WO2006002934A1

WO2006002934A1 - Blueprint biochips

Info

Publication number: WO2006002934A1
Application number: PCT/EP2005/007086
Authority: WO
Inventors: Jörn GLÖKLER; Philipp Angenendt; Jürgen KREUTZBERGER; Hans Lehrach
Original assignee: Rina Netzwerk Rna Technologien Gmbh
Priority date: 2004-06-30
Filing date: 2005-06-30
Publication date: 2006-01-12
Also published as: EP1763589A1

Abstract

This invention relates to a method for producing a surface with one or more products attached thereto, comprising the steps of: (a) putting a first and a second surface on top of each other, wherein one or more starting materials or one or more enzymes are attached to distinct sites on said first surface; (b) contacting said one or more starting materials attached to said first surface with one or more enzymes, or contacting said one or more enzymes attached to said first surface with one or more starting materials, wherein said contacting is effected under conditions permitting generation of one or more products by said enzyme(s) using said starting material(s); and (c) attaching said product(s) to distinct sites on said second surface, thereby obtaining said surface with one or more products attached thereto; wherein (i) step (a) is effected prior to step (b); (ii) step (b) is effected prior to step (a); or (iii) steps (a) and (b) are carried out simultaneously. In a preferred embodiment, said first surface with one or more starting materials attached thereto is a DNA chip. In a further preferred embodiment, said surface with one or more products attached thereto is a protein chip.

Description

Blueprint Biochips

In this specification, a number of documents is cited. The disclosure of these documents, including manufacturer's manuals, is herewith incorporated by reference in its entirety.

The biochip technology has gained importance in the recent years, and even more so since the sequence of the human genome is available. The term "biochip" embraces a miniaturized device (a chip) carrying one or more distinct biological species such as biological molecules attached to distinct sites of its surface. The arrangement of said sites may be regular, such arrangement being also referred to as an array. Accordingly, this type of biochip is also referred to as array or microarray. In a first step, DNA microarrays have been produced. Different DNA molecules, be it oligonucleotides or cDNA molecules, are fairly homogeneous with regard to their physicochemical properties as compared to peptides and proteins. This is a result of the limited repertoire of functional groups provided by the building blocks of DNA, the nucleotides, as opposed to amino acids. Secondly, DNA is either devoid of a defined tertiary structure or at least a tertiary structure, if present, is not relevant for the type of interaction typically assayed on a DNA microarray, viz. hybridisation. Furthermore, being a linear molecule, DNA may be immobilized via a terminal group on the surface of a chip without loss or without significant loss of the capability to hybridize. Finally, cDNA libraries are available as are methods for the generation thereof, which provide a fair coverage of the transcriptome of a given sample and permit, if needed, to perform a normalization, whereby the cDNA copies of a primary library are equalized so that each original mRNA transcript is represented in the normalized library to the same extent as all the rest of the clones. Manufacture of DNA arrays may be accomplished by performing on-chip synthesis of the DNA molecules. This is effected by a photolithographic process and is typically applied for oligonucleotides (Fodor et al. (1993), Pease et al. (1994)). Alternatively, and in particular when longer probes such as cDNAs are used, said longer probes are obtained, for example, from a library and subsequently transferred and attached to the surface of a carrier (Schena et al. (1995)). Transfer is typically performed by a printer, also referred to as arrayer. Attachment may involve the formation of covalent bonds or may be effected by non-covalent interaction. For the first process of manufacture, knowledge of the oligonucleotide sequence is a prerequisite, whereas in the second process, the identity (such as the clone the cDNA originates from), if not the sequence, is known. Also known (as a consequence of the process of manufacture) are the coordinates on the array where a given DNA is attached. DNA microarrays have been used to monitor the expression of cells, tissues and even entire organisms.

In the meantime, also protein and antibody chips are being used in order to study composition and function of the proteome (Glδkler and Angenendt (2003)). Proteins for microchips are typically expressed in simple recombinant organisms such as E.coli and subsequently purified. As many proteins are produced by the host in a non-soluble form, purification is effected under denaturing conditions in many cases. Even functional native proteins may suffer from immobilizing on a chip surface, from running dry of the surface they are attached to, or from long-term storage of the chip in that their activity is lost, decreased or modified.

Only a few enzymatic assays conducted on the surface of a microchip have been described so far. Typically, the substrate of the enzyme is immobilized and the enzyme is added in solution (MacBeath and Schreiber (2000), Zhu et al. (2000)). This allows for the study of a limited number of enzymes, as multiplexing, i.e., the addition of different enzymes to different substrates - as feasible with microtiter plates - is not possible. Methods using microwells permit to perform enzymatic reactions without immobilization, however, require complicated facilities for handling of the samples and further down-scaling is limited.

A first approach towards a multiplex enzyme assay on a microarray has been described by Angenendt et al. (2003). Repeated addressing of the same coordinate on the chip surface allows the free combination of components as opposed to a conventional microarray assay, whereby all probes are brought into contact with the same sample. In said multiplex assay, each coordinate has to be addressed individually, even if all reaction sites use the same component. If contact printing is used for the delivery of components to the surface of the chip, the method is rather time-consuming.

WO-A1 0170399 describes a microhybridization chamber. Aiming at improved throughput of chip experiments, preferably of DNA chip experiments, two chips are put on top of each other with a spacer in between, such that a chamber is formed, allowing the simultaneous hybridization of two chips with the same hybridization cocktail. A transfer of material from on chip to the other is not envisaged.

WO-A1 0214860 relates to a method of producing a protein array starting from DNA (or mRNA) using cell-free in vitro synthesis of the protein. The protein is generated in situ in wells or on the surface of beads. Capture of the protein occurs on the surface of the same well, or the synthesized protein is transferred to a well on another surface and immobilized there. Transfer using a gridding robot is envisaged. A transfer by direct superposition of two surfaces is not envisaged. US-A1 20030087292 describes methods and apparatus for promoting interactions between an array of probes deposited on a microarray substrate and target molecules in a target liquid. The apparatus comprises a microarray and a cover forming a reaction chamber, whereby the microarray and the cover movable relative to each other. Moving the cover and the microarray relative to each other brings the target liquid more efficiently into contact with the probes on the microarray.

Wang (2004) describes a method an antibody array is put on top of a second support with cells bearing antigens of interest attached thereto. Antibodies may dissociate from the array and bind to their cognate antigens and may be subsequently detected. An intermediate enzymatic process leading to the formation of material to be attached to a second support from starting material attached to a first support is not envisaged.

Although expanding, the technology of protein arrays is less developed than that of DNA microarrays (Wang (2004)), as many of the conditions, which apply for DNA and are described above, do not hold for proteins. Among others, protein chip technology is facing in particular the following problems: (i) the provision of an expression library with a sufficient number of different clones for the recombinant expression of proteins, (ii) adequate protein expression in recombinant organisms and purification to homogeneity for the purpose of immobilizing the proteins on microchips, (iii) structural differences between proteins and different optimal conditions of different proteins, noting that uniform conditions are applied to all samples during a conventional microarray assay, and (iv) limited stability of proteins, which may be further reduced by attaching proteins to the surface of a chip.

In view of the limitations of the methods described in the prior art, the technical problem underlying the present invention was therefore the provision of a method of making a surface with enzymatic reaction products attached thereto.

Accordingly, this invention relates to a method for producing a surface with one or more products attached thereto, comprising the steps of: (a) putting a first and a second surface on top of each other, wherein one or more starting materials or one or more enzymes are attached to distinct sites on said first surface; (b) contacting said one or more starting materials attached to said first surface with one or more enzymes, or contacting said one or more enzymes attached to said first surface with one or more starting materials, wherein said contacting is effected under conditions permitting generation of one or more products by said enzyme(s) using said starting material(s); and (c) attaching said product(s) to distinct sites on said second surface, thereby obtaining said surface with one or more products attached thereto; wherein (i) step (a) is effected prior to step (b); (ii) step (b) is effected prior to step (a); or (iii) steps (a) and (b) are carried out simultaneously.

The surfaces according to the invention may be any surface. The surface materials may be the same for both the first and the second surface, or they may be distinct. The surface may be a coating applied to a carrier, or the surface of the carrier itself may be used. Carrier materials commonly used in the art and comprising glass, plastic, gold and silicon are envisaged for the purpose of the present invention. Coatings according to the invention, if present, include poly-L-lysine- and amino- silane-coatings as well as epoxy- and aldehyde-activated surfaces.

The phrase "putting a first and a second surface on top of each other" denotes an arrangement whereby two surfaces are superimposed in such a manner that (i) the first surface with one ore more starting materials or one or more enzymes attached thereto faces the second surface where the products are to be attached to and, as a consequence, (ii) the arrangement of said distinct sites on said second surface is a mirror image of the arrangement of said distinct sites on said first surface. The term "mirror image" refers to an arrangement with the same spacings between the centers of distinct sites on both surfaces. At the same time, a mirror image on the second surface of an individual distinct site on the first surface and the site on the first surface may be, but are not necessarily of the same size. If both surfaces are of the same size and shape, a sandwich-like arrangement is obtained upon putting the two surfaces on top of each other (see also Example enclosed herewith). The second surface with one or more products attached thereto could also be referred to as a blueprint of the first surface with one or more starting materials or enzymes attached thereto, whereby it is of note that there is an intermediate enzymatic reaction converting starting materials into products. The preferred distance between the two surfaces put on top of each other will depend on the nature of the surfaces (e.g. smooth or structured) and the amount of substance attached thereto. Preferred distances may be implemented by the use of spacers of defined thickness.

The term ..distinct sites" denotes spatially separated regions of said surfaces. These sites may be adjacent, virtually without surface patches not carrying material in between, or the distinct sites may be separated from each other by uncharged surface patches.

The surfaces may both be planar. Alternatively, the surfaces may exhibit a curvature. For example, the first surface may be convex, such as a cylinder, sphere or microsphere, and the second surface may be concave.

The term "product" according to the invention relates to one or more molecular species formed from one or more molecules of the starting material(s) by the action of one or more enzymes. Enzymes according to the invention may be any enzyme. The enzymes according to the invention may fall in any of the established categories consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.

Depending on the starting material(s), one or more enzymes may be used for the method of the invention. If more than one enzyme is being used, the enzymes may fall into the same category or into different categories.

The skilled person is aware of conditions appropriate for the formation of product(s) from starting material(s) and enzyme(s).

These conditions include temperature, pH, ionic strength as well as the presence or absence of accessory molecules required for enzymatic reaction to occur. It is understood that the term "starting material" according to the invention is not exhaustive, i.e., the addition of further reactants or educts may be necessary in order to establish the conditions permitting generation of product.

Preferably, incubation times are adjusted to the times needed for enzymatic reaction to occur or to essentially reach equilibrium and are in the range between 1ms and 1 day, preferably between 0.1 sec and 2 hours. Reaction may be performed in the dark, or at daylight, or under irradiation with electromagnetic radiation. Preferred wavelengths of said electromagnetic radiation are between 1 and 10000 nm, preferably between 10 and 2000 nm, and most preferred between 200 and 1000 nm. The reaction temperature is between 250 and 400 K, preferably between 273 and 313 K, and most preferred in the interval between 290 and 310 K, i.e., comprising ambient temperature. Alternatively, and if enzymes(s) and/or starting material(s) originating from psychrophilic, thermophilic or hyperthermophilic organisms are used, the preferred temperature interval may be centered around the optimal growth temperature of the source organism. Temperature is maintained by means of a thermostat. Alternatively, and for example in case the temperature is ambient temperature, fluctuations of the temperature may occur. Said fluctuations, as measured on the timescale of the reaction, do not exceed 2 degrees, preferably 0.5 degree, and most preferred 0.1 degree from the average temperature. The reaction may be exposed to air, or performed under a protective atmosphere provided by inert gas, such as nitrogen or argon. In a further preferred embodiment, further reactants may be provided which are gaseous, such as NO or acetylene.

Conditions in step (b) preferably comprise an aqueous solution. More preferred, said aqueous solution is buffered. Buffers are well known in the art and the skilled person is aware of appropriate buffers in dependency of enzyme(s), starting material(s) and product(s). Common buffers comprise (pK_a values in brackets) H₃PO₄ / NaHaPO₄ (pK_a,i = 2.12), Glycine (pK_a,i = 2.34), Acetic acid (4.75), Citric acid (4.76), MES (6.15), Cacodylic acid (6.27), H₂CO₃ / NaHCO₃ (pK_a,i = 6.37), Bis-Tris (6.50), ADA (6.60), Bis-Tris Propane (pK_a,i = 6.80), PIPES (6.80), ACES (6.90), Imidazole (7.00), BES (7.15), MOPS (7.20), NaH₂PO₄ / Na₂HPO₄ (pK_a,₂ = 7.21), TES (7.50), HEPES (7.55), HEPPSO (7.80), Triethanolamine (7.80), Tricine (8.10), Tris (8.10), Glycine amide (8.20), Bicine (8.35), Glycylglycine (pK_a,₂ = 8.40), TAPS (8.40), Bis-Tris Propane (pK_a,2 = 9.00), Boric acid (H₃BO₃ / Na₂B₄O₇) (9.24), CHES (9.50), Glycine (pK_a,₂ = 9.60), NaHCO₃ / Na₂CO₃ (pK_a,₂ = 10.25), CAPS (10.40) and Na₂HPO₄ / Na₃PO₄ (pK_a,3 = 12.67).

Furthermore, ionic strength may be adjusted, e.g., by the addition of sodium chloride and/or potassium chloride. The concentration of sodium chloride is between 0 and 2 M, preferably between 100 and 200 mM. Examples comprise PBS (phosphate buffered saline) containing 1.37 M NaCI, 27 mM KCI, 43 mM Na₂HPO₄-7H₂O and 14 mM KH₂PO₄ in the 10-fold aqueous stock solution, which is adjusted to pH 7.3; SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold aqueous stock solution, which is adjusted to pH 7.0; and STE (Saline Tris EDTA) containing 10 mM Tris base, 10 mM NaCI and 1mM ETA (acid). Alternatively, sodium chloride is absent 5 from the buffer preparation. Examples for common buffer preparations without sodium or potassium chloride are TAE (Tris acetate EDTA) containing 2 M Tris acetate and 0.1 M EDTA in the 50-fold aqueous stock solution at pH 8.5; TBE (Tris borate EDTA) containing 0.89 M Tris base, 0.89 M Boric acid and 0.02 M EDTA in the 10-fold aqueous stock solution at pH 8.0; and TE (Tris EDTA) containing 10 mM 10 Tris base and 1 mM EDTA (acid) at pH 7.5.

For enzymatic reactions in many cases the presence of further substances, including salts other than sodium chloride, trace elements, amino acids, vitamins, growth factors, ubiquitous co-factors such as ATP or GTP, is required. Said further 15 substances may either be added individually or provided in complex mixtures such as serum. These and further accessory substances are well known in the art as are suitable concentrations.

It is preferred that the conditions permitting generation of one or more products are

20 established concomitantly with said contacting. Alternatively, and also embraced by the present invention are methods, wherein the conditions necessary for reaction to occur are established after contacting has occurred. For example, further reactants or educts needed to generate product may be added at a later stage. Similarly, the temperature required for the enzymatic reaction to occur may be adjusted after

■25. contacting.

The term "attaching" in step (c) of the method of the invention refers to any method of attaching. It may involve the formation of one or more covalent bonds between the product and the surface. Alternatively, it may involve no bond formation and be 30 effected by non-covalent interactions. Attachment may involve a specifically derivatized surface on the one side and/or a tagged product on the other side. Alternatively, sufficient attachment may be provided by unspecific interaction between product and surface. Attachment by also be effected by surface tension, i.e., by adhesion of liquid or solution comprising the product to the surface. The first and the second surface according to the invention may be put on top of each other in a first step, followed by delivery of enzyme(s) or starting material(s) to the volume between the two surfaces. Alternatively, enzyme(s) or starting material(s) may be brought into contact with the first surface with starting material(s) or enzyme(s) attached thereto prior to putting the second surface on top of this arrangement.

In a further alternative, a second surface may be covered with enzyme(s) or starting material(s), either at distinct sites or throughout, and then put on top of the first surface with starting material(s) or enzyme(s) attached thereto, which amounts to performing steps (a) and (b) simultaneously. Preferably, said enzyme(s) or starting material(s) are attached to distinct sites forming a mirror image of the pattern of distinct sites in the first surface or part thereof. This embodiment permits multiplexing, i.e. different treatment of different enzyme(s) and/or starting material(s), thereby allowing to account for different optimal conditions for activity of different enzymes.

The direct attachment of product(s) to said first surface avoids cumbersome transfer steps such as re-arraying. Accordingly, an easy, fast and cost-effective method for producing a surface with enzymatic reaction products attached thereto is provided.

In a preferred embodiment of the method of the invention, said starting material(s) serve(s) as (a) template(s). The term "template" refers to a molecular species which determines the chemical nature of the product(s). A template is usually not subjected to change, or subjected only to a transient change, during an enzymatic reaction. A template is not converted into product, but governs the de-novo synthesis of product from educts, for example building blocks such as monomers, in that it determines the type of monomer to be used at any given position of a product to be made from building blocks. Usually, the number of building blocks constituting the template and the number of building blocks constituting the product are the same or substantially the same. The building blocks constituting the template on one side and the product on the other side may be of the same compound class or belong to different compound classes. It is understood that the conditions permitting the generation of one or more products according to the invention involve the addition of further reactants or educts such as the building blocks needed for the de novo synthesis of product using the template as guide.

In a more preferred embodiment, said templates are attached to said first surface.

In a further preferred embodiment, steps (a) to (c) are effected more than once with the same first surface with one or more starting materials or one or more enzymes attached thereto, thereby obtaining multiple surfaces with one or more products attached thereto. Preferably, said starting materials attached to the first surface are templates, which are read multiple times, whereby steps (c), i.e. attachment of product to the second surface, occur between subsequent readings of the templates. If enzymes are attached to the first surface, starting materials, which for example are educts to be converted by the enzymes, may be added multiple times.

In a further preferred embodiment, steps (a) to (c) are effected more than once and said surface with one or more products attached thereto obtained in a step (c) serves as a first surface with one or more starting materials or one or more enzymes attached thereto in a subsequent step (a). This embodiment relates to a chain of two or more blueprint processes, wherein the one or more enzymes and/or the one or more starting materials used in a particular blueprint process may be distinct from the one ore more enzymes and/or the one or more starting materials used in a previous blueprint process. For example, the products of a previous blueprint process may be enzymes to be used in the subsequent blueprint process. Alternatively, the products of a previous blueprint process may serve as templates for a subsequent blueprint process. For example, DNA chips can be copied in a blueprint process according to the invention to yield another DNA chip. The DNA obtained thereby can be used to create an RNA chip. The latter can be used to make a protein chip. Depending on the fidelity of the blueprint process, several such processes can be performed in a series before spots start to blur significantly.

Preferably, the generation of one or more products in step (b) involves an amplification. This embodiment relates to methods wherein the starting materials serve as templates. By providing sufficient amounts of educt such as building blocks needed for the synthesis of product, the templates may be read multiple times, thereby obtaining more than one product molecule per template molecule during one blueprint process. Preferably, the amplification factor achieved is at leastiO.

Preferably, said starting material is DNA. Accordingly, it is preferred that said first surface with one or more starting materials attached thereto is a DNA chip. The DNA according to the invention may be genomic DNA or cDNA. Also envisaged are derivatives of DNA, such PNAs or DNA-PNA chimera. DNA-PNA chimera are molecules comprising one or more DNA portions and one or more PNA portions. PNA stands for "peptide nucleic acid". In brief, a PNA is nucleic acid, wherein the sugar-phosphate backbone has been replaced with an amide backbone. PNA oligomers are synthetic DNA-mimics with an amide backbone (Nielsen et al. (1991), Egholm et al. (1993)) that exhibit several advantageous features. They are stable under acidic conditions and resistant to nucleases as well as proteases. Their electrostatically neutral backbone increases the binding strength to complementary DNA compared to the stability of the corresponding DNA duplex. Thus, PNA oligomers can be shorter than oligonucleotides when used as hybridisation probes. On the other hand, mismatches have a more destabilising effect, thus improving discrimination between perfect matches and mismatches. For its uncharged nature, PNA also permits the hybridisation of DNA samples at low salt or no-salt conditions, since no inter-strand repulsion as between two negatively charged DNA strands needs to be counteracted. As a consequence, the target DNA has fewer secondary structures under hybridisation conditions and is more accessible to the probe molecules.

In a more preferred embodiment, said DNA is an aptamer. Aptamers are DNA (or RNA) molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/.

In a preferred embodiment of the method of the invention using DNA as starting material, said enzyme(s) comprise(s) (an) enzyme(s) catalyzing the replication of DNA. Preferably, said enzyme(s) is/are (a) DNA polymerase(s). The use of any DNA polymerase is envisaged. More preferred, said DNA polymerase(s) is/are selected from the group consisting of thermophilic DNA polymerases, mesophilic DNA polymerases derived from phages like phi29, T7, T4, and E.coli polymerases.

In a further preferred embodiment of the method of the invention using DNA as starting material, said enzyme(s) comprise(s) (an) enzyme(s) transcribing DNA into RNA. This embodiment relates to in vitro transcription of DNA. Also preferred are embodiments of the method of the invention using DNA as starting material, wherein said enzymes comprise (an) enzyme(s) transcribing DNA into RNA and (an) enzyme(s) translating RNA into protein. An example of the latter is the in vitro mix known in the art which comprises enzymes and educts for both in vitro transcription and in vitro translation. The latter embodiments provide a plurality of recombinant proteins or (poly)peptides, whereby an expression library is not needed. As the proteins or (poly)peptides are synthesized in vitro, problems pertinent to protein expression in recombinant organisms, such as formation of inclusion bodies, are avoided.

More preferred, said enzyme(s) transcribing DNA into RNA is/are (a) RNA polymerase(s). Yet more preferred, said RNA polymerase(s) is/are (a) viral RNA polymerase(s) such as SP6 RNA polymerase or T7 RNA polymerase.

Also yet more preferred are embodiments, wherein said RNA polymerase is RNA polymerase III. The use of RNA polymerase III is particularly envisaged for applications of the method of the invention, wherein small interfering RNAs (siRNAs) are to be generated. siRNA molecules are short (typically 21 to 23 nucleotides long) double-stranded RNA molecules acting as mediators of RNA silencing (Tijsterman et al. (2002)). They are capable of reducing or causing to virtually vanish the amount of their cognate transcript. RNA polymerase III transcribes small, noncoding transcripts that are not capped or polyadenylated at the 5' and 3' ends, respectively. Furthermore, RNA polymerase III initiates transcription at defined nucleotides, and terminates transcription when it encounters a stretch of four or five thymidines. Consequently, it is possible to design small RNAs synthesized by RNA polymerase III that carry 3' overhangs of one to four uridines, a structural feature resembling that defined for siRNAs to be effective in vitro (see also Shi (2003)). For applications of the method of the invention relating to RNA interference it is preferred that double-stranded RNA is formed during or upon transcription, and that said enzymes comprise furthermore an endonuclease such as RNase III. Means for ensuring the formation of double-stranded RNA are known in the art and reviewed in Shi (2003). RNase III (Dicer) cleaves the double-stranded RNA to yield siRNA molecules.

In a further more preferred embodiment, said surface with one or more products attached thereto is a siRNA chip.

The siRNA chips obtainable by the method of the invention may be used directly after generation. Problems arising from limited stability of siRNAs attached to a surface are thereby obviated. siRNA chips may be used for studying the phenotypes of genes knocked down by RNAi. For this purpose, cells to be transfected with the siRNA molecules may be provided in arrayed form and brought into contact with a siRNA chip according to the invention, thereby obtaining a transfected cell array.

In a further preferred embodiment, said starting material is RNA.

In a more preferred embodiment, said enzyme(s) comprise(s) (an) reverse transcriptase(s). This embodiment of the method of the invention yields cDNA, when mRNA is provided as a template.

In another more preferred embodiment, said RNA is an aptamer.

In a further more preferred embodiment, said enzyme(s) comprise(s) (an) enzyme(s) translating RNA into protein. This embodiment is directed to in vitro translation of RNA. Yet more preferred, said enzyme(s) translating RNA into protein is/are comprised in extracts from E.coli, wheat germ, or rabbit reticulocytes, or are purified recombinant components of the translational apparatus.

In a preferred embodiment, said surface with one or more products attached thereto is a protein chip. Also embraced are (poly)peptide chips. The term "(poly)peptide" as used herein describes fragments of proteins as described herein above and refers to a group of molecules which comprise the group of peptides, consisting of up to 30 amino acids, as well as the group of polypeptides, consisting of more than 30 amino acids. The term ,,protein" as used herein refers to any protein. It includes soluble proteins, for example enzymes such as kinases, proteases, phosphatases, oxidases and reductases as well as non-enzyme soluble proteins, for example soluble proteins comprising immunoglobulin domains such as soluble antibodies. Membrane proteins, comprising peripheral membrane proteins and integral membrane proteins are also included. Examples of membrane proteins include receptors. Receptors include growth factor receptors, G-protein coupled receptors and receptors with seven, but also with less or more than seven transmembrane helices. The term "protein" refers to single-domain proteins as well as to multi-domain proteins. Further embraced are embodiments, wherein the protein in its natural environment functions as part of a metabolic pathway and/or a signal transduction pathway. The protein may consist of or comprise protein domains known to function in signal transduction and/or known to be involved in protein-protein interaction. Examples for such domains are Ankyrin repeats; arm, Bcl-homology, Bromo, CARD, CH, Chr, C1 , C2, DD, DED, DH, EFh, ENTH, F-box, FHA, FYVE, GEL, GYF, hect, LIM, MH2, PDZ, PB1, PH, PTB, PX, RGS, RING, SAM, SC₁ SH2, SH3, SOCS, START, TIR, TPR, TRAF, tsnare, Tubby, LJBA, VHS, W, WW, and 14-3-3 domains. Further information about these and other protein domains is available from the databases InterPro (http://www.ebi.ac.uk/interpro/, Mulder et al., 2003), Pfam

(http://www.sanger.ac.uk/Software/Pfam/, Bateman et al., 2004) and SMART (http://smart.embl-heidelberg.de/, Letunic et al., 2004).

DNA chips can be stored effectively over long time periods, which is in general not possible for protein chips without loss of activity or function. The protein or (poly)peptide chips obtainable by the method of the invention may be used directly after generation. Problems arising from limited stability of proteins or (poly)peptides attached to a surface are thereby obviated.

More preferred, the generated protein comprises an affinity tag. The affinity tag allows specific and affine attachment. Attachment via a tag may involve covalent and/or non-covalent interactions. Alternatively, tags are absent from the proteins and attachment is unspecific.

Yet more preferred, said affinity tag is selected from the group consisting of a His tag, a streptavidin binding tag, a cellulose binding domain, glutathione-S-transferase, cutinase or a tag comprising the sequence LPXTG, wherein X is any one of the naturally occurring L-amino acids. Accordingly, said attaching in step (c) is preferably effected by Ni-NTA, streptavidin, cellulose, glutathione, phosphonate or oligo-gycine. Ni-NTA designates nickel which is chelated by nitrilotriacetic acid (NTA), which is similar to EDTA. LPXTG is a sequence recognised by sortase. Sortase is an enzyme capable of catalyzing a transpeptidation reaction and has been described in the patent application W0-A2 0062804. First, a protein or (poly)peptide carrying a recognition site with the consensus sequence LPX₁X₂G, wherein Xi is any one of the naturally occurring L-amino acids, and X₂ is selected from the group consisting of threonine, alanine and serine, is cleaved between X₂ and G, and a thioester bond is formed with the enzyme, yielding an enzyme-linked intermediate. In a second step, the N-terminal portion of the protein or (poly)peptide, ending with residue X₂ of the recognition site, is transferred from the enzyme to a second protein or (poly)peptide, and a peptide bond is formed. The second protein or (poly)peptide envisaged by the inventors of WO-A2 0062804 is a pentaglycine peptide with a free N-terminus and part of a peptidoglycan occurring in the cell wall of a Gram-positive bacterium. The recognition motif LPXiX₂G is sufficiently rare such that unwanted cleavage hardly occurs. Therefore, tagging of the proteins or (poly)peptides obtained by the enzymatic reaction with said motif is envisaged. Other enzymes may be employed for cross-linking of the proteins or (poly)peptides to appropriately prepared surfaces. For example, transglutaminase can be used for linking proteins or (poly)peptides containing the amino acid glutamine to a surface carrying primary amino groups.

In a more preferred embodiment, said protein chip is an antibody chip or antibody array.

Said antibody, which is monoclonal antibody, polyclonal antibody, single chain antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, a F(ab₂)', Fv or scFv fragments etc., or a chemically modified derivative of any of these (all comprised by the term "antibody"). Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kδhler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO89/09622. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741 , WO 94/02602, WO 96/34096 and WO 96/33735. Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit..

The term "monoclonal" or "polyclonal antibody" (see Harlow and Lane, (1988), loc. cit.) also relates to derivatives of said antibodies which retain or essentially retain their binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.

The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments. Preferably, the antibody, fragment or derivative thereof according to the invention specifically binds the target protein, (poly)peptide or fragment or epitope thereof whose presence or absence is to be monitored.

The term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (polypeptides of similar structures. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, (1988), loc. cit.) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides.

In a particularly preferred embodiment of the method of the invention, said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

The term "humanized antibody" means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity. Optionally, the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO90/07861.

In a further preferred embodiment, said starting material(s) is/are (a) substrate(s) of said enzyme(s) and said enzyme(s) convert(s) said substrate(s) into said product(s). For example, enzymes like horseradish peroxidase may convert starting materials like thyramide into reactive species which may be conjugated to a second surface, for example a second surface with tyrosine groups attached thereto (thyramide signal amplification).

In a further preferred embodiment, the method of the invention further comprises prior to steps (a) and (b): (a¹) attaching to a first surface one or more starting materials or one or more enzymes, thereby obtaining said first surface with one or more starting materials or one or more enzymes attached thereto. In a more preferred embodiment, said attaching comprises transfer of said starting material(s) or said enzyme(s) to said first surface using an arrayer, wherein distinct pins of the arrayer transfer distinct solutions of said starting material(s) or of said enzyme(s). The term "arrayer" designates an apparatus comprising an array of pins attached to a robot capable of taking up a plurality of different solutions with different pins and delivering them to a surface, thereby generating an arrangement of distinct sites on the surface with different solutions being delivered to different sites. The pins may be arranged in a way such that solutions from the wells of a microtiter plate may be taken up. An arrayer may be operated manually or computer-controlled or by a combination of both.

In an alternative more preferred embodiment said starting material(s) or enzyme(s) are provided in a solution which is diluted such that molecules of said starting material(s) or enzyme molecules are separately attached to said first surface. It is envisaged that said first surface as a whole or significant parts thereof are suitable for the attachment of starting material(s) or enzyme(s), and that starting material(s) or enzyme(s) are delivered to the entire surface or significant parts thereof. In order to ensure that distinct molecular species of the starting material(s) or enzyme(s) are attached to distinct sites on the first surface, the solution of the starting material(s) or enzyme(s) is diluted such the probability of attachment of distinct molecular species at a distance below a certain threshold is sufficiently low. The distance threshold depends on the fidelity of the blueprint process, in particular with regard to the size and spacing of sites with distinct molecular species attached thereto. The distance threshold furthermore depends on the envisaged use of the surface with one or more products attached thereto, for example, on the chosen method for detecting products attached to said surface. It is understood that the arrangement of attached starting material(s) or enzyme(s) obtained thereby may not be regular, but random.

In a further alternative more preferred embodiment, said first surface comprises an array of locations suitable for attaching a molecule of said starting material or an enzyme molecule, separated by areas not suitable for attaching. Thereby, an arrayed arrangement of starting material or enzyme is achieved, whereby no arrayer is used for that purpose. This embodiment allows the use of higher concentrations than the previous embodiment. The latter two embodiments comprise a method of making a biochip, i.e., of a first surface with starting material(s) or enzyme(s) attached thereto, without the need for a library of, for example, clones, whereby different clones are stored in different containers. It is understood that the blueprint method according to the invention may be used to amplify the single molecules of template attached to distinct, but randomly located sites of the first surface. Said template molecules may be DNA molecules, which may subsequently be amplified using a DNA polymerase. This process can be considered as being analogous to, but more efficient than the plating and subsequent growth of colonies of a library on agar. In further blueprint process(es), RNA may be generated and/or a protein chip be obtained therefrom. At all levels, i.e. after DNA amplification and/or after transcription and/or translation, an identification of the DNA, either directly or via the transcription or translation product may be effected by methods known in the art.

In a preferred embodiment of the method of the invention, both surfaces are smooth. The term "smooth" denotes a surface which is unstructured, i.e., compartments such as wells or the like are absent from said surface. The distance between the two surfaces is preferably below 100 μm in order to avoid convection phenomena. At distances below 100 μm dilution occurs mostly owing to diffusion only. The experiments shown in the Examples enclosed herewith have been performed with a distance less than 30 μm between the two surfaces.

In a more preferred embodiment of the method of the invention using smooth surfaces, said distinct sites are droplets. The droplets provide compartmentalization of the reaction sites and attachment to the smooth surface. The cohesion of the droplet provides compartmentalization, whereas the adhesion to the surface provides attachment. Adhesion on a normal glass or plastic surface is sufficient to keep the droplets in place and to prevent flowing together and/or spill-over. The droplets are droplets of a liquid or solution comprising the starting material(s) or the enzyme(s). In embodiments of the method of the invention comprising attaching to a first surface one or more starting materials or one or more enzymes, said attaching may be achieved by the spotting of said droplets. The term "spotting" designates the localized delivery of small amounts of liquid to a surface. Devices for spotting, including the simultaneous spotting of multiple samples, wherein said multiple samples, for example, have been picked up from one or more microtiter plates, are well known in the art. Delivery of material by spotting may involve a direct contact between the spotting pin and the surface. Alternatively, and similar to inkjet printers, such contact is not required. Preferably, spotting is done with a spotting robot comprising the spotting device and a computer controlling the function of the spotting device and providing a user interface. Distances between droplets are for example about 20 μm when working with contact printing arrayers. It is understood that the use of smooth surfaces and droplets requires a precise adjustment of the distance between the surfaces when the surfaces are put on top of each other. A distance too large would not permit the transfer of product to the second surface, whereas a distance to small would render the droplet confluent. Appropriate distances depend on the volume of the droplets and the surface properties, for example the surface tension of the droplets on said surface.

In a yet more preferred embodiment, the method of the invention using droplets comprises the following further step after step (a¹) and prior to steps (a) and (b): (a") spotting of further droplets between droplets spotted in step (a'), such that droplets spotted in step (a') which are in the neighbourhood of said further droplets flow together. Alternatively, step (b) may be performed in a way that step (a") is effected concomitantly, i.e., the step of contacting involves the spotting of starting material(s) or enzyme(s) in such a manner that flowing together of droplets of enzyme(s) or starting material(s), respectively, is achieved. It is envisaged that said flowing together may be confined to a subset of said droplets of starting material(s) or enzyme(s). Thereby, microfluidics structures are created on the surface without necessitating arduous surface modifications such as etching commonly performed in the manufacture of microfluidics devices. For example, gradients may be established by making a row or column of droplets confluent, whereby the first droplet contains a solution of a compound, which is absent from the remainder of the droplets in that row or column.

In a more preferred embodiment of the method of the invention, at least one of said surfaces has compartments. Preferably, said compartments are wells, for example, the wells of a microtiter plate. In a further more preferred embodiment, said surface has hydrophobic and hydrophilic regions.

In a further preferred embodiment, a grid is placed between the surfaces put on top of each other. It is envisaged that said grid provides compartmentalization during the transfer of product from the first to the second surface. This embodiment of the method of the invention may be used when an arrayed arrangement of transferred product(s) is to be generated from a random arrangement of starting material(s) or enzyme(s). Preferably, the grid is finer than the expected dot size. This would result in a pixel-like resolution of the surface with positives situated in a neighbourhood.

In a more preferred embodiment, the spacing of said grid matches the spacing of locations on said first surface with said starting material(s) or enzyme(s) attached thereto. This embodiment relates a method wherein the arrayed arrangement on the first surface is to be maintained during a transfer of product(s) to the second surface under conditions where flowing together of the distinct sites might occur.

In a further preferred embodiment, different locations on said first surface with said one or more starting materials attached thereto are provided with different enzymes, or wherein different locations on said first surface with said one or more enzymes attached thereto are provided with different starting materials. This embodiment relates to a multiplex version of the method of the invention. The term "multiplex" indicates that a plurality of sites on said surface can be addressed and handled individually.

The present invention also embraces a surface generated by the method of any of the preceding claims. The Figures show:

Figure 1 : "Donor" chip carrying template DNA of GFP. The template DNA is immobilized to the donor chip and the expressed protein is detected both on the donor chip and the "acceptor" chip (shown in Figure 2) using GFP-specific polyclonal antibodies.

Figure 2: "Acceptor" chip obtained by a blueprint process according to the invention involving protein expression. The acceptor chip is coated with Ni-NTA. Bound GFP protein is detected using GFP-specific polyclonal antibodies.

The following examples illustrate the invention but should not be construed as being limiting.

Example 1

Material

Glass slides with an amine surface were obtained from Telechem Inc. (Sunnyvale, CA, USA) and nickel chelate slides from Xenopore Corp, Hawthorne, NJ, USA. The RTS 100 E. coli HY kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany), the polyclonal anti-GFP antibody (TP 401) from Acris Antibodies GmbH, Hiddenhausen, Germany) and the Cy3-labelled polyclonal anti-rabbit antibodies from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Methods

The control plasmid supplied by the cell-free transcription and translation mix was spotted in four columns per concentration on onto the amine glass slides at concentrations ranging from 100 μg/ml to 500 μg/ml using a QArray Spotting robot (Genetix, New Milton, UK). Additionally a negative control without DNA was performed.

The cell-free transcription and translation mix was prepared as recommended by the manufacturer. A nickel chelate slide was placed in a humidified chamber and 100 μl of the diluted cell-free transcription and translation mix was dispensed onto the surface, avoiding the formation of bubbles. The slide carrying the DNA was placed DNA-side down onto the nickel chelate slide and the chamber was closed and incubated at 3O⁰C for 4 hours. After expression of the proteins, the slide sandwich was opened in TBS containing 0.1 % Tween-20 (TBS-T) and both slides were rinsed with TBS and blocked in 3% (w/v) fat-free milk powder dissolved in TBST for 30 min. Next, the slides were incubated in 0.3 μg/ml polyclonal anti-GFP antibodies derived from rabbit in blocking solution for 30 minutes at 4⁰C. After rinsing both slides with TBS, the slides were incubated in 0.1 mg/ml Cy3-labelled polyclonal anti-rabbit antibodies dissolved in blocking solution for 30 min at 4°C. The slides were rinsed with TBS and washed twice in TBS-T for 15 minutes each. Finally, the slides are rinsed again with TBS, dried by pressurized air and scanned.

Results

The results shown in Figures 1 (donor chip) and 2 (acceptor chip) clearly show a DNA-dependent expression of GFP. Although no decrease of signal intensity can be observed with decreasing DNA concentration, a mirror image of the spotting pattern can be seen on the acceptor chip, thereby providing a proof-of-principle of the method of the invention. Deficiencies caused by the expression and diffusion and visible in the upper part of the scans are likely to be solved, e.g. a reduction of the incubation time and/or by covalent immobilization of the DNA template.

Further References

Angenendt et al. (2003). 3D protein microarrays: performing multiplex immunoassays on a single chip. Anal. Chem. 75, 4368-72.

Eqholm et al. (1993). PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature, 365, 566-568.

Fodor et al. (1993). Multiplexed biochemical assays with biological chips. Nature 364, 555-6.

Glόkler and Angenendt (2003). Protein and antibody microarray technology. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 797, 229-40.

MacBeath and Schreiber (2000). Printing proteins as microarrays for high-throughput function determination. Science 298, 1760-3.

Nielsen et al. (1991). Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science, 254, 1497-1500.

Pease et al. (1994). Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. U S A 91, 5022-6.

Schena et al. (1995). Quantitative monitorino of gene expression patterns with a complementary DNA microarray. Science 270, 467-70.

Shi (2003V Mammalian RNAi for the masses. TRENDS in Genetics 19, 9-12.

Tiisterman et al. (2002). The genetics of RNA silencing. Annu. Rev. Genet. 36, 489- 519.

Wang (2004). lmmunostaining with dissociable antibody microarrays. Proteomics 4, 20-6. Zhu et al. (2000). Analysis of yeast protein kinases using protein chips. Nat. Genet. 26, 283-9.

Claims

1. A method for producing a surface with one or more products attached thereto, comprising the steps of: (a) putting a first and a second surface on top of each other, wherein one or more starting materials or one or more enzymes are attached to distinct sites on said first surface;

(b) contacting said one or more starting materials attached to said first surface with one or more enzymes, or contacting said one or more enzymes attached to said first surface with one or more starting materials, wherein said contacting is effected under conditions permitting generation of one or more products by said enzyme(s) using said starting material(s); and

(c) attaching said product(s) to distinct sites on said second surface, thereby obtaining said surface with one or more products attached thereto; wherein

(i) step (a) is effected prior to step (b); (ii) step (b) is effected prior to step (a); or (iii) steps (a) and (b) are carried out simultaneously.

2. The method of claim 1, wherein said starting material(s) serve(s) as (a) template(s).

3. The method of claim 2, wherein said templates are attached to said first surface.

4. The method of any one of claims 1 to 3, wherein steps (a) to (c) are effected more than once with the same first surface with one or more starting materials or one or more enzymes attached thereto, thereby obtaining multiple surfaces with one or more products attached thereto.

5. The method of any one of claims 1 to 4, wherein steps (a) to (c) are effected more than once and wherein said surface with one or more products attached thereto obtained in a step (c) serves as a first surface with one or more starting materials or one or more enzymes attached thereto in a subsequent step (a).

6. The method of any one of claims 2 to 5, wherein said generation of one or more products in step (b) involves an amplification.

7. The method of any one of claims 1 to 6, wherein said starting material is DNA.

8. The method of any one of claims 1 to 7, wherein said first surface with one or more starting materials attached thereto is a DNA chip.

9. The method of claim 7 or 8, wherein said DNA is genomic DNA or cDNA.

10. The method of any one of claims 7 to 9, wherein said enzyme(s) comprise(s) (an) enzyme(s) catalyzing the replication of DNA.

11. The method of claim 10, wherein said enzyme(s) is/are (a) DNA polymerase(s).

12. The method of claim 11 , wherein said DNA polymerase(s) is/are selected from the group consisting of thermophilic DNA polymerases, mesophilic DNA polymerases derived from phages like phi29, T7, T4, and E.coli polymerases.

13. The method of any one of claims 7 to 9, wherein said enzyme(s) comprise(s) (an) enzyme(s) transcribing DNA into RNA.

14. The method of any one of claims 7 to 9, wherein said enzymes comprise (an) enzyme(s) transcribing DNA into RNA and (an) enzyme(s) translating RNA into protein.

15. The method of claim 13 or 14, wherein said enzyme(s) transcribing DNA into RNA is/are (a) RNA polymerase(s).

16. The method of claim 15, wherein said RNA polymerase(s) is/are (a) viral RNA polymerase(s) such as SP6 RNA polymerase or T7 RNA polymerase.

17. The method of claim 15, wherein said RNA polymerase is RNA polymerase III.

18. The method of claim 13 or 15 to 17, wherein double-stranded RNA is formed during or upon transcription, and wherein said enzymes comprise furthermore an endonuclease such as RNase III.

19. The method of claim 18, wherein said surface with one or more products attached thereto is a siRNA chip.

20. The method of any one of claims 1 to 6, wherein said starting material is RNA.

21. The method of claim 20, wherein said enzyme(s) comprise(s) (an) reverse transcriptase(s).

22. The method of claim 20, wherein said enzyme(s) comprise(s) (an) enzyme(s) translating RNA into protein.

23. The method of claim 14 to 16 or 22, wherein said enzyme(s) translating RNA into protein is/are comprised in extracts from E.coli, wheat germ, or rabbit reticulocytes, or are purified recombinant components of the translational apparatus.

24. The method of any one of claims 14 to 16, 22 or 23, wherein said surface with one or more products attached thereto is a protein chip.

25. The method of any one of claims 14 to 16 or 22 to 24, wherein the generated protein comprises an affinity tag.

26. The method of claim 25, wherein said affinity tag is selected from the group consisting of a His tag, a streptavidin binding tag, a cellulose binding domain, glutathione-S-transferase, cutinase or a tag comprising the sequence LPXTG.

27. The method of claim 26, wherein said attaching in step (c) is effected by Ni- NTA, streptavidin, cellulose, glutathione, phosphonate or oligo-gycine.

28. The method of claim 1 , wherein said starting material(s) is/are (a) substrate(s) of said enzyme(s) and said enzyme(s) convert(s) said substrate(s) into said product(s).

29. The method of any one of the preceding claims, further comprising prior to steps (a) and (b): (a¹) attaching to a first surface one or more starting materials or one or more enzymes, thereby obtaining said first surface with one or more starting materials or one or more enzymes attached thereto.

30. The method of claim 29, wherein said attaching comprises transfer of said starting material(s) or said enzyme(s) to said first surface using an arrayer, wherein distinct pins of the arrayer transfer distinct solutions of said starting material(s) or of said enzyme(s).

31. The method of claim 29, wherein said starting material(s) or enzyme(s) are provided in a solution which is diluted such that molecules of said starting material(s) or enzyme molecules are separately attached to said first surface.

32. The method of claim 29, wherein said first surface comprises an array of locations suitable for attaching a molecule of said starting material or an enzyme molecule, separated by areas not suitable for attaching.

33. The method of any one of claims 1 to 32, wherein both surfaces are smooth.

34. The method of claim 33, wherein said distinct sites are droplets.

35. The method of any one of claims 1 to 32, wherein at least one of said surfaces has compartments.

36. The method of claim 35, wherein said compartments are wells.

37. The method of claim 35, wherein said surface has hydrophobic and hydrophilic regions.

38. The method of any of the preceding claims, wherein a grid is placed between the surfaces put on top of each other.

39. The method of claim 38, wherein the spacing of said grid matches the spacing of locations on said first surface with said starting material(s) or enzyme(s) attached thereto.

40. The method of any of the preceding claims, wherein different locations on said first surface with said one or more starting materials attached thereto are provided with different enzymes, or wherein different locations on said first surface with said one or more enzymes attached thereto are provided with different starting materials.

41. A surface generated by the method of any of the preceding claims.