluoresced light intensity as a function of location on said substrate, said means for storing connected to said means for translating and said means for detecting.
The invention also provides a system for determining binding of a fluorescently labeled receptor to a ligand comprising: a) a substrate bearing a plurality of different polymer sequences 'coupled to a surface of said substrate, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of l0-2 cm2 or less b) means for applying light to said surface of said substrate, said means for;appl.ying light providing simultaneous illumination at a plurality of said known locations; and c) an array of detectors for detecting light fluoresced at said plurality of known locations upon binding of a fluorescently labeled receptor to said polymer sequences.

7a The invention also provides an apparatus for detection of fluorescently marked locations on a substrate comprising: a) a substrate bearing a plurality of different polymer sequences coupled to a surface of said substrate, wherein said plurality of different polymer sequences comprises a plurality of :different nucleic acid sequences, each of said different polymer sequence being coupled in a different known location 'of said surface, each of said known locations having an area of 10~Z cm2 or less; b) a light source for directing, light at a surface of said substrate;
c) a detector for detecting light fluoresced from a fluorescent label bound to the polymer sequences on said surface in response to said light source; d) a translator for translating said substrate relative to said light source; and e) a data storage system for storing fluoresced light intensity as a function of location on said substrate, said data storage system connected to said translator and said detector.
The invention ai.so,provides an apparatus for detection of fluorescently marked locations on a surface of a substrate, comprising: °a point light source for generating an excitation light; a substrate bearing a plurality of different polymers coupled to a surface of said substrate, wherein said plurality of different polymers comprises a plurality of different nucleic acid sequences, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less; an objective lens for focusing said point light source at said surface of said substrate, whereby locations, upon binding of a fluorescent label to the polymer sequences coupled therein, emit a fluoresced light in response to said excitation light; an x-y translation stage for moving said substrate 7b relative to said excitation light,~ a dichroic mirror for reflecting light having a wavelength of said excitation light and passing light having a wavelength of said fluoresced light; a photomultiplier and photon counter for detecting said fluoresced light; and an appropriately programmed computer for recordingsaid fluoresced light as a function of a position on said surface of said substrate from which said fluoresced light was emitted.
The invention also provides a method of detecting the presence of a fluorescent marker on a surface of a substrate, the method comprising: directing an excitation light at the surface of the substrate; and detecting light fluoresced from the surface of the substrate; wherein said surface of said substrate!bears a plurality of different nucleic acids covalently bound thereto, each of said different nucleic acids being bound at a different known location on the substrate; each of said known locations having an area of less than l0-2 cm2 and said fluorescent marker comprises a fluorescently labeled target nucleic acid that is capable of hybridizing with'one or mare of said plurality of different nucleic acids.
The invention also provides a method of determining whether a fluorescently labeled ligand binds to one or more of a plurality of different polymer sequences, wherein said fluorescently labeled ligand comprises a fluorescently labeled target nucleic acid, and said plurality of different polymer sequences on the surface of a substrate comprises a plurality of different nucleic acids, comprising: providing a plurality of different polymer sequences covalently bound to a surface of a substrate, each of said different polymer sequences being bound at a known location on the surface of the substrate, each of said known locations having an area of less than 10-2 cm2; contacting the surface of the substrate with the fluorescently labeled ligand; washing the surface to remove unbound fluorescently labeled ligand from the surface of the substrate; and detecting binding between the fluorescently labeled ligand and the one or more polymer sequences, said detecting step comprising directing an excitation light at the surface of the substrate, and detecting light fluoresced from the surface of the substrate:
The invention also provides a nucleic acid analysis apparatus comprising: a substrate bearing a plurality of different: nucleic acids; each of said different nucleic acids being attached to a different known location of the surface of said substrate, each of said known locations having an area of l0-2 cm2 or less, said substrate comprising more than l0 of such nucleic acids, at least some of said nucleic acids coupled to fiuorescently labeled target molecules; a light source for directing light at a surface of said substrates a detector for detecting light fluoresced from said surface in response to said light source; a translator for -translating said substrate relative to said light source; and a data storage system for storing fluoresced light intensity as a function of location on said substrate, said data storage system coupled to said translator and said detector.
The invention also provides an apparatus for detection of labeled locations on a substrate comprising:
a) a light source capable!of directing light at a surface of a substrate, said subs rate bearing a plurality of different polymer sequences attached to a surface of said substrate, each of said different polymer sequences occupying a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less, and at least one of the polymers capable of being coupled to a labeled 7d receptor; b) a detector to detect light emitted from said surface in response to said light source to indicate which polymers) are coupled to the labeled receptor.
The invention also provides a method of detecting the presence of a fluorescent marker on a surface of a substrate, the method comprising: (1) directing an excitation light at a surface of a substrate, said substrate bearing a plurality of different polymer sequences attached to a surface of said substrate, each of said different polymer sequences- occupying a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less, and at least one of the polymers capable of being coupled to a fluorescently labeled receptor; and (2) detecting light fluoresced from the surface of the substrate.
Our copending Canadian Application No. 2,278,883, also divided out of parent'Application No. 2,054,706 provides a reactor for facilitating reaction of chemical compounds, said reactor comprising: a body having a sealed cavity therein, said cavity being less than about 1,000 um deep; and an inlet port and an outlet port, said inlet pbrt and said outlet port being in fluid communication with said cavity for flowing fluid comprising nucleosides into and through said cavity.
The parent application, Canadian Application No.

2,054,706, provides a method of forming an array of diverse polymers on a substrate, a surface of said substrate comprising at least first and second known locations having polymer molecules thereon, said polymer molecules comprising a protective group at an active site, said method comprising the iC
steps of: removing said:protective group from polymer molecules in said first known location of said substrate to expose said active site, but not rerciosring said protect.i:ve group from polymer molecules in said second known location; exposing said first and second known locations of said surface to first selected monomer molecules, to couple said first selected monomer molecules in said firs known location but not said second known location, said first selected monomer molecules comprising a protective group at an active site; removing a protective group from at least a portion of said first selected monomer molecules in said first known location to expose an active site on said at least a portion of said first selected monomer molecules; and exposing said first and said second known location to second selected monomer molecules, forming polymer molecules in said first known location having a different monomer sequence than monomers in said second known location.
Furthermore, another embodiment of the invention of the parent application provides an apparatus for investigating by receptorlligand binding a nucleotide sequence, which apparatus .., comprises a substrate with a surface, said surface comprising at least 103 known locations, said known locations containing different polynucleotide sequences thereon, said known locations each occupying an area of less than 2.5 x 10'3 cm2.

Ir A further understanding of the nature and advantages of the inventions of the divisional applications and the parent application herein may be realized by reference to the remaining portions of t:~e specification and the attached S drawings.
BRIEF DESCRIPTION OF THE FTGURES
Fig. 1 illustrates masking and irradiation of a substrate at a first location. The substrate is shown in cross-sect~.an;
Fig. 2 illustrates t:3e substrate after application of a monomer "A" ;
Fig. 3 illustrates irradiation of the substrates at a second location;
Fig. 9 illustrates trie substrate after application of monomer "B" ;
Fig. 5 illustrates i:r'radiation of the "A" monomer;
Fig. 6 illustrates the substrate after a second application of "B" ;
Fig. ~ illustrates a completed substrate;
Figs. 8A end 8B illustrate alternative embodiments of a reactor system for forming;a glurality of polymers on a substrate;
Fig. 9 illustrates a detection apparatus for locating fluorescent markers on thz substrate;
Figs: l0A-lOM illustrates the method as it is applied to the production of the trimers of monomers "A" and "B":

Figs. 11A, 11S and I~C arp fluorescence traces fcr standard fluorescent beads;
Figs. 12A and 12B are fluorescence curves for NVOC slides not exposed and erposed to light respectively;
E"igs. 13A and 13B illustrate formation of a slide with a checkerboard pattern of Y~GFL and GGFL exposed to labeled Herz antibody; and $ .
Figs. 14A and 14B illustrate the mapping of szxteen sec_uences synthesized on two different glass slides.
DE'_T'AILED DESCRIPTION OF THE P:~.EFERRED EMBODIi~NTS
GONTEH'TS
I. Glossary II. General la III. Polymer Synthesis ZV. Details of One Embodiment of a Reactor System V: Details of One Embodiment of a Fluorescent Detection Device 13 VI. Determination of Relative Binding Strength of Receptors VII. Examples A. Slide Preparation 20 B. synthesis of Eight Trimers of ~~Au and ~gf~

C: Synthesis of a Dimer of an Aminopropyl Group and a Fluorescent Group D. emonstration of Signal Capabzlzty E. Determination of the Number of Molecules Per Unit Area F. Removal of NVOC and Attachment of a Fluorescent Marker 30 G: U.se of a Mask in Removal of NVOC

H: Attachment of YGGFL and Subsequent Exposure to Here Antibody and Goat Apt in~ous a I. Monomer-by-Monomer Formation of YGGFL and Sub equent Exposure to 'S Labeled Antibody CONTENTS
(Coat'd) J. Monomerby-Monomer Synthesis of YGGiL and PGGFL
K. Monomer-by Monomer Synthesis of YGGFI~ and YPGGFL
L. Synthesis of an Array of Sixteen Different Amino Acid Sequences and Estimation of Relative Binding Affiniay to Herz Antibody 1~ VII2.. Illustrative Alaernative Embodiment Ix. Conclusion I. Glossa~-v The following terns are intended to nave the following general meanings as they are used herein':
1. Complementary: Refers to the Lopvlogical compatibility or matching together of interacting surfaces of a ligand molecule and its receptor.
Thus, the receptor and its ligand can be described as complementary; and furthermore, the contact surface characteristics are complementary to each other.
'r 2. ypitone: The portion of an antigen molecule which is delineated by~the area of interaction with the subclass of receptors-known as antibodies.

3. ' and: A ligand is a molecule that is recocnized by a particular receptor: Examples of ligands that can be investigated by this invention include, but are not-restricted to, agon'2sts and antagonists fog cell meanbrane receptors, toxins and venoms, viral epitopes, hornones (e. g., opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme Substrates, cofactors, drltgs, lectins, sugars, oligonucleotides, Nucleic acids, oligosaccharides, proteins, and monocianal antibodies.

4. ~ionomer~. A member of the set of small molecules which can be joined together to form a polymer. The set of monomers includes but is not restricted to, .
far example, the set of common L-amino acids, the set of D-amino acids,. the set of synthetic amino acids, the set of nucleotides and the set o:
10 pentoses and h~xoses. As used herein, monoae_s refers to any member of a basis set for synthesis of a polymer. For example; dimers of L-amino acids form a basis set of 40o monomers for synthesis of polypeptides: Different basis sets of monomers may be used at successive steps in the synthesis of a polymer.
a t' e: A polymer in which the monomers are aloha amino acids and which are joined together through a amide bonds and alternatively referred to as a polypeptide. In the context of this specification it should be appreciated that the amino acids may be the L-optical iso~aer or the D-optical isomer.
Peptides a.a more than t;~o amino acid monomers long, and often more than 20 amino acid monomers long.
Standard abbreviations for amino acids are used (e.c., P for proline:). These abbreviations are included in 8tr~ter;,Biochemstrv, Third Ed., 1988 6. Radiation: Energy which may be selectively applied including energy having a wavelength of between 10w4 and l0' meters including, for example, electron bear radiation, gammai radiation, x-ray radiation, ultra-vielet radiationy, visible light, infrared radiation, microwave radiation, and radio waves. "Irradiatie::"
refers to the appl~c3ticn of radiation to a surface.
7. Recsotor: A molecule,that has an affinity for a given ligand. Receptors may be naturally-occuring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently; to a binding member, either directly or via a specific bending substance. Examples of receptors which can be employed by this invention include, but are not restricted to, antibodies, cell membrane re;c~ptor~, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cell s or other materials), drug , polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cel3s, cellular membranes, and organelles. Receptors are sometimes referred to in the art as anti-ligands. As the terra receptors is used herein, no difference in meaning is intended.
A "Ligand Receptor Pair'! is formed when two macromolecules have combined through molecular recognition to form a complex.
Other examples of receptors which can be investigated by this invention include but are nct restricted to:
a) Microoraani~m r~ceators:, Determination of ligands which bind to receptors, such as specific transport proteins or enzymes essential t~ survival of mieroorganisms, is useful in a new class of antibiotics. Of particular value wouldbe antibiotics against opportunistic fungi, protozoa, and those bacteria resistant'to the antibiotics in current use.

b) Enzymes: For instances; the binding site of enzymes such as the enzymes responsible for cleaving neurotransatitters; determination of ligands whic:~ bind to certain receptors~to modulate the action of the enzymes which cleave the different neurotransmitters is useful in the development of drugs which can be used in the treatment of disorders of neurotransmission.

14 c) Artibcdies: Fcr instance, the inveht~on may be useful in investigating the liganc-binding site on the antibody molecule which combines with the epitope of an antigen of interest;

determining a s~cruence that mimics an antigenic 15 epitoce may lead to the development cf vaccines of which the immunogen is based on one or more of such sequences or Lead to the development of relatEd diagnostic agents or campounds useful in therapeutic treatments such as for auto-immune diseases (e. g., by blocking the binding of the "sel " antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized to establish DNA or RNA binding sequences:

Z3 a) Cata~.ytic Polvuetti~ies: Polymers, preferably polypegtides, which are capable of promoting a chemical reaction involving the conversion of one or more reactants to one or more products.

Such polypeptides generally include a binding 30 site specific for at least one reactant or reaction intermediate and an active functionality proximate to the binding site, which functionality is capable of chemically modifying the bound reactant.

f) F~ormone receptors: For instance, the receptors far insulin and growy-~h hormone. Determination of the ligands which bind with high affinity to a receptor is useful in the development of, for example, an oral replacement of the daily inj~ctiors which diabetics must take to re?ieve the svmptons cf dabetss, and ir. the cthe;
case; a replacement for the scarce human growth hornone which can only be obtained from cadavers or by recombinant DNA technology.
Other examples are_the vasoconstrictive hormone receptors: deterainat'ion of those ligands yhich bind to a receptor may lead to t:~e development of drugs to c~ntrol,blood pressure.
g) Oviate receata=s: Determination of ligards which bind to the opiate receptors in the brain is useful in the development of less-addictive replacements for morphine and related drugs.
8. Substrate: A material having a rigid or semi-rigid surface. In many embodments, at least- one surface of the substrate will, be substantially flat, although in some embodiments it maybe desirable to physically separate,synthesis regions for different polymers with, for example, wells, raised regions, etched trenches, or the like. According to other embodiments, smal3 beads may be provided on the surface which may be released upon completion of the syntr.es is .
9. Protective G~c~ut: A material which 3s bound to a monomer unit and which may be spatially removed upon selective exposure to an activator such as electromagnetic radiation.. Examples of protective groups with utility herein include Nitroveratryloxy 14 .
carbonyl, Ni robenzyloxy carbonyl,. Dimethyl dimethoxybenzyloxy carbonyl, 5-3romo-7-nitroindolinyl, o-Fiydroxy-a-methyl cinnamoyl, and 2-Oxymethylene anthraquinone. Other examples of activators include icn beams, electric fields, magnetic fields; electron beams, x-ray, and the like.
10. Predefined Region: A predefined region is a IO localized area on a surface Which is, was, or is intended to be activated for formation of a pclvmer.
The predefined region may have any convenient shape, e.g:, circular, rectangular, elliptical, wedge-shaped, etc. For the sake of brevity herein, "predefined regions" are sometimes referred to simply as "regions."
11. Substantially Pure: A polymer is considered to be ~~substantialLy pure" within a predefined region of a substrate when it exhibits characteristics that distinguish it from other predefined regions.
Typically, purity will be measured in terms of biological activity or function as a result of uniform sequence. Such characteristics will typically be measured by way of binding with a selected ligand or receptaz.
II. Ge a a The present invention provides methods and apparatus for the preparation and use of a substrate having a plurality of polymer ser~uences in predefined regions. The invention is desczibed herein primarily with regard to the preparation of molecules containing sequences of amino acids, but, could readily be applied in the preparation of other polymers. Such polymers include, for example; both linear and cyclic polymers of nucleic acids, polysaccharides, phospholipids, and i~
peptides having either a-, p-, or w-amino acids, hetare-polymers in which a kncwn drug is covalently bound to a.~.y of the above, polyurethanes, polyesters, polycarbcnates, poiyureas, polyamides; polyethyleneiraines, polyarylene S sulfides, polysiloxanes, polyimides, polyacetates, cr other polymers which will be apparent upon review cf this disclosure. In a preferred embodiment, the inventicn herein is used in the synthesis of peptides.
The prepared substrate may, for example, be 1~ used in screening a variety of polymers as ligands for binding with a recepto=, although it will be apparent that the invention cou3d be used for the synthesis of a receptor for binding with a ligand. The substrate disclosed herein will have a wide variety of other uses.
1S Merely by way of example; the invention herein can be used in detenaining peptide, and nucleic acid seQUences which bind to proteins, finding seguence-specific binding drugs, identifying ep.itopes recogni2ed by antibodies, and evaluation of a variety of drugs for clinical and 20 diagnostic applications, as well as combinations of the above.
The invention preferably provides for the use of a substrate "S" with a surface. Linker molecules "L"
are optionally provided on a surface of the substrate.
23 The purpose of the linker: molecules, in some embodiments;
is to facilitate receptor recognition of the synthesized polymers.
Optionally, the linker molecules may be chemically protected for storage purposes. A chemical 30 storage protective group such as t-3oC (t-butaxycarbonyl) maybe used in some embodiments. Such chemcal protective group would be chemically removed upon exposure to, for example; acidic solution and °;aould serve to protect the surface during storage and be 3S removed prior to polymer preparation.
On the substrate or a distal end of the linker molecules, a functional group with a protective group P

is provided.: The protective greup Po may be remcved upcn expcsure to radiation, electric fields, electric currents, or ether activators to expose the functional group.
In a preferred embodiment, the radiation i:s ultraviolet (W), infrared (IR;, or visible light. As more fully described below, the protective g=oun may altzrnatively bean electrochemically-sensitive group which may be removed in the presence of an electric field. In still further a-lternative embodiments, ion beams, electron beams, or the like may be used fcr deprotection.
In some embod'lmentg,, the exposed regions and, therefore, the area upon which each distinct polymer sequence is synthesized are- smaller than about 1 cm2 cr less than 1 mmz. Ln preferred emoodiments the exposed area is less than about 10,000 ~cmz or, more preferably, less than 100 ~,m2 and may, in some embodiments, encompass the binding site for as few as a single molecule. Within these regions, each polymer is preferably synthesized in a substantially pure form:
Concurrently or after exposure of a known region of the substrate to light, the surface is contacted with a first monomer unit M, which reacts with the functional group which has been expo ed by the deprotection step. The first monomer includes a protective group P1. PI may or may not be the same as Po.
Accordingly, after a first cycle, known first regions of the surfacemay comprise the sequence:
S-L-Mi'pi while remaining regions of the,surface comprise the sequence:
S-L-Po .

i7 Thereafter, second regions of the surface (which may include the first region) are exposed to light and con-tacted with a second monomer M2 (which may or may not be the same as M1) having a protective group P2. P~,mav or may not be the same as Po and PI. After this second cycle, different regions of the substrate may ccznprzse one or more of the following sequences:
S'L_Mi'~'f z-Pz S-Ia-Mz-P~
S-L-Mi-pi and/or S-L-Po .
Tre above process is repeated until the substrate includes desired po3ymers of desired lengths. 3y controlling the locations of the substrate exposed to light and the reagents exposed to the substrate following exposure, the location of each sequence will be known.
. Thereafter, the,pzotective groups are removed from some or all of the substrate and the sequences are, optionally, capped with a capping unit C. The process results in a substrate having a surface with a plurality of polymers of the following general formula:
S-(L)-(Mi)-(M~)-(Mk) ... (Mx)-(C~
where square brackets indicate optional gr3ups, and Mi...M~ indicates any sequence of monomers. The number of monomers could cover a wide variety of values, but in a preferred. embodiment they will range from 2 to 100.
Zn some embodiments a plura3:ity of locations on the substrate polymers'are to contain a common monomer subsequence. For examcle, it may be desired to synthe-size a seguence S-MI-Mz'-M3 at first locations and a sequence S-M,~-Mz-M3 at second locations. The process would commence with irradiation of the first locations followed by c~ntac'ing with Ml-P, resulting in the sequence S-MI-P at the first location. The second locs-tions would then be iradiated and contacted with M4-P, resulting in the sequence S-?~4-P at the second locations.
Thereafter both the first and'second locations would be irradiated and contacted with the direr M2-?~3, resulti~g in the sequence S-Ml-Zri2-M3 at the first locaticns and S-M~-M2-M3 at the second locations. Of course, common subsequences of any length could be utilized including those in a range of 2 or-more monomers, 2 to 100 monomers, 2 to 20 monomers, and a mcst preferred range of 2 to 3 monomers.
According to other.smbodiments, a set of masks is used for the fiast monomer layer and, thereafter, is varied light wavelengths-are used for selective deprotection. For examp~:e, in the: process discussed above, first regions are first exposed through a mask and reacted with a first monomer having a first protective graup PI, whic:~ is, removable upon exposure to a first wavelength of light (e.g., IR). Second regions are masked and reacted with a second monomer having a second protecive group PZ, which is removable upon exposure to a second wavelength of light (e. g:, W). Thereafter; masks become unnecessary in the synthesis because the entire substrate may be exposed alternatively to the first and secand wavelengths of 'light in the deprotection cycle.
The polymers prepared on a substrate according to the above methods will have a variety of uses includ ing, for example, sc=eening for biological activity. In such screening activities, the substrate containing the seruences is expesed to an unlabeled or 3.abeled receptor such as an antibody, receptor on a cell, phospholipid vesicle, or any one of a variety of other receptors. In one preferred embodiment the polymers- are exposed to a first, unlabeled receptor of interest and, thereafter, exposed to a labeled receptor-specific recognition element, which is, for example, an antibody. This prvcass will provide signal amplification in the detection stage.
The reeeator molec~;xles gay bind with one o.
more polymers on the'substrate. The presence of~the labeled receptor and; therefore, the presence of a sequence which binds whh the receptor is detecte~, in a preferred embodiment through the use of autozadiography, detection of fluorescence with a charge-coupled device, fluorescence micrascopy, or the like. The sequence of the polymer at the locations where the receptor binding is detected may be used to determine all or part of a sequence which is complementary to the receptor.
Use of the inventy~n herein is illustra;.ed primarily with reference to screening for biological activity. The invention will; however, find many othe-r uses. For example, the invention may be used in infcz:nation storage ('e.g., on optical disks), production of molecular electronic devices, production of stationary phases in separation sciences, production of dyes and brightening agents, :photography, and in immobilization of cells, proteins, hectins; nucleic acids, polysaccharides and the like in patterns on a surface via molecular recognition of specific polymer sequences. By synthesizing the same compound in adjacent, progressively differing concentrations, a gradient will be established to control chemotaxis or to develop diagnostic dipsticks which, for example, titrate an antibody against an increasing amount of antigen. By synthesizing sev< ~al catalyst molecules in close proximity, more efficient multistep conversions may be achieved by ~~coordinate immobilization." Coordinate immobilization also may be used for e3ectron transfer systems, as well as to provide both structural integrity and other desirable properties to materials such as lubrication, wetting, etc.
According to alternative embodiments; molecular bindistribution or pha=macokinetic progerties may be examined. For example, to assess resistance to intestinal or serum,proteases, polymers may be capped with a fluorescent tag and exposed to biological fluids of interest.
~~I. P,id~,E?r SVZlthe515 Fig. l illustrates one embodiment of the invention disclosed herein in which a substrate 2 is shown in cross-section. Essentially, any conceivable substrate may be employed in the invention. The l~ substrate may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries; pads, slices, filers, plates, slides, etc. The substrate may have any ,5 convenient shape, such as a dis;., square, sphere; circle, etc. The substrate is preferably flat but may take on a variety of alternative surface configurations. For , example, the substrata may contain raised or depressed regions on which the synthesis takes place. The 2p substrate and its surface preferably form a rigid support on which to carry out the reactions described herein.
The substrate and its surface is also chosen to prcvide appropriate light-absorbing characteristics. ror instance, the substrate may be a po3ymerized Langmui~
25 Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, Si02, SiN4, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetraflucro-ethylene, (poly)viny3idenedifluoride; polystyrene, poiycarbonate, or combinations thereof. other substrate 3p materials will be readily apparent to those of skill in the art upon review of'this disclosure. In a preferre3 embodiment the substrata is flat glass or single-crystal silicon with surface relief features of less than 10 A.
According to some embodiments, the surface of 35 the substrate is etched using we31 known techniczues to provide for desired surface faatures. For example, by way of he formation of trenches, v-grooves mesa st.~,:ctures, or the like, the synthesis regions aay be more closely placed within the focus point of impingi.~.7 light, be provided with reflective "mirror" str~,:ctures far maximization of light collection from fluorescent v sources, or the hike.
Surfaces on the solid substrate will usually, though not always, be composed of tie same material as the substrate. Thus, the surface may be composed of any of a wide variety of materials, for example; polymers, plastics, resins, polysaccharides, silica or siliea-based materials, car:~on, metals; inorganic glasses, membranes, or any of the above-listed substrate materials. In some embodiments the surface may provide for the use of caged binding members which are attached fircly to the surace is of the substrate, Prieferabiy, the surface will contain reactive groups, which could be carboxyl, amino, hydroxyl, or the like: Most preferably, the surface will 2Q be optically transparent and will have surface Si-OH
functicnalities, such as, are found on silica surfaces.
The surface 4 of the substrate is preferably provided with a layer of linker molecules 6, although it will be understood that the linker molecules are not re 25 quired elements of the invention. The linker molecules are preferably of sufficient length to permit polymers in a completed substrate to into=act freely with molecules exposed to the substrate. The linker molecules should be 6-50 atoms long to provide sufficient exposure. The 30 life= molecules may be, for example, aryl acetylene, ethylene glycol oligomers captaining 2-l0 monomer uni s, diamines, diacids; amino acids, or combinations thereof.
Other linker molecules may be used in light of this disclsoure.
;S According to alternative embodiments, the linker molecules are selected based upon their hydrophilic/hydrophobic-properties to improve presentation of ynthesized polymers to certain receptors: For example, in the case of a hydrophilic receptor, hydrophilic linker molecules wi.Il be preferred so as to pex-mit the receptor to more closely approach the synthesi2ed polymer:
According to another alternative embcdiment, linker molecules are also provided with a photocleavabls group at an intermediate position. The photocleavable group is prefe=ably cleavable at a wavelength different from the protective group. This enables removal of the various polymers following completion of the synthesis by way of exposure to the different wavelengths of light.
The linker molecules can be attached to the substrate via carbon-carbon bonds using, for example, 35 (Poly)trifluorochloroethylene surfaces, or preferably, by siloxane bonds (using;:foz example, glass or silicon oxide ssrfaces). Siloxane bonds with the surface of the substrate may be formed in one embodiment via reactions of linker molecules bearing trichlorosilyl groups. The linker molecules may optionally be attached in an ordered array, i.e., as parts of the head groups in a polymerized Langmuir Blodgett film. In alternative embodiments, the linker molecules are adsorbed to the surface of the substrate.
The linker molecules and monomers used herein are provided with a functional group to which is bound a protective group. Preferably, the protective group is on the distal or terminal end of the linker molecule opposite the substrate. The protective group may be either a negative protective group (i.e.,,the p=otective group renders the linker molecules less reactive with a monomer upon exposure) or a positive protective group (i.e., the protective group renders the linker molecules more reactive with a monomer upon exposure). In the case of negative protective groups an additional step of reactivation will be reqtai=ed: In some embodiments, this will be done by heating.

The prctective group on the linker .~"oiecules may be selected from a.wide variety of positive night-reactive groups preferably -including vitro aromatic compounds such as o-nitrobenzyl derivatives or benzylsul-fonyl. In a preferred embodiment, s-nitrove=atryloxy-ca=bonyl (NVOC), 2-niarobenzyloxycarbonyl (NBOC) or r~,a-dimethyl-dimethoxybenzyioxycarbonyl (DDZ) is used:
In one embodiment, a vitro aromatic compound containing a benz~~' is hydr age:i ortho to t.:e vitro group is usad, i . a . , a c:~emical o f the f ora R~ RS O

H O ...
15 Rz ~ ~NO2 where RI is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen: RZ is alkoxy, alkyl, halo, azyl; vitro, or' 20 hydrogen: R3 is alkoxy, alkyl; halo; vitro, aryl, or hydrogen: Rs iS alkoxy,'alkyl, hydrogen, aryl, halo, or vitro; and RS is-alkyl, alkynyl, cyano, alkoxy, hydrogen, halo, ary3, or alkenyl. Other materials whicz may be used include o-hydroxy-a-methyl cinnamoyl der watives:
25 photoremovable protective gsoups are described in; fvr example, Patchornik, J: Fpm. Chem. Soc. (1910) 92:6333 and Amit et al., J. ora. Chem. (1974y ~.9:192a In an alternative embodiment the pcsitive 30 reactive group is activated for reaction with reagents in solution. For example, a, 5-bromo-7-nitre indoline group, when bound to a carbonyl, undergoes reaction upon exposure to light at 420 nm:
In a second aLternativ~ embodiment, the reactive group on the Tinker molecule is selected from a wide variety of'negatiVe light-reactive groups including a cinammate group.

Alternatively; the reactive group is activated or deactivated by electrcn beam lithography, x-ray lithography, or any other radiation. Suitable reac;.ive groups for el~ctran beam lithography include su?fonyl.
other methods may be used inc3uding, for example., expo-sure to a currant source. Other reactive groups and methods of activation may be used in light of this disclosure.
As shown in Fig. 1, the linking molecules ire pr-'e~abiy exposzd to; for example, light throuch a suitable mask 8 using photolithographic techniaues cf the type known in the semiconductor industry and described in, for example, Sze, VLSI Technoloav, McGraw-Hill (3983), and Mead et a-?., Introduce=cn tc zorc-Systems, Addison-Wesley (1880).
The 1 i-gh ~ may be directed at either the surface containing the g~otec~we groups or at the bac3c of the substrate, so long as the substrate is transparent to the wavelength of light needed for removal of the protective groups. In the embodiment shown in Fig: 1, light is directed at tie surface of the substrate captaining the protective groups. Fig. l illustrates the use of such masking techniczues as they are applied to a positive reactive group so as to activate linking molecules and expose functional groups in areas 10a and 10b:
The mask 8 !is in one embodiment a transparent support material selectively coated with a layer of opaque material. Portions of the opaque material are removed, leaving opaque>mat~rial in the precise pattern desired on the substrate surface. The mask is brought into cJ:ose proximity with, imaged on, or brought directly into contact with the substrate surface as shown in Fig. 1. "flpenings'! in the mask correspond to locations on the substrate where it is desired to remove photoremovable protective-groups from the substrate.
Alignment may be pe=formed using conventional alignment z5 techniques in which alignment r~ayks (not shown) ara used to accurately overlay successive masks with previous patterning steps, Or more sophisticated techniuaes ~av be used. For example; inteyferorsetis tec:~niques such as the one described.in Flanders et a~., "A New Interf erometric Alignment Technique," Apo. phvs. Lptt.
(1977) 31:426-428. may be used.
To e:lhanc~ contrast cf lig, t acolied to 1Q the substrata, it is desira5le to provide contrast enhancement mate=ials between the mask and the suysrrate according tc, some embodiments . This cont; as t enhznce:~er, t layer may comoris~ a molecule which. is deco:aposad by Light suc:~ as cuinone diazid or a material whic:, is 1 ~ tr ans ier. tlv bleac:~ed a t ~~e wave l eng th o f inter es ;: .
Transient bleaching cf materials will allow grea~c=
penetration where light is~applied,hereby enr;anci~c contrast. Alternatively, contrast enhancement mzv be provided by way of a cladded fiber optic bundle.
20 The light may be~f.~om a conventional incandescent source, a laser; a lase-r diode, or the like.
If non-colli~ate~ sources of light are used it may be desirable to provide a thick- or mufti-layered bask to prevent spreading of the fight onto the substrate. it may, further, be desirable in some embodimentsto ~:t;li~e groups which are sensitive to different wavelengths tc control synthesis. For example, by using grou~s.whic:-:
are sensitive to different ~;ravelength5, it is possible to select branch positions in'the synthesis of a polymer c~
30 eliminate certain masking steps. several reactive g~eu~s along crith their corresponding wavelengths for deprotec~ion are provided in Table 1.

Table 1 Approximate Group ' Denrotzction Wave~encth Nitrove=atryloxy carbonyl (NVOC) W (300-40o nm) Nitrobenzyloxy carbonyl (NBOC) W (300-350 n:i) Dimethyl dimethoxybenzyloxy carbonyl W (280-300 nm) 5-Bromo-7-nitroindolinyl W (420 nm) o-Hydroxy-a-methyl c'innamoyl UV (300-350 nm) 2-oxymethylene anthraqu.none UV (35e nm) While the invention is illustrated primarily herein by way of the use of a mask to iiiuminata selected regions the substrate, other techniques may also be used.
For example, the substrate may be translated under a modulated laser or diode light source. Such technicues are discussed in, for example, U.S. Patent No. 4,7I9,6?5 _ ,. (Feyrer et al.), In alternative embodiments a laser galvanometric scanner is utilize. In other embodiments, the synthesis may take place on yr in contact with a conventional liquid crystal (referred to herein as a "light valve") o= fiber optic light sources. By appropriately modulating liquid c:ystals, Light may be selectively controlled so: as to permit light to contact selected regions of the ubstrate: Alternatively, synthesis may take place on the end of a series of optical fibers to which light is selectively applied.
Other means of controlling the location of light exposure will be apparent to those- of skirl in the art:
The substrate;may be irradiated either in contact or not in contact with a solution (not shown) and is, preferably, irradiated in contact with a 35 Solution. The solution'conta'1ns reagents to prevent the by-products formed by irradiation from interfering with synthesis of the polymer according to some embodiments.
Such by-products might include, ~or example, carbon Z' dioxide, nitrosacarbonyl compounds, styrene der.vatives, indoie derivatives, and products of their photochemical reactions. Alternatively, the solution may contain reagents used to match the index of refraction of t:~e substrate. Reagents added to t'~e solution may further include, for example; acidic or basic buffers, thiols, substituted hydrazines and hydroxylamines, reducing agents~(e.g:, NADH) or reagents known to react with a given functional group (e, g., aryl nitroso + glyoxylic acid ~» aryl formhydroxamate + Co2).
Either concurrently with or after the irradiation step, the linker molecules are washed or otherwise contacted with a fltst monomer, illustrated by "A" in regions 12a and 12b in Fig. 2. Tha first Monomer reacts with the activated functional groups of the linkage molecules which have been exposed to light.
The first monomer, which is preferably an amino acid, is also provided with a photaprotective group. The photoprotective g=oup on the monomer may be the same as or different than the protective group used in.the linkage molecules; and may be selected from any of the above-described protective groups. In one embodiment, the protective groups for the A monomer is selected from the group NBOC and NVOC.
As shown in Fig: 3, the process of irradiating is thereafter repeated, with a mask repositioned so as to remove linkage protective groups and expose functional groups in regions 14a and 14b which are illustrated as being regions which were protectea in the previous masking step. As an alternative to repositioning of the first mask, in many embodiments a second mask will be utilized. In other alternative embodiments, same steps may provide fo-r illuminating a common region in successive steps: As shown in Fig. 3, it may be desirable to provide separation between irradiated regions. For example, separation of about 1-5 um may be appropriate to account for alignment tolerances.

As shown in Fig. 4, the subsgate is then exposed to a second protzctad monomer "B," producing B regions 15a and 16b. Thereafter, the substrate is again masked so as to remove the protectlY2 groums a:~d expose reactive groups on A region 12a and B region 16b.
The substrate is again exposed to monomer B, resulting in the foraation of the structure shown in Fig. 6. The dimers B-A and B-B have been produced cn the substrate.
A subsequent series of masking and contacting steps similar to those described above with A (not shown) provides the strscture shown in Fig. 7. The process provides all possible dimers of B and A, i.e:, B-A, A-B, A-A, and H-B:
The substrate, the area of synthesis, and the lc area for synthesis of each individual polymer could be of any size or shape. For example, squares, ellipsoids, rectangles, triangles, circles; or portions thereof, along with irregular geometric shapes, may be utilized.
Duplicate synthesis areas may a3so be applied to a single substrate for purposes of redundancy.
In one embodiment the regions 12 and 16 on the substrate will have a surface area of between about 1 c:n2 and 10-1° cm2. In some embodiments the regions l2 and 16 have areas of less than about 10-j cm2; 10-2 cmz; 10-3 cmZ, IO-4 cm2, 10-5 cmz, I0-6 cm2; i0-~ cm2; 10-8 cmZ, or lOwo cm2.
In a preferred embodiment, the regions 12 and 16 are between about 10x10 ~Cm and 500x500 ~cm.
In some embodiments a single substrate supports more than about 10 differ=ent monomer sequences and perferably more than about IOO different monomer seguences, although in some embodiments more than about 103, TOj, 105, 10b, 101, or 108 different sequences are provided on a substrate. Of course, within a region of the substrate in which a monomer sequence is synthesized; it is preferred that the monomer sequence be substantially pure: Ln some embodiments, regions of the substrate contain polymer sequences which are at least about l%, 5%, 10%, 15%; 20%, .~.5%, 30%, 35%, ~0%, 45%, 50%, 60%, 70$, 80%, 90%, 95%, 96%, 97%, 98g, or 99% pure.
According to same embodiments, several.
sequences are intentiona3ly provided within a single region so as to provide an initial screening for biological activity, after which materials within rec:cns exhibiting significant binding are further evaluatec.
q IV. Details of One Eriboc~iment of a Reactor System Fig. 8A schematically illustrates a preferred embodiment of a reactor system l00 for synthesizing polymers an the prepared sub,~trate in accordance wit:: one aspect of the invention: The reactor system induces a body 102 with a cavity 104 on a surface thereof. In preferred embodiments the cavity 104 is between about 50 and 1000 ~cm deep with a depth of about 500 ~cm preferred.
The bottom of the cavity is preferably provided with an array of ridges 106 Which extend laoth into the 20 plane of the:Figure and parallel to the plane of the Figure. The ridges are preferably about 50 to 200 ~m deep and spaced at about 2 to 3mm. The purpose of the ridges is to generate turbulent flow for better mixir.G.
The bottom surtace of the cavity is preferably light ~5 absorbing so as to prevent reflection of impinging light.
A substrate 11:2 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 p substrate is preferably transparent to a wide spectrum of light; but in same 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 physica3 support: Preferably, the substrate is less than about 1 mm thick, more preferably less than 0.5 mm thick, :yore preferably less than 0.1 mm trick, and most preferably less than o.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 fort 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 wit:. two i0 concentric gas~sts and the intarvening space i,s held at vacuum to ensure mating of the substrate to the gaskets.
Fluid is pumz~ed through the inlet port into the cavity by way of a pump 116 ~thich may be, for example, a model no. H-120-S made by Elder Laboratories. Selected fluids are circulated into the cavity by the pump, through the cavity, and out the outlet for reei~culation or disposal. The reactor may be subjected to ultrasonic radiation and/or heated to aid in agitation in some embodiments.
Above the Substrate i12, a lent 120 is provided which may be, for example, a 2" lOOmm focal length fined silica lens. For the sake of a compact system, a reflective mirror l22 may be provided for directing light from a light source 124 onto the substrate. ~,igr~t source 124 may be, for example, a Xe(Hg) light source manufactured by Oriel and having~model no. 66024. A
second lens 12fi may be provided for the purpose of projectirc a mask image onto the substrate in combination with lens 120. This for~.a of lithography is referred to herein as projection'printing. As will be apparent from this disclosure, proximity printing and the like may also be used according to ame embodiments.
Light from the bight source is permitted to reach only selected locations on-the substrate as a result of mask 128. Mask IZ8 maybe, for example, a glass slide having etched chrome thereon. The mask 128 in one embodiment is provided with a grid of transparent locations andopaque'locations. Such masks may be manufactured by, for exaixple, Photo Sciences, Inca Ligh 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 expo ed to light.
As discussed above, light valves (LCD's}
may be used as an alternative to conventional masks to selectively expose regions of the substrate.
Alt~raatively, fiber opt.ic'faceplates such as those available from Schott Glass, Inc, may be used for the, purpose of contrast enhancement of the mask or as the sale means of restricting th~.region to which light is applied. Such f3caplates would be placed directly above IS or on the sub trate in the reactor shown in Fig. 8A: Ia still further embodiments; flys-dye lenses, tapered =fiber optic faceplates, or the like; may be used for contrast enhancement.
In order to provide for illumination of regicns smaller than a wavelength of light, more elaborate techniques may be utilized. For example, according to one preferred embodiment, light is directed at the substrata by way of molecular microcrysta~.s on the t~p of, for example, micropipettes. Such devices are 25 disclosed in 'Lieherman ,e~ a~., "A Light Source Smaller Than the Optical Wavelength;" Science (1990) 247;59-61>
In operation, the substrate i5 placed on the fl cavity and sealed thereto. A11 operations in the process cf preparing the substrate are carried out in a room lit primarily or entirely by light of a Wavelength outside of the light range at which the protective group is removed.
For example, in the case of NVOC, the room should be lit 35 With a.conventional darkroom light which provides little or no UV light. A11 operations are preferably conducted at about room temperature.

A first, deprotection fluid (Without a monomer;
is circulated through the cavity. The solution preferably is of 5 mM sulfuric acid in dioxane solution which serves to keea exposed amino groups protonated and decreases their reactivity with photolysis by-products.
Absorptive materia3:s such as N;N-diethylamino 2,4-dinitrobenzene, for example, may be included in the deprotection f3uid which serves to absorb light and prevent reflection and unwanted photolysis.
1Q The slide is, thereafter, positioned in a light raypath fram the mask such that first locations or, the substrate are illuminated and, therefore, deprotected.
In preferred embodi-~ents the substrate is illuminated for between about l and 15 minutes with a preferred 15 illumination time of about l0 minutes at 10-20 mW/c~n= wit'.~.
365 nm light. The slides are neutralized (i..e., brought to a pH of about 7) after photolysis with, for example, a.
solution of di-isopropylethylamine (DIEA) in methylene chloride for about 5 minutes.
The first monomer is then placed at the first locations on the substrate. After irradiation, the slide is removed, treated in bulk, and then reinstalled in the flow cell. Rlternatively, a fluid containing the first monomer ,preferably also protected by a protective group, is circulated through the cavity by way of pump 116. Lf, for example, it is: de fired to attach the amino acid Y to the substrate at the first locations; the amino acid Y
(bearing a p:otective group on its a-nitrogen), along with reagents used to render the monomer reactive, and/or p a carier, is circulated from a s orage container 11s, through the pump, through the cavity, and back to the inlet of the pump.
The monomer carrier aoiution is, in a preferred embodiment; formed b~ mixing of a first solution 3S (referred to herein as solution "A") and a second solution (raferrsd to herein as solution "H"): Table 2 provides an illustration of a mixture which :nay : a used for sarution A.
Tab3,e 2 reap t . ~ Ve t- arri er p: ti n ttA n LOO mg NVOC amino protected amino acid 37 mg Ii08T (1-Hydroxybenzotriazole) 250 ~1 DMF (D:i.methylformamzde) 86 u1 DIEA (Diisopropylethylamine) The compo ition of solution B is illus~razs~: in Table 3. Solutions A and H are mixed and allowec to react at soo:a temperature for about 8 minutes, t!~en diluted with 2 m1 of DMF, and 500 ~c1 are applied to the surface of the slide or the solution is circulated through the reactor system and allowed to react far about 0 2 hours at room temperature: The slide is then washed with DMF, methylene chloride and ethanol.
Reore ent~tive MQ;p;omer Carrier Sol~ut~ an 250 ~l DMF
111 mg BQP (Benzotriazolyl-n-oxy-tris(dimethylaminc) phosphoniumhexafluorophosphate) As the solution containing the monomer to be attached is circulated through the cavity the amino acid or other monomer will react at its carboxy teranus with amino groups on the regions of the substrate which have been deprotectad. Of course, while the invention is illustrated by way of circulation of the monomer through the cavity, the invention cou3d be practiced by way o' removing tae slide frcm the reactor and submersing i~.. in an appropriate monomer solution.
After addition of the first monomer, the solution containing the first amino acid is then purged from the system. Aftzr circulation of a sufficient amount of the DMF/methylene chloride Such. that removal of the amino acid can be assured (e.g., about 50x times the volume of the cavity and carrier lines), the mask or substrate is repositioned, or a new mask is utilized such la that second regions on the substrate will be exposed to light and the light l24 is engaged for a second expcsure.
This will deprotect second regions on the substrate and the process is repeated until the desired polymer sequences have been ynthesized. .
15 The entire derivatized substrate is then exposed to a receptor of interest, preferably labeled with, for example, a fluorescent marker, by circ~~laticn of a solution or suspension of the receptor thrbugh the.
cavity yr by contacting the surface of the slide in bulk.
The receptor will prefez8ntially bind. to certain regions of the substrate which contain complementary seguences.
Antibodies are typically suspended in what is commonly referred to as: "supercacktail,": whic:~ may be, for example, a solution of about I~ BSA (bovine seru:.~, albumin), o:5% T'ween'~in PHS (phosphate buffered sa?iney buffer. The antibodies are diluted into the supercacktail buffer to a final concentration of, for example, about 0.3. to 4 ~sg/ml.
Fig. 88 illustrates an alterna rive prat erred 3Q embodiment of the reactor shown in Fig. 8A. According to this embodi.went, the mask 128 is placed directly in contact with the substrate. Preferably, the etched portion of the mask is placed face down so as to reduce the effects of light dispersion: According to this 35 embodiment; the imaging lenses'120 and iZ6 are not necessari because the mask is brought into close proximity with the substrate.
* trade-mark j5 Fog pur~oses of increasing the signal -to-noise ratio of the tech.~.ique; some embodyments of the invention provide for exposure of the substrata to a first labeled or u~~.labeiec recsp~~r fllowed by exposure of a labeled, second receptor (e. g:, an antibody) which bfinds~at multirle sites or. she first receptor. If, for example, the first receptor is an antibody derived from a first species of an animal, t.'~e second receptor is an antibody derived from a second species directed to epitopes associated with the fist species. In the case of a mouse antibody, for example, fluorescently labeled goat antibody or antiserum which is antimouse may be used to hind at multiple sites an the mouse antibody, providing several times the fluor~scencs compared to the attachme.~.~
of a single mouse antfibody at each binding site. This process may be reaeate3 again with additional antibodies (e.g., goat-mouse-goat, etc.) for further signal amplification.
In preferred embodiments an ordered sequence of 2~y masks is utilized. Tn soiae embodiments it is possible to use as few as a single mask to synthesize all of the possible polymers of a given monomer set.
If, for example, it is desired to synthesiza all 16 dinuc3eotides from-four bases, a l cm scuare synthesis region is divided conceptually into 16 boxes, each 0.25 c~ wide: Denote the four monomer units by A, B, C, and D. The first reactions are carried out in four vent=cal columns, each 0.25 cm wide. The first mask exposes the legt~bost column of boxes, where A is 30 oouPled. The second mask exposes the next column;
where 8 is coupled; followed-by a third mask, for the C column: and a f::nal mask that exposes the right-most column, for D. The first, second, third, and fourth masks may be a single mask translated to different 35 locations.

The process is repeated in the horizontal directicn for the second unit of the dimer. This time, the masks allow exposure of horizontal rows, acain 0.25 cm wide. A, B; C, and D are sequentially coupled using masks that expose horizontal fourths of the reaction area. The resulting substrate contains all 16 dinucleotides of four bases:
The eight masks used to synthesize the dinucieotide are related to one another by trans?at.on c.
rotation. In fact, one mask can be used in all eight steps if it is: suitably rotated and translated. :or example, in the example above, a mask with a single transparent region could be sequentially used to expose each of the vertical columns, translated 90', and then ,s sequentially used to allow exposure of the horizontal rows.
Tables 4 and 5 provide a simple computer program in Quick Basic for planning a masking program and a sample output; respectively, for the synthesis of a p°lYmer chain of three monomers ("residues") having three-diffe=ent monomers in the first Ieyel, four different monomers in the second level, and five different monomers in the third le~ei in a striped pattern. The -output of t:~e program is the nurber of cells, the number of "stripes" (light regions) on each mask, and the amount of translation required far each exposure of the mask.

a 1e 4 Ma k ~t-ra~,egv Pro.~rar~.
DE:INT A-Z
DIM b(20), w(20), 1(500) F $ - "I:PT1: "
OPEN f$ FAR OUTPUT AS #1 jmax - 3 'Number of residues b(i) - 3: b(2) - 4: b(3) - 5 'Number of building blacks zor res 1,2,3 g - I: lmax(I) - l FOR j - 1 TO j max : g- g * b ( ~ ) : ' NEXT j w(0) - 0: w(1) - g / b(1) PRINT ~1, "M.ASK2 . BAS " DAT~$ , TI?YiE$ : PRINT #1, PRINT T1, USING ":lumber of residues:" ; fax FOR j ~ 1 TO jmax ?P,INT w l , USING " Residue ~ =~ building blocks" ; j ; b ( j ) NEXT j PRINT ,=1, "
PRINT T, L!SING "Number of cells-~x"; g: PRINT #l, FOR j - 2 TO jmax lmax(j) - lmax(j - 1) * b(j - 1) w(j ) - w(j - 1) / b(3 ) NEXT j FOR j - 1 TO j max PRT_NT #1; USING "Mask for residue ~"; j: PRINT #1, PRINT #L, USING " Numcer of stripes-"; lmax(j) PRINT =I, USING " Wfdth of each stripe-~a"; w(j) FOR 1 - 1 TO lmax(j) a - 1 + (1 - 1) * w(j - 1) ae - a + w(j) ~ I
PRINT ~1, USING " St:i;pe ~e begins at location and ends at ~"~ 1; a; ae PRINT =1, PRINT ~1, USING " For each of os# building blocks, translate mask by ~~
cells)"; b(j); w(j), .
PRINT #1, . PRINT #1, . PRINT #I, NEXT j m Copyright 1990, Affymax N.V.

Tab a Masking StrateQV Output 'lumber of residues- 3 Residue 1 3 building blocks Residue 2 4 building blocks Residue 3 5 building blocks Number of cells- 60 Mask for residue 1 Number o= stripes- 1 Width of each stripe- 20 Str~pe l begins at location 1 and ends at 20 For each of 3 building Mocks, translate mask by 20 cells) ::ask for residue 2 Number of stripes- 3 Width of each stripe- S
Stripe l begins at location 1 and ends at 5 Stripe 2 begins at location 21 and ends at 25 Stripe 3 begins at location 4l and ends at 45 For each of 4 building blocks; cransiate mask by 5 cells) Mask for residue 3 Number of stripes- 12 Width of each stripe- I
Stripe 1 begins at location I and ends at 1 S;.ipe 2 begins at location 6 and ends at 6 Stripe 3 begins at location I1 and ends at 11 Stripe 4 begins at location I6 and ends at 16 Stripe 5 begins at location 21 and ends at 2I
Stripe 6 begins at location 26 and ends at 26 Stripe 7 begins at location 31 and ends at 3I
Stripe 8 begins at location 36 and ends at 36 Stripe 9 begins at location 41 and ends at 4I
Stripe 1O begins at location 46 and ends at 46 Stripe 1l begins at location 51 and ends at 51 Stripe 12 begins at location 56 and ends at 56 For each of 5 building blocks, translate mask by l cells) ~ Copyrigh 1990, Affymax N.V:

V. Details of ~~e ~ ~odime.~.~ of A Fluor=_scfl.~.t -Detection Device Fig. 9 illustrates a fl~:orescent 'detectic;.
device far detecting fluorescantl:: ?abeied receptors on a substrate: A substrate 112 is placed on an x/_~
translation table 202. In a preferred e~odi:~2nz t'.-.=_ x/y transiaticn table i5 a model. no . ~ M5 0 0-yl man4f actor ;d by Newport Corporation. The x/y ~_anslaticn table connected to and controlled by an aapratriatev i0 Programmed digital camputer 204 which may be, =ar example, an apprapriately program.~ed LB:~ PC/Aor A
compatible coma~ter. Of cau=se, other ;:omputa= sys~_:~s, special purpose hardware, of the l;ke c:.;:ld reacilv ~' substituted for tie ~T. com~u,;.er used herein for 15 illus tra ripe . Computer sof triare f :,r the tr ars=a tic-and data coilac~ion functions described :~erei:: can ~~
provided based on commerciaz :.y ava~.l ~bi~ sof t.:are including, for example, "Lab Windc~rs~~ licensee by National Inst~ents':
The substrate and x/y translation table a.-=
planed under a microscape 206 which includes one or pore objectives 208. Light '(abo4t 488 em) fram a Laser 2=0, which in some embodiments is a model no. 2020-05 arccn ion laser manufactured by.Spectrapysics, is direcz=c at the substrate by a dichroic mirrc~ 207 which gasses greater than about 520 em i~ght bLt ref'ects ~38 em light. Dic:~roic mirror 207 may be; for example, a w~del no. FT510 manuzacturad by Carl Zeiss. Light reflec==:
from the mir;or then enters the m=croscope 206 whic:: :aay be, for example, a model no. Axioscov 20 manufactured by Carl Zeiss. Fluorescein-harked materials on the substrate will =luoresce >488 em light, and t::e fluflresced light will be co?lected by t:':e micrcsc,~,pe 3c and passed through the mirror. T:~e fluoresce~c lic::=
from the substrate is then directe3 thr;.ugh a ;~avele.-.gt:~
filter 209 and, thereafter throng:. an aperture plate 2I1.
* trade-mark wavelength filter 209 may be, for example, a model no. 0G~30 manufactured by Melles Griot and aperture plats 211 may be, for example, a model no. 477352/:~a7380 manufactured by Cari Zeiss.
The fluoresced lfight then enters a photomultipli~r tube 212 which in some embcdi:aents is a model no. 8943-02 manufactured by Hamamatsu, the sicnal is amplified in preamplifier 214 and~photons are counted by photon counter 216: The number of photons is recorded as a function of the location in t:~e computer 204.
Pre-Amp 2Z4 may be, for example, a model no. SR440 manufactured by Stanford Research Systems and photon counter 2I5 may be a model no. SR4D0 manufactured by Stanford Research Systems. The substrate is then moved to a subseauent location and the process is repeated.
In preferred embodiments the data are acquired every 1 to 100 ~cm with a data collection diameter of about 0:8 to 10 ~cm preferred. In embodiments with sufficiently high fluorescence, a CCD detector with broadfield illumination is utilized:
By counting the number of photons generated in a given area in response to the laser, it is possible to determine where fluorescent marked molecules are located on the substrate. Consequently, for a slide wh.ic:~ has a matrix of poiypeptidesfor example, synthesized on the surface hereof, it is possible to determine which of the polypeptides is complementary to a fluorescently marked receptor.
According to preferred embodiments, the intensity and duration of the light applied to the substrate is controlled by varying the laser power and scan stage rate foz improved signal-to-noise ratio by maximizing fluorescence emission-and minimizing background noise.
35 While the detection apparatus has been illustrated primarily herein with regard to t!~e detection of marked receptors, the invention will find application ~1 in other areas. For example, the detactian apparatus discicsed herein could be used in the fields of catalysis, DNA or protein gel scanning, and t:~e like.
VI . DeteT-mination of Rel ati.VA
~indincr Strength of ~,ecettors The signal=to-noise ratio of the present invention is sufficient?y high that not only can the presence or absence of a redeptor on a ligand be detected, but also the relative binding affini~~y of receptors to a variety of sequences can be deter";inec.
In practice it is found that a receator will bind to several peptide sequences in an array, but will bind muc:z more strongly; to some secxuences than others.
Strong binding affinity will be evidenced herein. by a strong fluorescent or radiographic signal since many receptor molecules will bind in a region of a strongly bound ligand. Conversely, a weak binding affinity will be evidenced by a weak fluorescent or radiographic signal due to the relatively small number of receptor molecules which bind in a particular region of a substrate having a ligand with a weak binding affinity, for the receptor.
Consat~uently; it becomes possible to determine =elative binding avidity (or affinity in the case of univalent l~ interactions) of a ligand herein by way of the intensify of a fluorescent or radiographic signal in a region containing that ligand.
Semiquantitative data on affinities might also be obtained by varying washing conditions and concentrations of the receptor. Thz would be done by comparison to known ligand receptor pairs, for example.
VII. Exam~Ies The following examples are provided t~
illustrate the efficacy of the inventions here n. All operations were conducted at about ambient temneratu=es and pressures unless indicated to the contrarv.

A. Slide Prpnaration Before attachment of reactive groups it is preferred to clean the substrate wrich is, in a preferred emi~cdiment a glass substrate such. as a microscope slide or cover slip. according to one embodiment the slide is soaked in an alkaline bath consisting of, for example, 1 liter of 95% ethanol with 120 ml of water ard:120 grams of sodium hydroxide for L2 hours. The slides are then washed under running water and allowed to air dry, anc i0 rinsed once with a solu~icn of 95% ethanol.
The slides are then aminated with, for exa:~ple, aminopropyitriethoxysilane for the purpose of attaching amino groups to the glass surface on linker molecules, although any omega funct~onalized silane could also be 1~ used for this purpose. in one embodiment 0.10 aminopropyltriethoxysilane is utilized, although solutions with concentrations from l0-'% to l0% may be used, with about IO-3% to 2% preferred. A 0:1% mixture is prepared by adding to 100 ml of a 95% ethanol/5% water 20 mixture, 10O microliters (~l) of aminopropyltriethoxy-silane. The mixture is agitated at about ambient temperature on a rotary shaker for about 5 minutes.
500 u1 of thismixture is then applied to the surface of one side of each cleaned slide. After 4 minutes, the 25 slides are decanted of this solution and rinsed three times by dipping in, for example, 100% ethanol.
After the plates dry, they are placed in a 110-120'C vacuum oven for about 20 minutes, and then allowed to cure at room temperature for about l2 hours 30 in an argon environment. The slides are then dipped into DMF (dimethylformamide) solution, followed by a thorough washing with methylene chloride:
The aminated surface of the Tide is then exposed to about 500 u1 of, for example; a 30 milli:~olar 25 (mM) solution of NVDC-GAGA (gamma amino butyric acid) NhS
(N-hydroxysuc~ini~ide) in DMF for attachment of a NVOC-GABA to each of the amino groups.

The surface is washed :pith, fir example, ~MF, methylene chloride, and etzanal.
Any u~rsacted a~inoprcpyl siiane on the surface--that is, these amino grcu~s which have nc:.':ad the NVOC-GAGA attached--are now capped with acct 1 ~r Y' ~ ours (to preventfurther'reaction) by exposure to a 1:3 mixture of acetic anhydride in pyridine for 1 hour.
other materials whicmay perform this residual capping function include tri;luorcacetic anhydride; formicacetic a~Ydride, or other reactive acyiating agents. Fi.~.ally, the slides are washed again with DMF, methylene c::'_~ride, and ethanol.
r T..;~.~,r.s Cr ttpn and tmt~
g. Svn~hesis of E_a;
F:ig. 10 illustrates a possible synthesis of the eight trimers of the t:ao-:~onc~z~r set: gly, phe (represented by ttAtt and t3, ~n respectively) . A gla=_5 slide bearing silane groups terminating in 6-nitre-veratryloxycarboxamide (NVOC-NH) residues is prepared as a substrate. Active esters (pentafluorophenyl, OBt, etc:) of gly and phe pratectad at the amino group ;pith NVOC are prepared as reagents:: While not pertiner~ to this example, if side chain protecting gsoups are rpcuired for the monomer set, these must not be 23 photoreactive at the wave?ength of light used to protect the primary chain.
For a monomer set of size n, n x E cycles are required to synthesize all possible sequences of length t. A cycle consists of:
30 1. Irradiation through ,an appropriate »:ask to expose t:'~e- amino groups at the si;.es where the next residue is to be added, with appropriate washes to remove t'.~.e by-products o~ the deprotection.
35 2. Addition of a single activated and protected (with t::e same phot~chem:cally-removablegroup) monomer, which wil'_ react only at the sites addressed in step 1; ::~th appropriate washes to remove the excess reagent f i ~m the surf ace .
The above cycle is repeated for.eacn member of the monomer set unti-1 each location on the surface has been extended by one residue i:~ one embodiment. In ct:~er embodiments, save-ral residues are sequentially added a~
one location before moving on to t2~.e next location:
Cycle times wil? generally be limitzd by the coupling reaction rate, now as short as 20 min in autematea peptide synthesizers. This step is optionally followed by addition of a protecting group to stabilize the ar=ay for later testing. For some types of polymers (e. g., peptides), a final deprotection of the entire ,5 surface (removal of photoprotective side chain groups) may be reauired.
More particularly, as shown in Fig. 20A, she glas 20 is provided with regions 22, 24, 26, 23, 30, 32, 34, and 36. Regions 30, 32, 34, and 36 are masked, as shown in Fig: 108 and the glass is irradiated and ex-posed to a reagent containg "A" (e.g., gly), with t~.e resulting structure shown in Fig. lQC. Thereafter, regions 22, 24; 26, and 28 are masked, the glass is irradiated (as shown in Fig. lOD~ and exposed to a 5 reagent containing "H" (e. g., phe), with the result_.a structure shown in Fig. IOE: The process pr4ceeds, consecutively masking and exposing the sections as s;~own until the structure shown in Fig. lOM is obtained. The glass is irradiated and the terminal groups are, optionally, capped by acetylation. As shown, all possible trimers of gly/phe are obtained.
In this example, no side chain protective group removal is necessary. If it is des iced, side chain deprotection may be accomplished by treatment with ethanedithiol and trifluoroacetic acid.
In general, the number of steps needed to obtain a particular polymer chain is defined by:

n x E (1) where:
n = the numrae~ of monomers i~ the basis set of monomers, and L = the number ~of monomer units in a pclymer chain.
Conversely; tie synthesi2ed number of szauences of length E will be:
nc. (2) Of course, greater diversity is obtained b_J
.- using masking strategies which will also include the synthesis of polymers having a length of less than E.
If, in the extreme case, all polymers having a lengt:: , less than or equal to E are synthesized, the number of polymers synthesized will be:
nt + nt:. + . . . + n1 : ( 3 ) The maximum number of lithographic steps needed will generally be n for'each "layer" of monomers, i.e., the total number of masks (and, therefore, t:~e number of lithographic steps) needed will be n x E. The size of the transparent mask regions will vary in accardanea with the area of the substrate availab3e for synthesis and the number of sequences to be formed. In general; the size 0 of the synthesis areas will be:
size of synthesis areas _ (A)/(S) where:
35 ,A is the total area available for synthesis;
and S is the number of sequences desired in to area.
I t will be aDarec'iated by those of skit 1 i~
the art that the above method could readily be used to simultaneously produca thousands or millions oz oligcmers on a substrate using the photolithographic tec:~nicues disclosed herein. Consea_uently, the methcd rssult in the ability to practically test large numi~ers of, for example, dl, ti, tetra; penta, hexa, hepta, octapeptides, dodecapeatides, er larger polypeptides (or coriespondinrly, polynucleotides).
The above examale has illustrated the me~::cd by ~aav of a manual example. It will of course be appreciated that autoaate:- ~r semi-automated methcds cou?d be used. The substrate would be mounted in a flow cell for automated addition and removal of reage.~.as, to minimize the volume of reagents needed, and to more carefully control reaction condit.ons. Successive masks Q . could be applied manually or automatically.
~, C. Synthesis of a Dimer of an Aminoaroavl Groua and a Fluorescent Groub In synthesizing the dim~r of an amiroar pyl group and a fluorescent-group, a functionalizec durapore*
membrane was used as a substrate: The duraaare me~~rane was a polyviny2idine difluoride with aminopropyl Groups.
The aminoprapyl groups were protected with the DDZ group by reaction of the carbonyl chloride with the amino groups, a reaction readily known to t,.'~ose of skill in the art. The surface bearing these groups was placee in a solution of THF and contacted with a mask bearing a checkerboard pattern of 1 mm opacrue and transparent regions. The mask was exposesi to ultraviolet light having a wavelength down to at least about 280 nm yor about 5 minutes at ambient temperature, although a wide range of exposure times and temperatures may be * trade-mark apcr cpr late in var ions embodiments of the inver. tio.~. , For example, iZ one ebbodiment, an exposure time of between about 1 and 5000 seco.~ds may be used at process temz~eratures of betwee:~ -70 and -~50 ~ C.
In one pref ery ed emnodianent , exposure times c f between about l and 500 seecnds at about ambient pressure are used. In some preferred embodiments, presssre above ambient is used to prevent evaporation.
Tre surface of the membrane was then washed fcr about l hour with a fluorescent label which irciuded an active ester bound to a chelate of a lanthanide. wash times will vary over a wide range of values frc:,i about a few minutes to a few hours. .These materials fluoresce in the red and the green visible region. Afte.'he reaction with the active ester in the fluoronhcre was complete, the locations in which the fluorophore was bound could be visualized by exposing them to ultraviolet light and observing-the-red and the green fluoresce.~.ce.
It was observed that the derivatized regions of the substrate closely corresponded to the original pattern of the mask.
D. Demonstration of Signal Canabilitv Signal detection capability was c3emcnstraZed I5 using a low-level standard fluorescent bead kit manufactured by Flow Cytomet~y Standards and having :~edel no. 824. This kit includes 5:8 ~cm diameter beads, each impregnated with a known number of fluorescein molecules.
One of the beads was placed in the illumination field on the scan stage as shown in Fig. 9 in a field of a laser spot which was initially shuttered. Alter being po itioned in the illumination field, the photon detection equipment was turned on;. The laser beam was unblocked and it interacted with the particle bead, which then fluoresced. Fluorescence curves of beads impregnated with 7,000 and 29,000 fluorescein r.:olecules, are shown in Figs: 11A and 1L3, respectively. On each curve, traces for beads without fluorescein molecules aye also shown. These experiments were performed wish 4$3 r.:a excitation, with 100 I~W of laser power. The light was focused throuch a 40 power 0.75 NA objective.
The fluorescence intensity in all cases'started off at a high valise and then decreased exponentially.
The fall-off in intensity is due to photableaching o the fluorescein molecules: The traces of beads without fluorescein molecules are used for background subtraction. The difference in the initial exponential decay between labeled and noniabe~;ed beads is integrated to give the total number of photcn counts, and this :number is related to the number of noiecules per bead.
Therefore; it is possible to deduce the number cf photcrs per fluorescein molecule that can be detected. :or t:e curves illust~at~ed in Fig. 1l, this calculation indicates the radiation of about 40 to SO photons per fluorescein molecule are detected.
p ~. Determination of the Number of Molecules Per Unit-Area Aminoprop~lated glass microscope slides pre~ared according to the methods discussed above were utilized in order to establish the density of labeling c_' the slides. The free amino termini of the slides were reacted with FITC (fluorescein isothiocyanatP) which forms a covalent linkage with the amino group. The slide is then Scanned to count the number of fluorescent photons generated in a region which, using the estimated 30 40-SO photons per fluorescent molecule, enables the calculation of the number of molecules which are on the surface per unit area.
A slide with aminaprapyl silane on its surface was immersed in a l mM solution of FITC in DMF for 35 1 hour at about ambient temperature. After reaction, the slide was washed twice with DMF and then washed with ethanol, water, and then ethanol again. It was then drie3 anci stored in the dark until it was react' to ~=
examined.
Through the use of curves similar to thcs2 shown in Fig. L1, and by integrating the fluoreseer~
G counts under the exponentially decaying signs:, the number of free amino groups on the surface after deri:vitization was determined. Z t was determined t'.-.at slides wit.' labeling densities of 1 fluoroscein per 103x103 to ~2x2 nm could be reproducibly made as the canc~ntration of aminopropyltriethoxysilane varied _=om IO-5% to 10-1% .
F, removal of NVOG and Attachment of A Fluorescent Marker NVOC-BABA groups were attached as descri'cec above. The entire surface of one slide was e,coos2c to light so as to expose a'free amino group at the enc of the gamma amino butyric acid. This slide, and a duplicate which was not exposed, were then exposed 20 to fluo~escein isothiocyanate:(FZTC).
Fig. 12A illustrates the slide whic:: was ::ot exposed to light, but which was exposed to FITC. T~:e units of the x axis are time and the units of the y axis 'are counts. The trace contains a certain amount of background fluoresc8nce. The duplicate slide was extosec to 35O nm broaaband illumination for about l ninut= .
(12 mW/cm2, -350 nm illumination), washed and react=d with FZTC. The fluorescence curves for this slide a~-e shown in Fig. 1ZH. A large increase in the level c=
p fluorescence is observed, which indicates photolysi_=
has expo ed a number of amino graups on the surface of the slides for attachment of a fluorescent marker.

G. Use of a Masl~ in Removal of NVOC' The next experiment was performed wit:: a C.1%
ami.~.corcpylated slide. Light from a Hg-Xe are lamp was imaged onto the subs rate through a laser-ablated chrome-on-glass mask in direct contact wi~,:h the substrate.
This slide was illuminated for approximately 5 minutes, with 12 mW of 350 nm broadband light and then eacted with the l mM FITC solution. It was put on the IO laser detection scanning.stage and a graph was plotted as a two-dimensional representation of position color-coded for fluorescence intensi y. The experiment was repeated a number of times through various masks. The fluorescence patterns for a 100x100 ~m mask; a 50 um 15 mask, a 20 ~cm mask, and a 10 um mask indicate that the mask pattern is distinct down to at least about l0 um squares using this lithographic technique.
H. Attachment of'YGGFL and Subsecruent ~xDOSUrp to 20 Herz'Antibodv and Goat A~ntimouse Zn order-to establish that receptors to a particular polypeptide sequence would bind to a surface-bound peptide and be defected; Leu enkephalin was coupled to the surface and recognized by an antibody. A slide was derivatized with0:1% amino-propyl-tr~ethoxysilane and protected with NVOC. A 500 ~m checkerboard mask was used to expose the slide in a flow cell using backside contact printing. The Leu enkephalin sequence (HZN-tyrosine,glycine,glycine,phenylalanine,leucine-CO=H, 30 o~erwise refer=ed to herein as YGGFL) was attached via its carboxy end to the exposed amino groups on the surfaceof theslide. The peptide was added in DMF
solution with the 80P/HDHT/D3EA coupling reagents and recirculat~d through the flow cell for 2 hours at room 35 temperature.
A'first antibody, known as the Herz antibody, was applied tb the surface of the slide for 45 minutes at 2 ug/ml in a sup~rcocktail (cAntaining I% BSA and 1% ova~.bumin a?so in this case). A second antibcdy, goat anti-mouse fluorescein canjugate,.was then adde3 at 2 ~cg/ml in the supercacktail buffer, and allowed to incubate for 2 hours.
The results of this experiment were plctted as fluorescence intensity as a function of position. This image was taken at IO um steps and showed that not only can deprot2ction be carried out in a well deffined pattern, but also that (1) the method provided for successful coupl:.ng of peptides to the surface of the substrate, (2) the surface of a bound,peptide was available for binding with a~, antibody, arid (3) that the detection apparatus capabilities were suf;icient 15- to detect binding of a receptor.
3. Manamer-by-Monomer Fornation of YGGFT and Sub,~eauent Exvosure to Labeled Ant~,~odv Monomer-by-monomer synthesis of YGGFL and GGFL
in alternate squares was performed on a slide in a checkerboard pattern and the resulting slide was exposed to the Herz antibody. This experimentis illustrated in Figs. I3A and i3H.
In Fig: 13A, a slide is shown which is derivatized with the aminopropyi group, protec.ed in this case with t-BOC (t-butoxycarbonyl), The elide was treated with TFA:to remove the t-HOC protecting group.
E-aminocaproic acid-, which was t-BOC protected at its ami:~o group, was then coupled onto the aminopropyl 0 groups. The aminocaproic acid serves as a spacer between the aminopropyl group and the peptide to be synthesized.
The amino end of the spacer was deprotected and coupled to NVOC-leucine: The entire slide was then illuminated with I2 mW of 325 nm broadband illumination. The slida 35 was then coupled with NttOC-phenylalanine and washed. The entire slide was again illuminated, then coup3ed to NVOC-glycine and washed. The slide was again illuminated and coupled to NVOC-g'.:y cine to fore the seauence shown i~
the last portion of Fig. 13A.
As shown in Fig. 138, altdrnating regions of the slide were then illuminated using a projecticn print using a 500x500 ~um checkerboard mask: thus, the amirc group of glycine was exposed only in the lighted areas.
When the next coupling chemistr-~r step was carried cut, NVOC-tyrosine was added; and it coupled only at those spots which had received illumination: The entire slide was then illuminated to remove all the NVOC groups, leaving a checkerboard of YGGFL in the lighted areas and in the other areas, GGFL. The Herz antibody (which recognizes the YGGr~L~ but not GGFL) was then added, followed by goat anti-mouse fluorescein conjugate.
,~ The resulting fluorescence scan showed dark areas containing the tetrapeptide GGF~L, which is not reccgrized by the Herz antibody (and thus there is nc binding of the goat anti-mouse antibody with fluorescein conjugate), and red areas in which YGGFL was present.
The YGGFL pentapeptide is recognized by the Herz antibcdy and, therefore; there is antibody in the lighted regions for the fluorescein-conjugated goat anti-rouse to recognize.
Similar patterns for a 50 ~,m mask used in G3 direct contact ("proximity print") with the substra;.e provided a pattern which was more distinct and the corners of the checkerboard pattern were touching as a result of the mask being placed in direct contact with the substrate (which reflects the increase in rasolLt~cn using this technique)., J. Monomer-bv-Monomer Synthesis of YGGFL and PGGFL
A synthesis using a 50 ~m checkerboard mask similar to that shorn in Fig. 13 was conducted. However, P was added to the GGFL sites on the substrate through an additional coupling step. P.was added by exposing protected GGFL to light through a mask, and subseguence exposure to P in the manner set forth above. Therefore, half of the regions on t.'~e substrato contained YGGFL and the remaining half contained PGGFL.
The fluorescence plot for this experimen;.
showed the regions are again readily discernable~bet-,aeen those in which binding did and d:d not occur. This experiment demonstrated that antibcdies are able to recognize a specific sec_uenc~ and that the re.ccgnition is not length-dependent.
'0 K. Monomer-bv-Monomer ~vnthesis of vGGFL and vpGG~'~
In order to further demonstrate t:~e cperability of the invention, a 50 ~;m checkerboard pattern of ,~ alternating YGGCL and YPGGFL was synthesized cn a substrate using technicues like-those set forth above.
The resulting fluorescence plot showed that the ant_bccv was clearly able to recognize the YGGFL sequence and did not bind significantly at the YPGGFL regions:
L. Synthesi of an Arrav of Sixteen Differ~nt Amino Acid Secruences and ~'stimation of Rep ativ~
Bindincr Affinity to HeTz Antibody Using techniques similar to those set forth above, an array of 16 different amino acid sacuences (replicated four times) was synthesized on eacof two glass substrates. The sequences were synthesized by attaching the sequence NVOC-GFL across the entire surface of the slides: Using;a series of masks, two layers of amino acids were then selectively applied to the substrate. Each region had dimensions of 0.25 cm x 0.0625 cm. The first slide ccn ained amino acid sequences containing only L amino acids while the second slide contained selected D amino acids. Figs. 14A
and 14B illustrate a map of the various regions on the first and second s ides, respectively. The patterns shown in Figs. i4A and 14B were duplicated four times on each slide. The slides were then, exposed to the rier~
antibody and fluorescein-labeled goat anti-:house.
A fluarescencs plat of the first slide, whic contained only L amino acids snowed red areas (indicating strong binding, i.e., 14,9,000 courts or more) and black areas (indicating little or no binding of the Herz antibody, i.e., 20;000 counts or less). The secruen,ce YGGFL was clearly most strongly recognized. The sequences YAGFL and YSGFL also exhibited strong recognition of he antibody. By contrast, most of t::e remaining sequences howed little or no binding. The four duplicate portions of the slide were extremely consistent in the amount of binding shown therein.
A fluorescence plat of the D amino acic slide indicated that strongest binding was exhibited by the YGGFL sequence. Significant binding was also detec;.ec to YaGFL, YsGFL, and YpGFL: The-remaining sequences snowed less binding with the antibody. Low binding efficiency of the sequence yGGFL was observed:
Table 6 fists the various sequences tested in order of relative fluorescence, which provides information regarding relative binding affinity.
~5 Table 6 Apaa~ent 8indina to I~erz Ab a.a. ce D_a:a'' get L -YGGFh ' YGGFL ' YAGF L YaGFL

YSGFL YsGFL

LGGFh YpGFL

FGGF L fGGFL

YFGF L yGGF L

I~IGi L f 3GFL

FAGFL wGGFL

wGGFL yaGFL

fpGF:

waGF L

VIII. Ill ustrative Alternative Embodiment According tc an alternative embodiment of t::e invention; the methods provide for attaching to the surface a caged binding member which in its caged fog has a relatively low aff;pity for other potentially binding species, such ~s receptors and speci~:c bir.;~g substances.

Accor3ing to this alternative embodi~ert, t:~e invention provides methods for forming predef ;ned regicns on a surface of a solid support, wherein the oredef_ned regions are capable of'immobiliting receptors. The methods make use of caged binding members attached ~~ the surface to enable selective activation of the prede==ned ~5 regions. The caged binding members are liberated t:, act as binding members ultimately capable of binding receptors upon se? ective act~.vation of the predefine reaiors: The activated binding members are then used to immobilize specific molecules such as receptors on the predefined region of the surface. The above procedure is repeated at'the same Qr different sites on the surface so as to provide a surface prepared with a piurali.ty of regions on the surface contain'lng; for example, the sa:~e or different receptors, when receators immobilized in this way have a differential affinity for one or more ligands, screenings and assays for the ligands can be conducted in the regions of the surface containing the receators.
The alternative embodiment may maKe use of novel caged binding members attached to the substrate.
CaCed (unactivated),meiabers have a relatively low 1~ affinit;r for receptors of substances that specifically bind to uncaged binding members when compared with the corresponding affinities of activated binding members.
Thus, the binding; members are protected from reaction until a suitable source of energy is applied to the regions of the surface desired to be activated. Upon applicationof a suitable'energy source, the caging groups labilize, thereby presenting the activated binding member. A typical energy source will be light.
Once the binding members on the surface are activated they maybe attached to a receptor: The receptor chosen may be a monoclonal antibody, a nucleic acid sequence, a drug receptor, etc. The receptor will usually, though not always, be prepared so as to permit attaching it; directly or indirectly, to abinding member. For example, a specific binding substance having a strong binding affinity for the binding member and a strong affinity for the receptor or a conjugate of the receptor may be used to act as a bridge between bindi~g members and receptors if desired. The method uses a ;5 receptor prepared such that the receptor retains its activity towar3 a particular ligand:

Preferably, the caged binding member attached to the solid substrate will be a photoactiJatable biotin complex, i.e., a biotin molecule that has been chemically, modified with photoactivatable protectirig.groups so that it has a significantly reduced binding affinity for avidin or avidin analogs than does natural biotin: In a preferred embodiment, the protecting groups localized in a predefined region of the surface will be removed upon application of a suitable source of radiation to give bindingmembers, that are biotin or a functionally analogous compound having substantially the same binding affinity for avidin or a'vidin analogs as does biotin.
In another preferred. embodiment, avidin or an avidin analog is incubated with activated binding members on the surface until the avidin binds strongly to the binding members.
The avid:in so immobilized on predefined regions of the surface can then be incubated with a desired receptor or conjugate of a desired receptor . The receptor will preferably be bi.otinylated, e.g., a biotinylated antibody, when avidin is immobilized on the predefined regions of the surface.
Alternatively, a preferred embodiment will present an av:idin/bi.otinylated receptor complex, which has been previously prepared, to activated binding members on the surface.

Il. Conclusion The inventions of the present application and related Canadian Application Nos: 2,27f,883 and 2,054,706 provide greatly improved methods and apparatus for synthesis of S polymers on substrates. Lt is to be understood that the above description is intended to be, illustrative and not restrictive.
Many embodiments will be apparent to those of skill in the art upon reviewing the above description. By way of example, the invention has been described primarily with reference to the use of photoremovable protective groups, but it will be readily recognized by those of skill in the art that sources of radiation other than light could also be used. For example, in some embodiments it may be de irable to use protective groups which are sensitive to electron beam irradiation, x-ray irradiation, in combination with electron beam lithograph, or x-ray lithography techniques: Alternatively, the group could be removed by exposure to an electric current. The scope of the inventions of the present application and related Canadian Application Nos: 2,278,883 and 2,054,706, should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims; along with the full scope of equivalents to which such claims are entitlsd.

Claims (52)

1. Apparatus for detection of fluorescently marked regions on a substrate comprising:

a) a substrate bearing a plurality of different polymer sequences coupled to a surface of said substrate, each of said different polymer sequences being coupled in a different known location of said surface, each of said known location having an area of 10-2 cm2 or less;

b) a light source for directing light at said surface of said substrate;

c) a means for detecting light fluoresced from a fluorescent label bound to the polymer sequences on said surface in response to said light sources;

d) means for translating said substrate from a first position to a second position relative to said light source and/or said means for detecting light; and e) means for storing fluoresced light intensity as a function of location on said substrate, said means for storing connected to said means for translating and said means for detecting.

2. A system for determining binding of a fluorescently labeled receptor to a ligand comprising:

a) a substrate bearing a plurality of different polymer sequences coupled to a surface of said substrate, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less;

b) means for applying light to said surface of said substrate, said means for applying light providing simultaneous illumination at a plurality of said known locations; and c) an array of detectors for detecting light fluoresced at said plurality of known locations upon binding of a fluorescently labeled receptor to said polymer sequences.

3. A system as recited in claim 2, wherein said means for applying light comprises a point light source and a cylindrical lens for focusing said point light source along a substantially linear path.

4. A system as recited in claim 2, wherein said array of detectors comprises a linear array.

5. A system as recited in claim 2, wherein said array of detectors comprises a linear CCD array.

6. An apparatus for detection of fluorescently marked locations on a substrate comprising:

a) a substrate bearing a plurality of different polymer sequences coupled to a surface of said substrate, wherein said plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less;

b) a light source for directing light at a surface of said substrate;

c) a detector for detecting light fluoresced from a fluorescent label bound to the polymer sequences on said surface in response to said light source;

d) a translator for translating said substrate relative to said light source; and e) a data storage system for storing fluoresced light intensity as a function of location on said substrate, said data storage system connected to said translator and said detector.

7. The apparatus as recited in claim 6, wherein said plurality of different nucleic acid sequences comprises greater than 10 different nucleic acid sequences on a surface of said substrate.

8. The apparatus as recited in claim 6, wherein said plurality of different nucleic acid sequences comprises at least 100 different nucleic acid sequences on a surface of said substrate.

9. The apparatus as recited in claim 6, wherein said plurality of different nucleic acid sequences comprises at least 1,000 different nucleic acid sequences on a surface of said substrate.

10. The apparatus as recited in claim 6, wherein said plurality of different nucleic acid sequences comprises greater than 10,000 different nucleic acid sequences on a surface of said substrate.

11. The apparatus a recited in claim 6, wherein said plurality or different nucleic acid sequences comprises greater than 100,000 different nucleic acid sequences on a surface of said substrate.

12. The apparatus as recited in claim 6, wherein each of said different nucleic acid sequences is in a known location on a surface of said substrate, said known location having an area of less than 10,000 µm2.

13. An apparatus for detecting fluorescently marked locations on a surface of a substrate, comprising:

a point light source for generating an excitation light;
a substrate bearing: a plurality of different polymers coupled to a surface of said substrate, wherein said plurality of different polymers comprises a plurality of different nucleic acid sequences, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10-2 cm2 or less;

an objective lens for focusing said point light source at said surface of said substrate, whereby locations, upon binding of a fluorescent label to the polymer sequences coupled therein, emit a fluoresced light in response to said excitation light;

an x-y translation stage for moving said substrate relative to said excitation light;

a dichroic mirror for reflecting light having a wavelength of said excitation light and passing light having a wavelength of said fluoresced light;

a photomultiplier and photon counter for detecting said fluoresced light; and an appropriately programmed computer for recording said fluoresced light as a function of a position on said surface of said substrate from which said fluoresced light was emitted.

14. The apparatus as recited in claim 13, wherein said plurality of different nucleic acid sequences comprises greater than 10 different nucleic acid sequences on a surface of said substrate.

15. The apparatus as recited in claim 13, wherein said plurality of different nucleic acid sequences comprises at least 100 different nucleic acid sequences on a surface of said substrate.

16. The apparatus as recited in claim 13, wherein said plurality of different nucleic acid sequences Comprises a least 1,000 different nucleic acid sequences on a surface of said substrate.

17. The apparatus as recited in claim 13, wherein said plurality of different: nucleic acid sequences comprises greater than 10,000 different nucleic acid sequences on a surface of said substrate.

18. The apparatus as recited in claim 13, wherein said plurality of different nucleic acid sequences comprises greater than 100,000 different nucleic acid sequences on a surface of said substrate.

19. The apparatus as recited in claim 13, wherein each of said different nucleic acid sequences is in a known location on a surface of said substrate, said known location having an area of less than 10,000 µm2.

20. A method of detecting the presence of a fluorescent marker on a surface of a substrate, the method comprising:

directing an excitation light at the surface of the substrate: and detecting light fluoresced from the surface of the substrate;

wherein said surface of said substrate bears a plurality of different nucleic acids covalently bound thereto, each of said different nucleic acids being bound at a different known location on the substrate, each of said known locations having an area of less than 10 -2 cm2 and said fluorescent marker comprises a fluorescently labeled target nucleic acid that is capable of hybridizing with one or more of said plurality of different nucleic acids.

21. A method of determining whether a fluorescently labeled ligand binds to one or more of a plurality of different polymer sequences, wherein said fluorescently labeled ligand comprises, a fluorescently labeled target nucleic acid, and said plurality of different polymer sequences on the surface of a substrate comprises a plurality of different nucleic acids, comprising:

providing a plurality of different polymer sequences covalently bound to a surface of a substrate, each of said different polymer sequences being bound at a known location on the surface of the substrate, each of said known locations having an area of less than 10 -2 cm2;

contacting the surface of the substrate with the fluorescently labeled ligand;

washing the surface to remove unbound fluorescently labeled ligand from the surface of the substrate; and detecting binding between the fluorescently labeled ligand and the one or more polymer sequences, said detecting step comprising directing an excitation light at the surface of the substrate, and detecting light fluoresced from the surface of the substrate.

22. The method as recited in claim 21, wherein the plurality of different nucleic acids comprises greater than different nucleic acids on a surface of said substrate.

23. The method as recited in claim 21, wherein the plurality of different nucleic acids comprises at least 100 different nucleic acids on a surface of said substrate.

24. The method as recited in claim 21, wherein the plurality of different nucleic acids comprises at least 1,000 different nucleic acids on a surface of said substrate.

25. The method as recited in claim 21, wherein the plurality of different nucleic acids comprises greater than 10,000 different nucleic acids on a surface of said substrate.

26. The method as recited in claim 21, wherein the plurality of different nucleic acids comprises greater than 100,000 different nucleic acids on a surface of said substrate.

27. The method as recited in claim 21, wherein each of the different nucleic acid sequences is in a known location on a surface of said substrate, said known location having an area of less than 10,000 µm2.

28. A nucleic acid analysis apparatus comprising:
a substrate bearing a plurality of different nucleic acids; each of said different nucleic acids being attached to a different known location of the surface of said substrate, each of said known locations having an area of 10-2 cm2 or less, said substrate comprising more than 10 of such nucleic acids, at least some of said nucleic acids coupled to fluorescently labeled target molecules;

a light source for directing light at a surface of said substrate;

a detector for detecting light fluoresced from said surface in response to said light source;

a translator for translating said substrate relative to said light source; and a data storage system for storing fluoresced light intensity as a function of location on said substrate, said data storage system coupled to said translator and said detector.

29. An apparatus for detection of labeled locations on a substrate comprising:
a) a light source capable of directing light at a surface of a substrate, said substrate bearing a plurality of different polymer sequences attached to a surface of said substrate, each of said different polymer sequences occupying a different known location of said surface, each of sand known locations having an area of 10 -2 cm2 or less, and at least one of the polymers capable of being coupled to a labeled receptor;

b) a detector to detect light emitted from said surface in response to said light source to indicate which polymer(s) are coupled to the labeled receptor.

30. The apparatus of claim 29, further comprising a translator to translate said substrate relative to said light source.

31. The apparatus of claim 29, wherein the receptor is fluorescently labeled and the emitted light is fluoresced light.

32. The apparatus of claim 31 further comprising a data storage system to store fluoresced light intensity as a function of location on said substrate.

33. The apparatus of any one of preceding claims 29 to 32, wherein said plurality of different polymer sequences comprises greater than 10 different nucleic acid sequences on a surface of said substrate.

34. The apparatus of any one of preceding claims 29 to 32, wherein said plurality of different polymer sequences comprises at least 100 different nucleic acid sequences on a surface of said substrate.

35. The apparatus of any one of preceding claims 29 to 32, wherein said plurality of different polymer sequences comprises at least 1,000 different nucleic acid sequences on a surface of said substrate.

36. The apparatus as recited in any one of preceding claims 29 to 32, wherein said plurality of different polymer sequences comprises greater than 10,000 different nucleic acid sequences on a surface of said substrate.

37. The apparatus as recited in anyone of preceding claims 29 to 32, wherein said plurality of different polymer sequences comprises greater than 100,000 different nucleic acid sequences on a surface of said substrate.

38. The apparatus as recited in any one of claims 29 to 37, wherein each of said different nucleic acid sequences is in a known location on a surface of said substrate; said known location having an area of less than 10 -4 cm2.

39. The apparatus of any one of preceding claims 29 to 38, wherein the light source is a point light source that directs an excitation light;

the translator is an x-y translation stage;

the detector comprises a photomultiplier and photon counter to detect said fluoresced light;

the data storage system comprises an appropriately programmed computer to record said fluoresced light as a function of a position on said surface of said substrate from which said fluoresced light was emitted;

and the apparatus further comprises:

an objective lens and a dichroic mirror to reflect light from the light source having a wavelength of said excitation light and to pass light having a wavelength of said fluoresced light to the detector.

40. The apparatus of claim 39, wherein the appropriately programmed computer further comprises a video display to display the recorded fluoresced light as a function of position.

41. The apparatus of claim 39 or 40, wherein the light source is a laser.

42. The apparatus of claim 39 ar 40, wherein the polymers are nucleic acids and the receptor is a labeled polynucleotide.

43. A method of detecting the presence of a fluorescent marker on a surface of a substrate, the method comprising:
(1) directing excitation light at a surface of a substrate, said substrate bearing a plurality of different polymer sequences attached to a surface of said substrate, each of said different polymer sequences occupying a different known location of said surface, each of said known locations having an area of 10 -2 cm2 or less, and at least one of the polymers capable of being coupled to a fluorescently labeled receptor; and (2) detecting light fluoresced from the surface of the substrate.

44. The method of claim 43 further comprising:

(1) translating the substrate relative to the light source; and (2) repeating, step (1).

45. A method of determining whether one or more of a plurality of different polymer sequences binds to a fluorescently labeled receptor, comprising:

(1) providing an apparatus comprising:

a) a substrate bearing a plurality of different polymer sequences attached to a surface of said substrate, each of said different polymer sequences occupying a different known location of said surface, each of said known locations having an area of 10 -2 cm2 or less;

b) a light source to direct light at a surface of said substrate;

c) a detector to detect light fluoresced from said surface in response to said light source; and d) a translator to translate said substrate relative to said light source;

(2) contacting the surface of the substrate with the fluorescently labeled receptor; and (3) detecting binding between the fluorescently labeled receptor and one or more different polymer sequences by directing an excitation light at the surface of the substrate, and detecting light fluoresced from the surface of the substrate.

46. The method of claim 45, further comprising washing the surface to remove unbound fluorescently labeled receptor from the surface of the substrate.

47. The method of claim 46, wherein the polymers are nucleic acids and he receptor is a fluorescently labeled nucleic acid.

48. The method as recited in claim 46, wherein the apparatus is as described in any one of claims 31 to 42.

49. The method as recited in any one of claims 20 to 27 and 43 to 48, further comprising recording the fluoresced light and storing the data therefrom in an appropriately programmed computer.

50. The method as recited in claim 49, wherein the method further comprises displaying the data derived from the method.

51. The apparatus of claim 1, wherein said polymer sequences on said substrate have a density of at least 1,000/cm2.

52. The apparatus of claim 6, wherein said polymer sequences on said substrate have a density of at least 1,000/cm2.