MXPA01006935A - Target molecule attachment to surfaces - Google Patents

Abstract

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

Description

UNION OF WHITE MOLECULES TO SURFACES TECHNICAL FIELD The present invention relates to methods for attaching target molecules such as oligonucleotides (oligos) to a surface, and to compositions for use in such methods. In another aspect, the invention relates to the resulting coated surfaces themselves. In yet another aspect, the invention relates to the use of photochemical and thermochemical means to attach the molecules to a surface.

BACKGROUND OF THE INVENTION The immobilization of deoxyribonucleic acid (DNA) on support surfaces has become an important aspect in the development of DNA-based assay systems as well as for other purposes, including the development of microfabricated arrays or matrices for the analysis of DNA See, for example, "Microchip Arrays Put DNA on the Spot", R. Service, Sci en 282 (5388): 396-399, October 16, 1998; and "Fomenting a Revolution, in Miniature," I. Amato, Science 282 (5388): 402-405, October 16, 1998. Ref.131615 See also, "The Development of Microfabricated Arrays of DNA Sequencing and Analysis", 0 'Donnell-Maloney et al., TIBTECH 14: 401-407 (1996). In general, such procedures are carried out on the surface of the plates of the microcavities, the tubes, the be the microscope slides, the silicon microcircuits or the membranes. Certain methods or approaches, in particular have been developed to make possible or improve the likelihood of the binding at the endpoint of a synthetic oligonucleotide to a surface. The binding at the endpoint (ie, with the nucleic acid sequence linked through one or the other terminal nucleotide) is desirable because the total length of the sequence will be available for hybridization to another nucleic acid sequence . This is particularly advantageous for the detection of single base pair changes under the conditions of agent hybridization. Hybridization is the method most routinely used to measure or label nucleic acids by pairing the bases with the probes immobilized on a solid support. When combined with simplification techniques such as polymerase chain reaction (PCR) or ligase chain reaction (LCR), hybridization assays are a powerful tool for diagnosis and research. Microcavity plates, in particular, are convenient and useful for testing or testing relatively large numbers of samples. Several methods have been used for the immobilization of nucleic acid probes on microcavity plates. Some of these involve the rption of the modified or unmodified oligonucleotides on the polystyrene plates. Others involve covalent immobilization. Several methods have been used to increase the sensitivity of hybridization assays. Polymer capture probes (also known as target molecules) and detection probes have been synthesized and used to obtain sensitivities below 107 DNA molecules / ml. Another method used branched oligonucleotides to increase the sensitivity of the hybridization assays. Yet another method used an improved method of multi-step antibody. Other types of nucleic acid probes such as ribonucleic acid (RNA), complementary DNA (cDNA) and peptide nucleic acids (PNA's) have also been immobilized on the plates of the microcavities for the hybridization of the PCR products in diagnostic applications. In addition, the PCR primers have been immobilized on the microcavity plates for solid phase PCR.

Only a relatively small amount of methods to immobilize DNA, to date, has found its form or manner in commercial products. One such product is known as "NucleoLink ™", and available from Nalge Nunc International (see, for example, Nuc Tech Note Vol. 3, No. 17). In this product, the DNA is reacted with a carbodiimide to activate the 5'-phosphate groups which then react with the functional groups on the surface. The disadvantages of this method or approach are that it requires the additional step of adding the carbodiimide reagent as well as a five hour reaction time for DNA immobilization, and is limited to a single type of substrate material. As another example, Pierce has recently introduced a proprietary DNA immobilization product known as "Reacti-Bind ™ DNA Coating Solutions" (see "Instructions - Reacti-Bind ™ DNA Coating Solution" 1/1997). This product is a solution that is mixed with DNA and applied to surfaces such as polystyrene or polypropylene. After incubation overnight, the solution is removed, the surface was washed with the buffer and dried, after which it is ready for hybridization. Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it has some limitations. For example, the Applicants were not able to demonstrate the useful immobilization of DNA on polypropylene using the manufacturer's instructions. In addition, this product requires large amounts of DNA. The instructions indicate that the DNA should be used at a concentration between 0.5 and 5 μg / ml. Similarly, Costar sells a product called "DNA-BIND ™" for use in attaching DNA to the surface of a cavity in a microcavity plate (see, for example, the "Application Guide" of DNA-BIND ™). ). The surface of the DNA-BIND ™ plate is coated with a non-polymeric, non-charged heterobifunctional reagent containing a reactive group of N-oxysuccinimide (NOS). This group reacts with nucleophiles such as primary amines. The heterobifunctional coating reagent also contains a photochemical group and a spacer arm which covalently bonds the reactive group to the surface of the polystyrene plate. After this, the amine-modified DNA can be covalently bound to the NOS surface. The DNA is modified by adding a primary amine either during the process of synthesis for the incipient oligomer or enzymatically to the preformed sequence. Since the DNA-BIND ™ product is based on polystyrene, it is of limited use for these applications that require high temperatures such as thermal cyclization. These various products may be useful for some purposes, or under certain circumstances, but all tend to suffer one or more disadvantages or restrictions. In particular, they either tend to require large amounts of oligonucleotides, causing the background noise levels to be inadequately high and / or lacking in versatility. The International Patent Application No.

PCT / US98 / 20140, assigned to the assignee of the present application, describes and claims, inter alia, a reagent composition for attaching a target molecule to the surface of a substrate, the reactive composition comprising one or more groups for binding the target molecule to the reagent, and one or more thermochemically reactive groups to form covalent bonds with the corresponding functional groups on the target molecule attracted. Optionally, the composition further provides photo-groups for use in binding the composition to a surface. In one embodiment, for example, a plurality of photo-groups and a plurality of cationic groups (in the form of the quaternary ammonium groups) are attached to a hydrophilic polymer backbone. This polymer can be coinmobilized with a second polymer backbone that provides the thermochemically reactive groups described above (for example, the N-oxysuccinimide ("NOS") groups) for the immobilization of the target molecules. Although reactive compositions having both attraction groups and thermochemically reactive groups, as described in the PCT application mentioned above, are still considered useful and preferred for many applications, Applicants also find that attraction groups can not be required under all the circumstances. For example, a suitable process for preparing activated slides for microarrays or micro arrays includes the steps of coating the slides with a reactive composition of a type described in the PCT application (and particularly, one having both attraction groups and thermo-chemically and photoreactive reactive groups). ). The polymers are attached to the slide by the activation of the photoreactive groups, following the application of small volumes (eg, several nanoliters or a smaller amount) of the target molecules (eg, the oligonucleotides) using the printing techniques of precision. Once applied, the solvent used to deliver the oligonucleotide is dried (when the oligonucleotides are attracted to the bound polymer), and the slide incubated under the conditions suitable to allow thermochemical coupling of the oligonucleotide to the bound polymer. After this, however, any unbound oligonucleotide is typically washed out of the slide. Applicants have found, however, that a detectable tail of unbound oligonucleotides, referred to as a "comet effect" that leads to a move away from the site or site, occasionally persists. This tail is presumably due to attractive forces within the bound polymer present on the surface of the slide surrounding the spot or spot, serving to restrain or immobilize the generally charged oligonucleotide in a negative manner when spot washing or spotting. This tail, in turn, can provide unduly high and undesirable levels of background noise. Applicants have found that under certain circumstances (for example, the application of small volumes directly to a generally flat surface) the polymeric reagents are preferably provided without the presence of such attraction groups (albeit with the thermochemically reactive groups and optional photograms). Suitable reagents of this type are described in the co-pending PCT application mentioned above. Such reagents, in turn, can be used to coat the oligonucleotides in a manner that provides an improved combination of such properties as a reduced background, a spot size or small spot (eg an increased contact angle), when compared with polymeric reagents that have charged attraction groups.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method and a reactive composition for the covalent attachment of target molecules on the surface of a substrate, such as microcavity plates, tubes, beads, microscope slides, silicon microcircuits or membranes. In one embodiment, the method and composition are used to immobilize nucleic acid probes on plastic materials such as microcavity plates, for example, for use in hybridization assays. In a preferred embodiment, the method and composition are adapted for use with substantially planar surfaces, such as those provided by the microscope slides and other supporting surfaces of the plastic, silicon hydride or silicon or glass slide. pretreated with organosilane. The reactive composition can then be used to covalently link a target molecule such as a biomolecule (e.g., a nucleic acid) which in turn can be used for specific binding reactions (e.g., to hybridize a nucleic acid a its complementary strand). The support surfaces can be prepared from a variety of materials, including but not limited to plastic materials selected from the group consisting of crystalline thermoplastics (e.g., low or high density polyethylenes, polypropylenes, acetal resins, thermoplastic nylons and polyesters) and amorphous thermoplastics (eg, polycarbonates and poly (methyl methacrylates).) Suitable glass or plastic materials provide a desired combination of properties such as stiffness, strength or strength, long-term deformation resistance , recovery of deformation during stress release, and resistance to thermal degradation A reactive composition of the invention contains one or more thermochemically reactive groups (i.e., the groups having a rate of the reaction dependent on the temperature.) The appropriate groups are selected from the group that Activated esters (eg, NOS), epoxide, azlactone, activated maleimide and hydroxyl groups. Optionally, and preferably, the composition may also contain one or more photoreactive groups. Additionally, the reagent may comprise one or more hydrophilic polymers, on which the photoreactive and / or thermochemically reactive groups may depend. The photoreactive groups (alternatively referred to herein as the "photo-groups") can be used, for example, to attach the reactive molecules to the support surface during the application of a suitable energy source such as light. The thermochemically reactive groups, in turn, can be used to form covalent bonds with the appropriate and complementary functional groups on the target molecule. In general, the reactive molecules will be first bound to the surface by the activation of the photo-groups, after which the target molecule, (for example, an oligonucleotide) is brought into contact with the bound reagent under the appropriate conditions to allow it to reach to a bonding proximity with the bound polymer. The target molecule is thermochemically coupled to the reactant bound by the reaction between the reactive groups of the attached reagent and the appropriate functional groups on the target molecule. The thermochemically reactive groups and the ionic groups can be either on the same polymer or, for example, on different polymers which are co-immobilized on the surface. Optionally, and preferably, the target molecules can be prepared or provided with functional groups adapted to the groups of the reactive molecule.

During their synthesis, for example, oligonucleotides can be prepared with functional groups such as the amines or sulfhydryl groups. The invention further provides a method for attaching a target molecule, such as an oligo, to a surface, using a reagent as described herein. In turn, the invention provides a surface having the nucleic acids bound thereto by means of such reagent, as well as a material (e.g., the microcavity plate) that provides such a surface. In yet another aspect, the invention provides a composition comprising a reagent (s) of this invention in combination with a target molecule that contains one or more functional groups reactive with the reactive group (s) (s). ) thermochemically of the reagent. Using such reagents, applicants have found that capture probes can be covalently immobilized to a variety of surfaces, including surfaces that might not otherwise adsorb probes (such as polypropylene and polyvinyl chloride). The resulting surfaces provide signals comparable to, or better than, those obtained with the modified oligonucleotides adsorbed within the polystyrene or polycarbonate.

The present immobilization reagent and method can be used in the simplification methods in a manner that is simpler than those previously reported, and can also provide improved surfaces for the covalent immobilization of nucleic acid derivatives with the nucleophile. In addition to the immobilized probes for the amplification methods and hybridization assays, the reagents of this invention can provide improved immobilization of the nucleic acid for solid phase sequencing and for the immobilization primers for PCR and other amplification techniques.

DETAILED DESCRIPTION A preferred reactive molecule of the present invention comprises a hydrophilic backbone carrying one or more thermochemically reactive groups useful for forming a covalent bond with the corresponding functional group of the target molecule, together with one or more photoreactive groups useful for the binding of the reagent to a surface. In another embodiment of the invention, it is possible to immobilize the nucleic acid sequences without using the photoreactive group. For example, the surface of the material to be coated can be provided with the thermochemically reactive groups, which can be used to immobilize the hydrophilic polymers having thermochemically reactive groups as described above. For example, a surface can be treated with an ammonia plasma to introduce a limited number of reactive amines onto the surface of the material. If this surface is then treated with a hydrophilic polymer having thermochemically reactive groups (for example, NOS groups), then the polymer can be immobilized by means of the reaction of the NOS groups with the corresponding amine groups on the surface. Preferably, the reactive groups on the polymer are in excess relative to the corresponding reactive groups on the surface to ensure that a sufficient number of these thermochemically reactive groups remain after immobilization to allow coupling with the nucleic acid sequence. Although not intended to be limited by theory, it appears that by virtue of the size of the small dot or macnha, as well as the kinetic and dynamic characteristics of the fluid found in the use of dot sizes or reduced spot, the oligonucleotide is capable of get to be in close proximity to the attached reagent without the need for the attraction groups described above. When they are used to prepare microgrids or microarrays, for example, to join the capture molecules (for example, oligonucleotides or cDNA) to the surface of the microarray or microarray, such capture molecules are generally supplied to the surface in a volume of less than about 1 nanoliter per spot or spot, using tips or printing terminals adapted to conform to the dots or spots in arrangements or networks that have a center-to-center space of approximately 200 μm to approximately 500 μm. Given their small volumes, the printed white matrices or networks tend to dry quickly, thus also affecting the efficiency and kinetic characteristics of the coupling or union. Unlike the DNA coupling of the solution and on the surface of the cavities of the coated microplates, the oligonucleotides printed on networks or matrices of extremely small spot size or spot tend to dry quickly, thereby altering the parameters that affect the manner in which the oligonucleotides make contact and are coupled to the support. In addition to the design and handling of printing tips or terminals, other factors may also affect the size of the spot or spot, and in turn, subsequent hybridization signals, including salt concentrations, type of salts and Wetting agents in the impression buffer; the hyhobic / hyhilic properties of the surfaces; the size and concentration of the oligonucleotide; and the drying environments. As will be described here (for example, in Examples 25, 26 and 28), the coatings of the reagents having both photo-groups and thermo-chemically reactive groups ("Photo-PA-PolyNOS"), as well as the reagents having these groups together with the attraction groups (a mixture of "Photo-PA-PolyNOS / Photo-PA-PolyQuat"), both provided the useful and specific immobilization of the DNA modified with the amine, with the choice between the two methods or approaches being widely dependent on the choice of the substrate (for example, the flat slide as opposed to the microcavities). In a preferred embodiment, the reactive composition can be used to prepare the activated slides having the reactive composition immobilized photochemically thereon. The slides can be stored stably and used at a later date to prepare micro arrays or microarrays by immobilizing the modified DNA with the amine. Coupling of the capture DNA to the surface is carried out at pH 8-9 in a humid environment following the printing of the DNA solution in the form of small spots or spots.

The activated slides of the present invention are particularly well suited for replacing conventional glass slides (eg silylated) in the preparation of microgrids or microarrays using manufacturing and processing protocols, reagents and equipment such as micromarking robots (for example, as they are available from Cartesian), and a micromanking device manufacturer of integrated circuits (for example, as available from TeleChem International). The equipment and protocols of the appropriate dialing equipment are commercially available, such as the ChipMaker 3"Arraylt" ™ marking device. This product is said to represent an advanced version of the initial micro-marking technology, which employs 48 tips or printing terminals to supply as many as 62,000 samples per solid substrate. The use of such an instrument, in combination with conventional slides (for example coated with poly-1-lysine), is well known in the art. See, for example, U.S. Pat. No. 3,087,522 (Brown et al.), "Methods for Fabricating Microarrays of Biological Samples," and the references cited therein, the descriptions of each of which are not incorporated herein for reference.

For example, the method and system of the present invention can be used to provide a substrate, such as a glass slide, with a surface having one or more micro arrays or microarrays. Each microarray or microarray preferably provides at least about 100 / cm2 (and preferably at least about 1000 / cm3) of different target molecules (e.g., polynucleotide or polypeptide biopolymers) in a surface area of less than about 1 cm2. Each different target molecule: 1) is placed in a defined, separate position in the network or matrix, 2) has a length of at least 10 subunits, 3) is present in a defined amount between approximately 0.1 femtomoles and. about 10 nanomoles, and 4) is deposited in the selected volume in the volume range of about 0.01 nanoliters to about 100 nanoliters. These regions (e.g., discrete spots or spots) within the network or array may generally be circular in shape, with a typical diameter of between about 10 microns and about 500 microns (and preferably between about 20 and about 200 microns). The regions are also preferably separated from the other regions in the network or matrix by approximately the same distance (e.g., center-to-center spaces of about 20 microns to about 1000 microns). A plurality of specific regions for the substance to be analyzed can be provided, such that each region includes a specific reagent of the substance to be analyzed, uniquely and preferably different ("target molecule"). Those skilled in the art, given the present disclosure, will be able to identify and select the appropriate reagents depending on the type of target molecule of interest. Target molecules include, but are not limited to, the plasmid DNA, the cosmid DNA, the bacteriophage DNA, the genomic DNA (including, but not limited to, yeast, viral, bacterial, mammalian, insect) ), RNA, cDNA, PNA, and oligonucleotides. A polymer backbone can be either naturally present or synthetic, and is preferably a synthetic polymer selected from the group consisting of oligomers, homopolymers, and copolymers resulting from the addition or condensation polymerization. Polymers that are naturally present, such as polysaccharides, and polypeptides, can also be used. Preferred skeletons are biologically inert, because they do not provide a biological function that is inconsistent with, or detrimental to, their use in the manner described.

Such polymer backbones can include acrylics such as those polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate, acrylamide and methacrylamide, vinyls such as polyvinyl pyrrolidone and polyvinyl alcohol, nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamine and polyhexamethylene dodecandiamine, polyurethanes and polyethers (for example, polyethylene oxides). The polymeric skeletons of the invention are chosen to provide hydrophilic backbones capable of carrying the desired number and type of the thermochemically reactive groups, and optionally the photo-groups, the combination depending on the selected reagent. The polymer skeleton is also selected to provide a spacer between the surface and the thermochemically reactive groups. In this way, the reagent can be attached to a surface or an adjacent reactive molecule, to provide the other groups with sufficient freedom of movement to demonstrate optimal activity.

The polymeric skeletons are preferably hydrophilic (for example, soluble in water), with polyacrylamide and polyvinylpyrrolidone which are particularly preferred polymers.

The reagents of the invention carry one or more pendent latent reactive groups (preferably photoreactive) covalently linked (directly or indirectly) to the polymer backbone. The photoreactive groups are defined herein, and the preferred groups are sufficiently stable to be stored under the conditions in which they retain such properties. See, for example, U.S. Pat. No. 5,002,582, the description of which is incorporated herein for reference. The latent reactive groups can be chosen to be responsible for several portions of the electromagnetic spectrum, with those responsible for the ultraviolet and visible portions of the spectrum (referred to herein as "photoreactive") that are particularly preferred. The photoreactive groups respond to the specific applied external stimuli so that they suffer the generation of the active species with the resulting covalent binding to an adjacent chemical structure, for example, as provided by the same molecule or a different one. Photoreactive groups are those groups of atoms in a molecule that have their covalent bonds without change under storage conditions but which, during activation by an external energy source, form covalent bonds without other molecules.

The photoreactive groups generate active species such as free radicals and particularly nitrenes, carbenes, and the excited states of ketones during the absorption of electromagnetic energy. The photoreactive groups can be chosen to function in response to various portions of the electromagnetic spectrum, and photoreactive groups that function in response for example, to the ultraviolet and visible portions of the spectrum, are preferred and can be referred to here occasionally as the "group". fotoquimico "or el" fotogrupo ". Photoreactive aryl ketones are preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles such as those having N, O, or S at position 10), or their substituted derivatives (e.g. , replaced in the ring). The functional groups of such ketones are preferred since they are readily capable of undergoing the activation / inactivation / reactivation cycle described herein. The benzophenone is a particularly preferred photoreactive portion, since it is capable of photochemical excitation with the initial formation of a single excited state that undergoes the crossing of the intersystem to the triplet state. The excited triplet state can be inserted into the carbon-hydrogen bonds by the abstraction of a hydrogen atom (from a supporting surface, for example), thus creating a pair of radicals. The subsequent collapse of the pair of radicals leads to the formation of a new carbon-carbon bond. If a reactive bond (eg, hydrogen carbon) is not available for binding, the ultraviolet-induced excitation of the benzophenone group is reversible and the molecule returns to the energy level of the basal state during the removal of the energy source . Photoactivatable aryl ketones such as benzophenone and acetophenone are of particular importance since these groups are subjected to multiple reactivation in water and therefore provide an increased coating efficiency. Accordingly, the photoreactive aryl ketones are particularly preferred. Azides constitute a preferred class of photoreactive groups and include arylazides (CeRsN3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides (-CO-N3) such as azide of benzoyl and p-methylbenzoyl azide, azide formates (0C0-N3) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (-S02-N3) such as benzenesulfonyl azide, and azides of phosphoryl (R0) 2P0N3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. The diazo compounds constitute another class of photoreactive groups and include diazoalkanes (-CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (-CO-CHN2) such as diazoacetophenone and l-trifluoromethyl-l-diazo-2-pentanone. , diazoacetals (-0-CO-CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta keto-alpha-diazoacetates (-CO-CN2-CO-0-) such as alpha-diazoacetoacetate t-butyl. Other photoreactive groups include diazirines (-CHN2) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes (-CH = C = 0) such as ketene and diphenyl ketene. Photoactivatable aryl ketones such as benzophenone and acetophenone are of particular importance since these groups are subjected to multiple reactivation in water and therefore provide an increased coating efficiency. During the activation of the photoreactive groups, the reactive molecules are covalently bound to each other and / or to the surface of the material by covalent bonds through the residues of the photoreactive groups. The exemplary photoreactive groups, and their residues during activation, are shown as follows.

Photoreactive Group Functionality of the residue Aryl arylamines R-NH-R 'azides of acyl amide R-CO-NH-R' azidoformates carbamate R-0-C0-NH-R 'azides of sulfonyl sulfonamide R-SO ^ -NH- R 'phosphorylamide phosphoramide (RO) 2P0-NH-R' diazoalkane new bond CC diazo ketones new link CC and ketone diazoacetates new link CC and ester beta-keto-alpha-diazoacetals new link CC and beta-ketoester azo aliphatic new link CC diazirines new link CC cetenos new link CC photoetched ketones new link CC and alcohol Those skilled in the art, given the present disclosure, will be able to identify and select suitable thermochemically reactive groups to provide covalent immobilization of the appropriately derived nucleic acid sequences. For example, a derived amino acid nucleic acid sequence will undergo a covalent coupling reaction with an activated ester such as a NOS ester to provide an amide linking group. Similar activated esters such as the p-nitrophenyl and pentafluorophenyl esters could also provide amide bonds when reacted with the amine groups. Those skilled in the art would also recognize numerous other amine-reactive groups such as isocyanates, thioisocyanates, carboxylic acid chlorides, epoxides, aldehydes, alkyl halides and sulfonate esters, such as mesylate, tosylate and tresylate, each of which could serve as the thermochemically reactive group. In another example, the nucleic acid sequence can be derived with a sulfhydryl group using techniques well known in the art. The corresponding thermochemically reactive group could be, for example, a maleimide ring structure or an a-iodoacetamide. Any of these structures could easily react to provide a covalent bond with the nucleic acid sequence derived with sulfhydryl. The functionalized polymers of this invention can be prepared by the appropriate derivatization of a preformed polymer or, more preferably, by the polymerization of a set of comonomers to give the desired substitution configuration. This latter approach or method is preferred because of the ease of changing the ratio of the various comonomers and because of the ability to control the level of incorporation into the polymer. A combination of these two approaches or methods can also be used to provide the optimal structures. In a preferred embodiment, for example, the monomers are prepared having a polymerizable group at one end of the molecule, separated by a spacer group from a thermochemically reactive or photoreactive group at the other end. For example, polymerizable vinyl groups such as acrylamides, acrylates, or maleimides can be coupled via the short hydrocarbon spacer to an activated ester such as a NOS ester or a photoreactive group such as a substituted benzophenone. These compounds can be prepared and purified using the techniques of organic synthesis well known to those skilled in the art. Some of the desired monomers are commercially available, such as MAPTAC, N- [3- (dimethylamino) propyl] methacrylamide hydrochloride (DMAPMA), and N- (3-aminopropyl) ethacrylamide (APMA), these compounds provide quaternary ammonium salts , tertiary amines, and primary amines respectively along the polymer backbone. The polymers and copolymers can be prepared from the above monomers also, using techniques well known to those skilled in the art. Preferably, these monomers and copolymers suffer from the free radical polymerization of the vinyl groups using the azo initiators such as 2,2'-azobisisobutyronitrile (AIBN) or the peroxides such as benzoyl peroxide. The monomers selected for the polymerization are chosen based on the nature of the final polymer product. For example, a photoreactive polymer containing a NOS group is prepared from a monomer containing the photoreactive group and a second monomer containing the activated NOS ester. The composition of the final polymer can be controlled by the molar ratio of the monomers charged to the polymerization reaction. Typically, these functionalized monomers are used at relatively low molar percentages of the total monomer content of the polymerization reaction with the rest of the composition consisting of a monomer which is neither photoreactive nor thermochemically reactive towards the nucleic acid sequence. Examples of such monomers include, but are not limited to, acrylamide and N-vinylpyrrolidone. Based on the relative reactivities of the monomers used, the distribution of the monomers along the skeleton is largely random. In some cases, the thermochemically reactive group on the polymer backbone can act on its own as a polymerizable monomer, if present during the polymerization thus requiring the introduction of this group in a second step following the initial polymer formation. For example, the preparation of a photoreactive polymer having the maleimide along the backbone can be effected by an initial preparation of a polymer containing both the photoreactive groups and the amine groups using the techniques described above, followed by the reaction of the amine groups with a heterobifunctional molecule containing a group of maleimide and an isocyanate connected by a short hydrocarbon spacer. A wide variety of such polymer modification techniques are available using the typical organic reactions known to those skilled in the art. The invention will be further described with reference to the following non-limiting Examples. It will be apparent to those skilled in the art that many changes can be made in the described embodiment without departing from the scope of the present invention. Accordingly, the scope of the present invention should not be limited to the modalities described in this application, but only by the modalities described by the language of the claims and the equivalents of these modalities. Unless stated otherwise, all percentages are by weight. The structures of several of the "Compounds" identified from beginning to end of these Examples can be found in Table 13 included later. The NMR analyzes were obtained on an 80 Mhz spectrometer unless otherwise stated.

EXAMPLES Example 1 Preparation of 4-benzoylbenzoyl chloride (BBA-C1) (Compound I) The 4-benzoylbenzoic acid (BBA), 1.0 kg (4.42 mol), is added to a 5 liter Morton flask equipped with reflux condenser and overhead stirrer, followed by the addition of 645 ml (8.84 mol) of thionyl chloride and 725 ml of toluene. Then 3.5 ml of dimethylformamide was added and the mixture was heated to reflux for 4 hours. After cooling, the solvents were removed under reduced pressure and the residual thionyl chloride was removed by three evaporations using 3 x 500 ml of toluene. The product was recrystallized from toluene: hexane 1: 4 to give 988 g (91% yield) after drying in a vacuum oven. The melting point of the product was 92-94 ° C. Nuclear magnetic resonance (NMR) analysis at 80 MHz (1H NMR (CDC13)) was consistent with the aromatic protons of the desired product 7.20-8.25 (m, 9H). All values of chemical shifts are in ppm downstream of an internal tetramethylsilane standard. The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for example, in Example 3.

Example 2 Preparation of N- (3-Aminopropyl) methacrylamide hydrochloride (APMA) (Compound II) A solution of 1,3-diaminopropane, 1910 g (25.77 moles), in 1000 ml of CH2C12 is added to a 12-liter Morton flask and cooled in an ice bath. Then a solution of t-butyl phenyl carbonate, 1000 g (5.15 mol), is added in 250 ml of CH2C12 by dripping at a rate which keeps the reaction temperature below 15 ° C. Following the addition, the mixture was warmed to room temperature and stirred 2 hours. The reaction mixture is diluted with 900 ml of CH2C12 and 500 g of ice, followed by the slow addition of 2500 ml of 2.2 N NaOH. After the test to ensure that the solution was basic, the product was transferred to a funnel of separation and the organic layer was removed and set aside as extract # 1. The aqueous phase was then extracted with 3 X 1250 ml of CH2C12, keeping each extraction as a separate fraction. The four organic extracts were then washed successively with a single portion of 1250 ml of 0.6 N NaOH starting with fraction # 1 and proceeding through fraction # 4. This washing procedure was repeated a second time with a fresh 1250 ml portion of 0.6 N NaOH. The organic extracts were combined and then dried over Na2SO4. Filtration and evaporation of the solvent at a constant weight gave 825 g of N-mono-t-BOC-1,3-diaminopropane which was used without further purification. A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020 ml of CHC13 was placed in a 12 liter Morton flask equipped with the overhead stirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as an inhibitor, followed by the dropwise addition of N-mono-t-BOC-1, 3-diaminopropane, 825 g (4.73 mol), in 825 ml of CHC13. The rate of addition was controlled to keep the reaction temperature below 10 ° C all the time. After the addition is complete, the ice bath was removed and the mixture is left stirring overnight. The product was diluted with 2400 ml of water and transferred to a separatory funnel. After complete mixing, the aqueous layer was removed and the organic layer was washed with 2400 ml of 2N NaOH, ensuring that the aqueous layer was basic. The organic layer was then dried over Na 2 SO and filtered to remove the drying agent. A portion of the CHC13 solvent was removed under reduced pressure until the combined weight of the product and the solvent was approximately 3000 g. The desired product was then precipitated by the slow addition of 11.0 liters of hexane to the stirred CHC13 solution, followed by overnight storage at 4 ° C. The product was isolated by filtration and the solid was rinsed twice with a solvent combination of 900 ml of hexane and 150 ml of CHC13. By drying the solid, 900 g of N- [N '- (t-butyloxycarbonyl) -3-aminopropyl] -3-aminopropyl] -methacrylamide were obtained., p.f. 85.8 ° C by DSC. The analysis on an NMR spectrometer was consistent with the desired product: XH NMR (CDC13), amide NH's 6.30, 6.80, 4.55-5.10 (m 2H), vinyl protons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90 -3.45 (m, 4H), methyl 1.95 (m, 3H), remaining methylene 1.50, 1.90 (m, 2H), and t-butyl 1.40 (s, 9H). A 2-liter, 2-liter round bottom flask was equipped with an overhead stirrer and a gas spray tube. 700 ml of methanol was added to the flask and cooled on an ice bath. While stirring, the HCl gas was bubbled into the solvent at a rate of about 5 liters / minute for a total of 40 minutes. The molarity of the final HCl / MeOH solution was determined to be 8.5 M per titration with 1 N NaOH using phenolphthalein as an indicator. The N- [N '- (t-butyloxycarbonyl) -3-aminopropyl] methacrylamide, 900 g (3.71 moles), is added to a 5 liter Morton flask equipped with a top stirrer and an outlet adapter for the gas, followed by by the addition of 1150 ml of methanol solvent. Some solids remained in the flask with this volume of the solvent. The phenothiazine, 30 mg, was added as an inhibitor, followed by the addition of 655 ml (5.57 moles) of the 8.5 M HCl / MeOH solution. The solids dissolved slowly with the evolution of the gas but the reaction was not exothermic. The mixture was stirred overnight at room temperature to ensure complete reaction. Any solids were then removed by filtration and an additional 30 mg of phenothiazine was added. The solvent was then distilled under reduced pressure and the resulting solid residue was converted to an azeotrope with 3 X 1000 ml of isopropanol with evaporation under reduced pressure. Finally, the product was dissolved in 2000 ml of refluxing isopropanol and 4000 ml of ethyl acetate were slowly added with stirring. The mixture was allowed to cool slowly and was stored at 4 ° C overnight. Compound II was isolated by filtration and dried to constant weight, yielding 630 g with a melting point of 124.7 ° C by DSC. The analysis on an NMR spectrometer was consistent with the desired product: XH NMR (D20) 5.60 vinyl protons, 5.30 (m 2 H), methylene adjacent to N amide 3.30 (t, 2H), methylene adjacent to N of the amine 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-210 (m, 2H). The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for example, in Example 3.

Example 3 Preparation of N- [3- (4-benzoylbenzamido) propyl] ethacrylamide (BBA-APMA) (Compound III) Compound II 120 g (0.672 moles), prepared according to the general method described in Example 2, was added to a dry, 2-liter, three-necked round bottom flask equipped with an overhead stirrer. Phenothiazine, 23-25 mg, was added as an inhibitor, followed by 800 ml of chloroform. The suspension was cooled down to 10 ° C on an ice bath and 172.5 g (0.705 mole) of Compound I, prepared according to the general method described in Example 1, were added as a solid. The triethylamine, 207 ml (1485 moles), in 50 ml of chloroform was then added dropwise over a period of 1-1.5 hours. The ice bath was removed and stirring was continued at room temperature for 2.5 hours, the product was then washed with 600 ml of 0.3 N HCl and 2 x 300 ml of 0.07 N HCl. After drying over sodium sulfate, it was removed the chloroform under reduced pressure and the product was recrystallized twice from toluene: chloroform 4: 1 using 23-25 mg of phenothiazine in each recrystallization to prevent polymerization. The typical yields of Compound III were 90% with a melting point of 147-151 ° C. The analysis on an NMR spectrometer was consistent with the desired product: XH-NMR (CDC13) aromatic protons 7.20-7.95 (m, 9H), amide NH 6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m , 2H), methylene adjacent to the N of amide 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H), and remaining methylene 1.50-2.00 (m, 2H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for example, in Examples 9-11.

Example 4 Preparation of N-succinimidyl 6-maleimidohexanoate (MAL-EAC-NOS) (Compound IV) A functionalized monomer was prepared in the following manner, and was used as described in Examples 9 and 12 to introduce the activated ester groups onto the backbone of a polymer. The 6-aminohexanoic acid, 100 g (0.762 mol), was dissolved in 300 ml of acetic acid in a 3-liter three-necked flask equipped with an overhead stirrer and a drying tube. The maleic anhydride, 78.5 g (0.801 mole), was dissolved in 200 ml of acetic acid and added to the solution of 6-aminohexanoic acid. The mixture was stirred one hour while heating on a boiling water bath, leading to the formation of a white solid. After cooling overnight at room temperature, the solid was collected by filtration and rinsed with 2 x 50 ml of hexane. After drying, the typical production of (Z) -4-oxo-5-aza-2-undecendioic acid was 158-165 g (90-95%) with a melting point of 160-165 ° C. The analysis on an NMR spectrometer was consistent with the desired product. 1H-NMR (DMS0-d6) amide proton 8.65-9.05 (m, 1H), vinyl protons 6.10, 6.30 (d, 2H), methylene adjacent to nitrogen 2.85-3.25 (m, 2H), methylene adjacent to carbonyl 2.15 (t, 2H), and remaining methylenes 1.00, 1.75 (m 6H). The (Z) -4-oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 mol), acetic anhydride, 68 ml (73.5 g, 0.721 mol), and phenothiazine, 500 mg, were added to a flask of round bottom of three necks of 2 liters equipped with a top agitator. Triethylamine, 91 ml (0.653 mol), and 600 ml of THF were added and the mixture was heated to reflux while stirring. After a total of 4 hours of reflux, the dark mixture is cooled to < 60 ° C and poured into a solution of 250 ml of 12 N HCl in 3 liters of water. The mixture is stirred 3 hours at room temperature and then filtered through a filter pad (Celite 545, J T. Baker, Jackson, TN) to remove the solids. The filtrate was extracted with 4 x 500 ml of chloroform and the combined extracts were dried over sodium sulfate. After adding 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from hexane: chloroform 2: 1 to give typical yields of 76-83 g (55-60%) with a melting point of 81-85 ° C. Analysis on an NMR spectrometer was consistent with the desired product: XH NMR (CDC13) maleimide protons 6.55 (s, 2H), methylene adjacent to nitrogen 3.40 (t, 2H), methylene adjacent to carbonyl 2.30 (t, 2H) ), and methylenes remaining 1.05-1.85 (m, 6H). The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100 ml of chloroform under an argon atmosphere, followed by the addition of 41 ml (0.47 moles) of oxalyl chloride. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure with 4 x 25 ml of additional chloroform used to remove the last of excess oxalyl chloride. The acid chloride was dissolved in 100 ml of chloroform, followed by the addition of 12 g (0.104 mole) of N-hydroxysuccinimide and 16 ml (0.114 mole) of triethylamine. After stirring overnight at room temperature, the product was washed with 4 x 100 ml of water and dried over sodium sulfate. Removal of the solvent gave 24 g of the product (82%) which was used without further purification. The analysis on an NMR spectrometer was consistent with the desired product: XH NMR (CDC13) maleimide protons 6.60 (s, 2H), methylene adjacent to nitrogen 3.45 (t, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), and remaining methylenes 1.15-2.00 (m, 6H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for example, in Examples 9 and 12.

Example 5 Preparation of N-Succinimidyl 6-methacrylamidohexanoate (MA-EAC-NOS) (Compound V) A functionalized monomer was prepared in the following manner, and was used as described in Example 11 to introduce the activated ester groups onto the backbone of a polymer. The 6-aminocaproic acid, 4.00 g (30.5 mmol), was placed in a dry round bottom flask equipped with a drying tube. The methacrylic anhydride, 5.16 g (33.5 mmol), was then added and the mixture was stirred at room temperature for 4 hours. The resulting thick oil was triturated three times with hexane and the remaining oil was dissolved in chloroform, followed by drying over sodium sulfate. After filtration and evaporation, a portion of the product was purified by flash chromatography on silica gel using a solvent system of methanol in 10% chloroform. The appropriate fractions were combined, 1 mg of the phenothiazine was added, and the solvent was removed under reduced pressure. The analysis on an NMR spectrometer was consistent with the desired product: 1H NMR (CDC13) carboxylic acid proton 7.80-8.20 (broad, 1H), amide proton 5.80-6.25 (broad, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.00-3.45 (m, 2H), methylene adjacent to carbonyl 2.30 (t, 2H), methyl group 1.95 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H). The 6-methacrylamidohexanoic acid, 3.03 g (15.2 mmol), is dissolved in 30 ml of dry chloroform, followed by the addition of 1.92 g (16.7 mmoles) of N-hydroxysuccinimide and 6.26 g (30.4 mmoles) of the 1, 3- dicyclohexylcarbodiimide. The reaction is stirred under a dry atmosphere overnight at room temperature. The solid was then removed by filtration and a portion was purified by flash chromatography on silica gel. The non-polar impurities were removed using a chloroform solvent, followed by elution of the desired product using a 10% tetrahydrofuran solvent in chloroform. The appropriate fractions were pooled, 0.2 mg of the phenothiazine was added, and the solvent was evaporated under reduced pressure. This product, which contains small amounts of 1,3-dicyclohexylurea as an impurity, was used without further purification. The analysis on an NMR spectrometer was consistent with the desired product: XH NMR (CDC13) amide proton 5.60-6.10 (amp., 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.05 -3.40 (m, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), methyl 1.90 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for example, in Example 11.

Example 6 Preparation of 4- [Bromomethyl] benzophenone (BMBP) (Compound VI) The 4-methylbenzophenone, 750 g (3.82 mol) is added to a 5 liter Morton flask equipped with an overhead stirrer and dissolved in 2850 ml of benzene. The. The solution is then heated to reflux, followed by the dropwise addition of 610 g (3.82 moles) of bromine in 330 ml of benzene. The rate of addition was about 1.5 ml / minute and the flask was illuminated with a 90-watt halogen lamp (90 joules / sec) to initiate the reaction. A timer was used with the lamp to provide a duty cycle of 10% (lit 5 seconds, off 40 seconds), followed in one hour by a duty cycle of 20% (lit 10 seconds, off 40 seconds). At the end of the addition, the product was analyzed by gas chromatography and found to contain 71% of the desired Compound VI, 8% of the dibromo product, and 20% of 4-methylbenzophenone that did not react. After cooling, the reaction mixture was washed with 10 g of sodium bisulfite in 100 ml of water, followed by washing with 3 x 200 ml of water. The product was dried over sodium sulfate and recrystallized twice from toluene: hexane 1: 3. After drying under vacuum, 635 g of Compound VI are isolated, yielding 60% yield and having a melting point of 112-114 ° C. The analysis on an NMR spectrometer was consistent with the desired product: XH-NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H) and benzyl protons 4.48 (s, 2H). The final compound was stored for use in the preparation of a photoactivatable chain transfer agent as described in Example 7.

Example 7 Preparation of N- (2-mercaptoethyl) -3,5-bis (4-benzoylbenzyloxy) benzamide (Compound VII) The 3,5-dihydroxybenzoic acid, 46.2 g (0.30 mmol), was weighed into a 250 ml flask equipped with a Soxhiet extractor and condenser. Methanol, 48.6 ml, and concentrated sulfuric acid, 0.8 ml, were added to the flask and 48 g of 3A molecular sieves were placed in the Soxhiet extractor. The extractor was filled with methanol and the mixture was heated to reflux overnight. Chromatographic analysis of the gas of the resulting product showed a 98% conversion to the desired methyl ester. The solvent was removed under reduced pressure to give approximately 59 g of the crude product. The product was used in the next step without further purification. A small sample was previously purified for NMR analysis, leading to a spectrum consistent with the desired product: 1H NMR (DMS0-d6) aromatic protons 6.75 (d, 2H), and 6.38 (t, 1H), and methyl ester 3.75 (s, 3H). The complete methyl ester product above was placed in a 2 liter vessel with an overhead stirrer and condenser, followed by the addition of 173.25 g. (0.63 moles) of Compound VI, prepared according to the general method described in Example 6, 207 g (1.50 moles) of potassium carbonate, and 1200 ml of acetone. The resulting mixture was then refluxed overnight to give the complete reaction as indicated by thin layer chromatography (CCD). The solids were removed by filtration and the acetone was evaporated under reduced pressure to give 49 g of the crude product. The solids were diluted with 1 liter of water and extracted with 3 x 1 liter of chloroform. The extracts were combined with the acetone-soluble fraction and dried over sodium sulfate, yielding 177 g of the crude product. The product was recrystallized from acetonitrile to give 150.2 g of a white solid, a yield of 90% for the first two steps. The melting point of the product was 131.5 ° C (DSC) and the analysis on an NMR spectrometer was consistent with the desired product: XH-NMR (CDC13) aromatic protons 7-25-7.80 (m, 18H), 7.15 (d , 2H), and 6.70 (t, 1H), benzylic protons 5.05 (s, 4H), and methyl ester 3.85 (s, 3H). The methyl 3, 5-bis (4-benzoylbenzyloxy) benzoate, 60.05 g (0.108 mol), are placed in a 2-liter flask, followed by the addition of 120 ml of water, 480 ml of methanol, and 4.68 g ( 0.162 moles) of sodium hydroxide. The mixture is refluxed for three hours for complete hydrolysis of the ester. After cooling, the methanol was removed under reduced pressure and the sodium salt of the acid was dissolved in 2400 ml of hot water. The acid was precipitated using concentrated hydrochloric acid, filtered, washed with water, and dried in a vacuum oven to give 58.2 g of a white solid (99% yield). The melting point on the product was 188.3 ° C (DSC) and the analysis on the NMR spectrometer was consistent with the desired product: 1H-NMR (DMSO-d6) aromatic protons 7.30-7.80 (m, 18H), 7.15 ( d, 2H), and 6.90 (t, 1H), and benzyl protons 5.22 (s, 4H). The 3,5-bis (4-benzoylbenzyloxy) benzoic acid, 20.0 g (36.86 mmol), was added to a 250 ml vessel, followed by 36 ml of toluene, 5.4 ml (74.0 mmoles) of thionyl chloride, and 28 μl of N, N-dimethylformamide. The mixture is refluxed for four hours to form the acid chloride. After cooling, the excess solvent and thionyl chloride were removed under reduced pressure. The residual thionyl chloride was removed by four additional evaporations using 20 ml of chloroform each. The crude material was recrystallized from toluene to give 18.45 g of the product, 89% yield. The melting point on the product was 126.9 ° C (DSC) and the analysis on an NMR spectrometer was consistent with the desired product: 1H NMR (CDC13) aromatic protons 7.30-7.80 (m, 18H), 7.25 (d, 2H), and 6.85 (t, 1H), and benzylic protons 5.10 (s, 4H). The 2-aminoethiol hydrochloride, 4.19 g (36.7 mmol), was added to a 250 ml vessel equipped with an overhead stirrer, followed by 15 ml chloroform and 10.64 ml (76.5 mmole) triethylamine. After cooling the amine solution on an ice bath, a solution of 3,5-bis (4-benzoylbenzoyloxy) benzoyl chloride, 18.4 g (32.8 mmol), in 50 ml of chloroform is added dropwise over a period of time. 50 minutes Cooling on ice was continued for 30 minutes, followed by heating to room temperature for two hours, the product was diluted with 150 ml of chloroform and washed with 5 x 250 ml of 0.1 N hydrochloric acid. The product was dried over sulfate of sodium and recrystallized twice from toluene: hexane 15: 1 to give 13.3 g of the product, a yield of 67%. The melting point on the product was 115.9 ° C (DSC) and the analysis on an NMR spectrometer was consistent with the desired product: XH-NMR (DMSO-de) aromatic protons 7.20-7.80 (m, 18H), 6.98 ( d, 2H), and 6.65 (t, 1H), NH amide 6.55 (broad t, 1H), benzyl protons . 10 (s, 4H), methylene adjacent to N amide 3.52 (c, 2H), methylene adjacent to SH 2.10 (c, 2H), and SH 1.38 (t, 1H). The final compound was stored for use as a chain transfer agent in the synthesis of photoactivatable polymers as described, for example, in Example 12.

Example 8 Preparation of N-Succinimidyl, l, 1- (4-benzoylbenzamido) undecanoate (BBA-AUD-NOS) (Compound VIII) Compound I (50 g, 0.204 mole), prepared according to the general method described in example 1, was dissolved in 2500 ml of chloroform, followed by the addition of a solution of 43.1 g (0.214 mole) of acid 11- aminoundecanoic and 60.0 g (1.5 moles) of sodium hydroxide in 1500 ml of water. The mixture was stirred vigorously for one hour in a 5 liter Morton flask to secure the. complete mixing of the two layers. The mixture was acidified with 250 ml of the concentrated hydrochloric acid and stirred for an additional 30 minutes. The organic layer was separated and the aqueous layer extracted with 3 x 500 ml of chloroform. The combined organic extracts were dried over sodium sulfate, filtered, and evaporated to give a solid. The product was recrystallized from toluene to give 68.37 g (82%) of 11- (-benzoylbenzamido) undecanoic acid with a melting point of 107-109 ° C. The analysis on an NMR spectrometer was consistent with the desired product: 1H-NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H), amide NH 6.30 (broad t, 1H), methylene adjacent to N of amide 3.35 (m, 2H), methylene adjacent to carbonyl 2.25 (t, 2H), and remaining methylenes 1.00-1.80 (m, 16H). The 11- (4-benzoylbenzamido) undecanoic acid, 60.0 g (0.146 mol), was dissolved with heating in 1200 ml of anhydrous 1,4-dioxane in a 2000 ml flask dried in an oven. After cooling to room temperature, 17.7 g (0.154 mol) of N-hydroxysuccinimide and 33.2 g (0.161 mol) of 1,3-dicyclohexylcarbodiimide were added to the solution and the mixture is stirred overnight under a dry atmosphere. . The solids were then removed by filtration, rinsing the filter cake with 1,4-dioxane. The solvent was then removed under vacuum and the product was recrystallized twice from ethanol. After complete drying in a vacuum oven, 53.89 g (73% yield) of a white solid with a melting point of 97-99 ° C were obtained. The analysis on an NMR spectrometer was consistent with the desired product. XH-NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H), NH-amide 6.25 (broad t, 1H), methylene adjacent to N-amide 3.35 (m, 2H), methylenes in the succinimidyl ring 2.75 ( s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), and remaining methylenes 1.00-1.90 (m, 16H).

Example 9 Preparation of Acrylamide Copolymer, BBA-APMA, and MAL-EAC-NOS (Photo PA Random-PolyNOS) (Compounds IX, A-D) A photoactivatable copolymer of the present invention was prepared in the following manner. The acrylamide, 4,298 g (60.5 mmol), was dissolved in 57.8 ml of tetrahydrofuran (THF), followed by 0.219 g (0.63 mmol) of Compound III; prepared according to the general method described in Example 3, 0.483 g (1.57 mmoles) of Compound IV, prepared according to the general method described in Example 4, 0.058 ml (0.39 mmoles) of N, N, N ', N'-tetramethylethylenediamine (TEMED), and 0.154 g (0.94 mmoles) of 2,2'-azobisisobutyronitrile (AIBN). The solution was deoxygenated with a helium spray for 3 minutes, followed by an argon spray for an additional 3 minutes. The sealed container was then heated overnight at 60 ° C to complement the polymerization. The solid product was isolated by filtration and the filter cake was rinsed thoroughly with THF and CHC13. The product was dried in a vacuum oven at 30 ° C to give 5.34 g of a white solid. The NMR analysis (DMS0-d6) confirmed the presence of the NOS group at 2.75 ppm and the charge of the photo-group was determined to be 0.118 mmoles BBA / g of the polymer. The MAL-EAC-NOS composed 2.5 mol% of the polymerizable monomers in this reaction to give Compound IX-A. The above procedure was used to prepare a polymer having 5 mol% of Compound IV. Acrylamide, 3.849 g (54.1 mmol), was dissolved in 52.9 ml of THF, followed by 0.213 g (0.61 mmol) of Compound VI, prepared according to the general method described in Example 3, 0.938 g (3.04 mmol) of Compound IV , prepared according to the general method described in Example 4, 0.053 ml (0.35 mmole) of TEMED and 0.142 g (0.86 mmole) of AIBN. The resulting solid, Compound IX-B, when isolated as described above, gave 4.935 g of the product with a photogram loading of 0.101 mmol BBA / g of the polymer. The above procedure was used to prepare a polymer having 10 mol% of compound IV. Acrylamide, 3.241 g (45.6 mmol), was dissolved in 46.4 ml of THF, followed by 0.179 g (0.51 mmol) of Compound III, prepared according to the general method described in Example 3, 1579 g (5.12 mmol) of the Compound IV, prepared according to the general method described in Example 4, 0.047 ml (0.31 mmol) of TEMED and 0.126 g (0.77 mmol) of AIBN. The resulting solid, Compound IX-C when it was isolated as described above, gave 4.785 g of the product with a photogram loading of 0.098 mmol BBA / g of the polymer. A procedure similar to the previous procedure was used to prepare a polymer having 2.5 mol% of Compound IV and 2 mol% of Compound III. Acrylamide, 16.43 g (231.5 mmol); compound III, prepared according to the general method described in Example 3, 1.70 g (4.85 mmol); Compound IV, prepared according to the general method described in Example 4, 1.87 g (6.06 mmol), and THF (222 ml) were stirred in a round bottom flask with an argon spray at room temperature for 15 minutes. TEMED, 0.24 ml (2.14 mmol), and AIBN, 0.58 g (3.51 mmol), were added to the reaction. The reaction was then refluxed for 4 hours under an argon atmosphere. The resulting solid, Compound IX-D, when isolated as described above, gave 19.4 g of the product with a photographic filler of 0.23 mmole of BBA / g of the polymer.

Example 10 Preparation of Acrylamide Copolymer, BBA-APMA, and [3- (methacryloylamino) propyl] trimethylammonium Chloride (Photo PA- Random PolyQuat (Compounds X, A-B) A photoactivatable copolymer of the present invention was prepared in the following manner. The acrylamide, 10,681 g (0.150 moles), was dissolved in 150 ml of dimethyl sulfoxide (DMSO), followed by 0.592 g (1.69 mmoles) of Compound III, prepared according to the general method described in Example 3, 3727 g. (16.90 mmol) of [3- (methacryloylamino) propyl] trimethylammonium chloride (MAPTAC), supplied as 7.08 ml of a 50% aqueous solution, 0.169 ml (1.12 mmole) of TEMED and 0.333 g (2.03 mmole) of AIBN. The solution was deoxygenated with a helium spray for 4 minutes, followed by an argon spray for an additional 4 minutes. The sealed container was then heated overnight at 55 ° C to complement the polymerization. The DMSO solution was diluted with water and dialyzed against deionized water using 12,000-14,000 molecular weight intercalation tubing. Lyophilization of the resulting solution gave 14.21 g of a white solid. The NMR analysis (D20) confirmed the presence of the methyl groups on the quaternary ammonium groups at 3.10 ppm and the charge of the photo-group was determined to be 0.101 mmol of BBA / g of the polymer. Compound III constituted 1 mol% of the polymerizable monomer in this reaction to give Compound X-A. The above procedure was used to prepare a polymer having 2 mol% of Compound III. The acrylamide, 10,237 g (0.144 moles), was dissolved in 145 ml of DMSO, followed by 1148 g (3.277 mmoles) of Compound III, prepared according to the general method described in Example 3, 3.807 g (17.24 mmoles) of MAPTAC, supplied as 7.23 ml of a 50% aqueous solution, 0.164 ml (1.09 mmoles) of TEMED and 0.322 g (1.96 mmoles) of AIBN. The treatment as described above gave 12.54 g of the product (Compound X-B) with a photogram loading of 0.176 mmole of BBA / g of the polymer.

Example 11 Preparation of Acrylamide Copolymer, BBA-AMPA, MA-FAC-NOS, and [3- (methacryloylamino) propyl] trimethylammonium Chloride (Photo PA-PolyNOS-PolyQuat) (Compound XI) A photoactivatable copolymer of the present invention was prepared in the following manner. Water in the commercially available 50% aqueous MAPTAC was removed by azeotropic distillation with chloroform. The aqueous MAPTAC solution, 20 ml containing 10.88 g MAPTAC, was diluted with 20 ml of DMSO and 100 ml of chloroform. The mixture is refluxed in a liquid-liquid extract heavier than water containing anhydrous sodium sulfate for a total of 80 minutes. A slow flow of air was maintained during reflux to inhibit the polymerization of the monomer. At the end of the reflux, the excess chloroform was removed under reduced pressure to leave a DMSO solution of MAPTAC at a concentration of approximately 352 mg / ml. The acrylamide, 1.7 g (23.90 mmoles), was dissolved in 57.7 ml of dimethyl sulfoxide (DMSO), followed by 0.215 g (0.614 mmoles) of Compound III, prepared according to the general method described in Example 3, 1.93 ml (0.677 g, 3.067 mmol) of the previous MAPTAC / DMSO solution, 0.91 g (3.068 mmole) of Compound V, prepared according to the general method described in Example 5, and 0.060 g (0.365 mmole) of AIBN. The solution was deoxygenated with a helium spray for 4 minutes, followed by an argon spray for an additional 4 minutes. The sealed container was then heated overnight at 55 ° C to complement the polymerization. The polymer was isolated by pouring the reaction mixture into 600 ml of diethyl ether. The solids were separated by centrifugation and the product was washed with 200 ml of diethyl ether and 200 ml of chloroform. Evaporation of the solvent under vacuum gave 3.278 g of the product with a photocharge of 0.185 mmoles of BBA / g of the polymer.

Example 12 Copolymer of Acrylamide and MAL-EAC-NOS using N- (2-mercaptoethyl) -3,5-bis (4-benzoylbenzoyloxy) benzamide (Diphoto PA-PolyNOS of the end point) (Compound XII) A photoactivatable copolymer of the present invention was prepared in the following manner. The acrylamide, 3.16 g (44.5 mmol), was dissolved in 45.0 ml of tetrahydrofuran, followed by 0.164 g (1 mmol) of AIBN, 0.045 ml (0.30 mmol) of TEMED, 0.301 g (0.5 mmol) of Compound VII, prepared from according to the general method in Example 7, and 1539 g (5 mmoles) of Compound IV, prepared according to the general method described in Example 4. The solution was deoxygenated with a helium plug for 4 minutes, followed by a sprayed argon for about 4 additional minutes. The sealed container was then heated overnight at 55 ° C to complement the polymerization. The precipitated polymer was isolated by filtration and washed with chloroform. The final product was dried in a vacuum oven to provide 4,727 g of the polymer having a photo-group charge of 0.011 mmole of BBA / g of the polymer.

Example 13 Copolymer of N- [3- (dimethylamino) propyl] ethacrylamide and BBA-APMA (Photo Poly Tertiary Random Aramid) (Compound XIII) A photoactivatable copolymer of the present invention was prepared in the following manner. The N- [3- (dimethylamino) propyl] methacrylamide, 33.93 g (0.2 mol), is dissolved in 273 ml of DMSO, followed by 16.6 ml of concentrated HCl and 6.071 g (17.3 mmol) of Compound III, prepared in accordance with the general method described in Example 3. Finally, 0.29 ml (1.93 mmoles) of TEMED, 0.426 g (2.6 mmoles) of AIBN, and 100 ml of water were added to the reaction mixture. The solution was deoxygenated with a helium spray for 10 minutes and the top space was then filled with argon. The sealed container was heated overnight at 55 ° C to complement the polymerization. The product was then dialyzed against deionized water for several days using 12,000-14,000 M CO tubing. The product was filtered after dialysis to remove any solids and lyophilized to give 47.27 g of the solid product. The polymer was finished having a photocharge of 0.33 mmoles of BBA / g of the polymer.

Example 14 Preparation of N-succinimidyl 5-oxo-6-aza-8-nonenoate, (Allyl-GLU-NOS) (Compound XIV) A functional monomer was prepared in the following manner, and was used in Example 15 to introduce the activated ester groups onto the polymer backbone. The glutaric anhydride, 20 g (0.175 moles), was dissolved in 100 ml of chloroform. The glutaric anhydride solution was cooled to < 10 ° C using an ice bath. The allyl amine, 10 g (0.177 moles), was dissolved in 50 ml of chloroform and added to the cooled glutaric anhydride solution with stirring. The rate of addition of the allylamine was adjusted to maintain the reaction temperature < 10 ° C. After the addition of the allylamine was completed, the reaction solution was allowed to come to room temperature while stirring overnight. After removal of the solvent, the isolated 5-oxo-6-aza-8-nonenoic acid was quantified in 31.4 g (105% crude) with a double DSC melting point of 35.1 ° C and 44.9 ° C. The 300 MHz NMR analysis was consistent with the desired product: 1H NMR (CDC13) amide proton 6.19 (amp., 1H), vinyl protons 5.13, 5.81 (m, 3H), methylene adjacent to N of amide 3.85 (m, 2H), methylene adjacent to carbonyls 2.29, 2.39 (t, 4H), and central methylene 1.9 (m, 2H). The 5-oxo-6-aza-8-nonenoic acid, 20.54 g (0.12 mol), N-hydroxysuccinimide (NHS), 15.19 g (0.13 mol), and 204 ml of dioxane were placed in a 3-necked round bottom flask. 1 liter necks equipped with a top stirrer and an addition funnel. The dicyclohexylcarbodiimide ("DCC"), 29.7 g (0.144 mol), was dissolved in 50 ml of dioxane and placed on the addition funnel. The DCC solution was added with stirring to the acid / NHS solution for 20 minutes, and the resulting mixture is allowed to stir at room temperature overnight. The reaction mixture is filtered on a Büchner funnel to remove dicyclohexylurea (DCU). The solid was washed with 2 x 100 ml of dioxane. The solvent was evaporated to give 41.37 g of the residue, which was washed with 4 x 75 ml of hexane. After the solvents were removed, the yield of the crude NOS ester was 41.19 g. Recrystallization of the crude NOS product from toluene gave a 60% yield with a melting point of 90.1 ° C. The 300 MHz NMR analysis was consistent with the desired product: XH NMR (CDCl 3) proton of amide 6.02 (amp., 1H), vinyl protons 5.13, 5.80 (m, 3H), methylene adjacent to N of the amide 3.88 (m, 2H), protons of succinimidyl 2.83 (s, 4H), methylenes adjacent to carbonyls 2.31, 2.68 (t, 4H), and central methylene 2.08 (m, 2H). The final compound was stored for use in the synthesis of photoactivatable polymers as described in Example 15.

Example 15 Preparation of the copolymer of Vinylpyrrolidinone BBA-AMPA and Alil-GLÜ-NOS (Photo PVP-Random PolyNOS) (Compound XV) A photoactivatable copolymer of the present invention was prepared in the following manner. Vinylpyrrolidinone, 4.30 g (38.73 mmol), was dissolved in 5.2 ml of DMSO in the company of 0.14 g (0.41 mmol) of compound III, prepared according to the general method described in Example 3, 0.55 g (2.06 mmol) of the compound XIV, prepared according to the general method described in Example 14, by the combination of 0.08 g (0.49 mmole) of AIBN and 0.005 ml (0.033 mmole) of TEMED. The solution was deoxygenated with a helium spray for 3 minutes. The upper space was replaced with argon, and the container was sealed for heating overnight at 55 ° C. The viscous solution was diluted with 15 ml of chloroform, and then precipitated by pouring into 200 ml of diethyl ether. The precipitate was dissolved in 15 ml of chloroform, and precipitated a second time in 200 ml of ether. The product was dried in a vacuum oven at 30 ° C to give 4.79 g of a white solid. The NMR analysis (CDC13) confirmed the presence of the NOS group at 2.81 ppm and the charge of the photo-group was determined to be 1.1 mmol of BBA / g of the polymer. The alyl-GLU-NOS composed 5.0 mol% of the polymerizable monomers in this reaction to give the compound XV.

Example 16 Comparison of Random Photo PA-PolyNOS (Compound IX-C) with Random Photo PA-PolyNOS-PolyQuat (Compound XI) on Polystyrene Microcavity Plates (PS) Compound IX-C and Compound XI were dissolved separately in deionized water at 5 mg / ml. The PS plates (PS, Binding Media, Corning Costar, Cambridge, MA) containing 100 μl of Compound IX-C and Compound XI in the separate wells were illuminated with a Dymax lamp (model No. PC-2, Dymax Corporation, Torrington, CT) which contained a Heraeus bulb (WC Heraus GmbH, Hanau, Federal Republic of Germany). The duration of the illumination was for 1.5 minutes at an intensity of 1-2 m / cm2 in the wavelength range of 330-340 mm. The coating solution was then discarded and the cavities were air dried for two hours. The plates were then illuminated for an additional minute. The coated plates were immediately used to immobilize the oligonucleotides stored in a sealed pouch for up to 2 months. The capture probe of the 50-base oligomer (-mer) 5'-NH2-GTCTGAGTCGGAGCCAGGGCGGCCGCCAACAGCAGGAGCAGCGGGGCGG-3 '(ID 1) (synthesized with a 5'-amino modifier containing a spacer with C-12) at 10 pmol / cavity it was incubated in PS cavities in a 50 mM phosphate buffer solution, pH 8.5, 1 mM EDTA at 37 ° C for one hour. Hybridization was carried out as follows using the detection probe of the 5 'Biotin CCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCCTGGCTCCGACTC AGAC-3' (ID 3) complementary or the oligo 5'-Biotin-CGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACA CAGAA-3 '(ID 4), both of which were synthesized with a 5'-biotin modification. The plates with the immobilized capture probe were washed with a salted solution buffered with phosphate (PBS, 10 M Na2P04, 150 mM NaCl, pH 7.2) containing 0.05% of Tween 20 using a Microplate Auto Washer (model EL 403H, Bio -Tek Instruments, Winooski, VT). The plates were then blocked at 55 ° C for 30 minutes with the hybridization buffer, which consisted of 5X SCC (0.75 M NaCl, 0.075 M citrate, pH 7.0), 0.1% lauroyl sarcosine, 1% casein, and dodecyl 0.02% sodium sulfate. When the detection probe was hybridized to the capture probe, 50 fmoles of the detection probe in 100 μl were added per cavity and incubated for one hour at 55 ° C. The plates were then washed with 2X SSC containing 0.1% sodium dodecyl sulfate for 5 minutes at 55 ° C. The bound detection probe was evaluated by adding 100 μl of a conjugate of streptavidin and horseradish peroxidase (SA-HRP, Pierce, Rockford, IL) at 0.5 μg / ml and incubated for 30 minutes at 37 ° C. The plates were then washed with PBS / Tween, followed by the addition of the peroxidase substrate (H202 and tetramethylbenzidine, Kirkegard and Perry Laboratories, Gaithersburg, MD) and measurement at 655 nm on a microcavity plate reader (model 3550, Bio -Rad Labs, Cambridge, MA). The plates were read at 10 minutes. The results listed in Table 1 indicate that microcavity plates coated with Compound IX-C do not effectively immobilize capture probes derived from amine. However, by comparison with compound XI, as a coating, they provide significant binding and good hybridization signals. Compound IX-C reactive most likely passive surfaces and prevented the association of capture oligos. In contrast when Compound XI was used, the oligonucleotide was attracted to the surface by the ionic interactions where it could then be covalently linked with the NOS groups.

Table 1: Hybridization Signals (A65s) of PS Microcavity Plates Coated with Compound IX-C and Compound XI.

Example 17 Recubrim ent of Various Microcavity Plates with a Mixture of Random PA-PolyNOS Photo (Compound IX-B) and Photo PA-Po Random yuat (Compound X-B) A coating solution containing a mixture of 5 mg / ml of compound IX-B and 0.5 mg / ml of Compound X-B was prepared in deionized water. This mixture was used to treat multi-cavities of polypropylene (PP, Corning Costar, Cambridge, MA), PS, polycarbonate (PC, Corning Costar, Cambridge, MA) and polyvinyl chloride (PVC, Dynatech, Chantilly, VA) as described in Example 16. A 30-mer 5 'NH2 capture oligonucleotide GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC-3' (ID 2), (synthesized with a 5'-amino modifier containing a C-12 spacer) at 0.03, 0.1, 0.3 , 1, 3, or 10 pmol / cavity was incubated at 4 ° C overnight. Hybridization was performed as previously described in Example 16 using the complementary detection oligonucleotide ID 3 or the non-complementary ID 4 oligo. Since the PP plates were not optically clear, the contents of each cavity were transferred to the PS cavities after a 20 minute incubation with the chromogenic substrate. Hybridization signals were measured on the PS plates. The other plates were read without transfer at 10 minutes. The levels of the signal are only comparable within the same group of substrates due to the different geometries of the microcavity plates made of different materials. Table 2 lists the hybridization signals and shows the relationship between the intensity of the hybridization signals and the amount of the capture probe applied to several microcavity plates coated with a mixture of compound IX-B and Compound X-B. On the plates of PP and PVC, the adsorption of the probes was very low and the coatings with the polymeric reagents improved the signals dramatically. The signal was increased with the increasing capture probe added to the coated cavities, but completely leveled to approximately a capture of 3 pmol / cavity. The plateau in the amount of signal generated was not due to a saturation level of the hybridization, but rather to the limits of the color change reaction in the colorimetric assay. The oligonucleotide derivatives are efficiently adsorbed onto the uncoated PS and PC microcavity plates and lead to specific hybridization signals. Cros et al. (U.S. Patent No. 5,510,084) also reported that amine-functionalized oligonucleotides were successfully adsorbed onto polystyrene microcavities plates by unknown mechanisms. However, there was a marked variability in the amount of absorption on the PS plates not coated between the different groups or sets (Chevier et al., FEMS 10 245, 1995).

Table 2: Hybridization (Aess) Signs of Various Materials of the Microcavity Plaque Coated With a Mixture of Compound IX-B and Compound X-B.

Aggregate Capture Oligonucleotides (pmoles / cavity) 0.03 0.1 0.3 1 3 10 Comp NC Comp NC NC Comp NC Comp NC Comp NC Comp NC PP Uncoated 0.083 0.082 0.076 0.072 0.0760.074 0.088 0.07 0.070 0.067 0.078 0.073 Coated 0.541 0.099 1.070 0.099 1.769 0.091 2.283 0.094 2.582 0.141 2.490 0.320 PVC Uncoated 0.074 0.079 0.081 0.075 0.097 0.078 0.137 0.076 0.215 0.081 0.337 0.092 Coated 0.423 0.1160.875 0.110 1.326 0.112 1.583 0.142 1.628 0.186 1.60 0.332 PS Uncoated 0.235 0.099 0.435 0.091 0.827 0.090 1.205 0.093 1.380 0.093 1.404 0.136 Coated 0.435 0.121 0.801 0.105 1.177 0.116 1.401 0.132 1.470 0.132 1.487 0.302 PC Uncoated 0.6760.248 1.364 0.244 2.103 0.2562.701 0.2662.745 0.295 2.930 0.388 Coated 1.034 0.327 1.602 0.3062.1360.295 2.218 0.287 2.380 0.342 2.500 0.572 Comp .: The complementary detection probe was added for hybridization. NC: The non-complementary detection probe was added for hybridization.

Example 18 Evaluation of the Diphoto PA-polyNOS of the Final Point (Compound XII) and Photo PA-Poly uat at Random (Compound X-B) on Microcavities Plates of PP and PVC A coating solution containing a mixture of 5 mg / ml of Compound XII and 0.5 mg / ml of compound X-B was prepared with deionized water. This mixture of the two reagents was used to coat the microcavity plates of PP and PVC under conditions comparable to those described in Example 16. The capture oligonucleotide ID 2 from 30-mer to 0.03, 0.1, 1.3, or 10 pmol / cavity in 0.1 ml was incubated at 4 ° C overnight. Hybridization was carried out as described in Example 16 using the complementary ID 3 detection oligonucleotide or the non-complementary oligo ID 4. The hybridization signals listed in Table 3 demonstrate the relationship between the intensity of the hybridization signals and the amount of the capture probe applied to the microcavity plates of PP and PVC coated with a mixture of Compound XII and Compound X-B. The signal was increased with the increase of the capture oligonucleotides added to the coated cavities, but completely leveled out at approximately 1 pmol / cavity. The ratio of the signal with respect to the noise (from the complementary detection probes to the non-complementary ones) was as high as 26 and 11 for the coated PP and PVC surfaces, respectively.

Table 3: Hybridization Signals (A6ss) of the PP and PVC Plates Coated with the Mixture of Compound XII and Compound X-B. pmoles / cavity Microcavities plates Microcavities plates Aggregate capture of PVC PP Detection No comp. Detection No comp. Comp. Comp. 0.03 0.153 + 0.008 0.070 + 0.007 0.289 + 0.029 0.094 + 0.020 0. 1 0.537 + 0.042 0.075 + 0.009 0.759 + 0.054 0.104 + 0.014 0. 3 1.206 + 0.106 0.080 + 0.003 1.262 + 0.023 0.117 + 0.011 1 2.157 + 0.142 0.081 + 0.003 1.520 + 0.044 0.189 + 0.064 3 2.624 + 0.162 0.108 + 0.012 1.571 + 0.031 0.179 + 0.016 2.921 + 0.026 0.200 + 0.018 1.625 + 0.040 0.286 + 0.021 Example 19 Sequential Recubrimat with Random Photo PA-PolyQuat (Compound X-B) and BBA-AüD-NOS (Compound VIII) Compound X-B at 0.1 mg / ml in deionized water was incubated in PP and PVC cavities for 20 minutes. The plates were illuminated as previously described in Example 16 with the solution in the cavities for 1.5 minutes. The solution was discarded and the cavities were dried. Compound VIII at 0.5 mg / ml in isopropyl alcohol (IPA) was incubated in the cavities coated with Compound X-B for 5 minutes. The solution was then removed, the plate dried and illuminated as described in Example 16 for one minute after the cavities were dried. Capture oligonucleotide ID 2 from 30-mer at 0.03, 0.1, 0.3, 1, 3, or 10 pmol / well in 0.1 ml was incubated at 4 ° C overnight. Hybridization was carried out as described in Example 16 using the complementary ID 3 detection oligonucleotide or the non-complementary oligo ID 4. Table 4 contains the hybridization signals and shows the relationship between the intensity of the hybridization signals and the amount of the capture probe applied to the microcavity plates of PP and PVC coated with Compound XB followed by the coating with the Compound. VIII. The signal was increased with the increasing capture probe added to the coated cavities, but completely leveled to approximately 1 pmol / cavity of the capture oligo. The signals were up to 29 and 11 times higher for the coated PP and PCV surfaces, respectively, when compared to the uncoated controls.

Table 4: Hybridization Signs (A65s) of the Microcavities Plates of PP and PVC Coated with Compound X-B Followed by the Coating with Compound VIII.

Example 20 Comparison of Random Photo PA-PolyQuat (Compound X-A) with a Mixture of Random Photo PA-PolyNOS (Compound IX-A) and Random Photo PA-PolyQuat (Compound X-A) Compound X-A at 0.5 and 0.1 mg / ml was incubated in microcavity PP plates for 10 minutes. The plates were then illuminated as described in Example 16. A coating solution containing a mixture of Compound IX-A and Compound XA was prepared at two ratios, 5 / 0.5 mg / ml and 0.5 / 0.1 mg / ml of the Compound IX-A / Compound XA in deionized water to coat the microcavity plates of PP. The solution was incubated in the wells for 10 minutes and the wells were illuminated as described in Example 16. The ID-2 capture oligonucleotide from 30-mer to 1 pmol / well was incubated in each well at 37 ° C for one hour. hour. Hybridization was done as described in Example 16 using the oligonucleotide for the detection of complementary ID 3 or the non-complementary oligo ID 4. The results listed in Table 5 indicate that the coating containing the combination of compound IX-A and Compound X-A gave higher signals when compared to those of the coating of Compound X-A alone.

Table 5: Hybridization Signals (A6ss) of the Microcavities Plates of PP Coated with Compound X-A.

Example 21 Comparison of the Unmodified Oligonucleotide against Oligonucleotide Modified with Amine on Random Photo PA-PolyNOS (Compound IX-B) and Random Photo PA-PolyQuat (Compound X-B) on Coated Microcavities Plates A coating solution containing a mixture of Compound IX-B (5 mg / ml) and Compound X-B (0.5 mg / ml) is prepared in deionized water to coat the microcavity plates with PP, PS and PVC. The solution was incubated for approximately 10 minutes and illuminated as described in Example 16. The 5'-NH2-TTCTGTGTCTCC CGCTCCCAATACTCGGGC-3 '(ID 5) capture oligonucleotide of 30-me5r in 1 pmol / well was coupled to the cavities in the 50 M phosphate buffer, pH 8.5, 1 mM EDTA at 4 ° C overnight. Hybridization was performed as described in Example 16 using ID 4 of the oligonucleotide for the complementary detection or ID 3 of the non-complementary oligonucleotide. To determine the effect of the amine functionality of the capture oligo, a 30-mer 5 'capture probe -TTCTGTGTCTCC CGCTCCCAATACTCGGGC-3' unmodified (without amine) was also added to the coated surfaces and tested. The results shown in Table 6 indicate that when an oligonucleotide without the 5'-amino modification was used as the capture probe on surfaces coated with Compound IX-B / Compound XB, the hybridization signal was less than 30% of that with the modification of amine.

Table 6: Signals (A ^ s) Generated from the Hybridization Reactions with the Oligonucleotides either ID 5 or ID 6 on the Microcavity Plates Coated with Compound IX-B / Compound X-B.

No Aggregate Catch Unmodified Catch Modified amine Catch Detection Detection Detection Detection Detection Detection Comp. No Comp. Comp. Do not comp Comp. No Comp. PP Uncoated 0.032 + 0.001 0.036 + 0.004 0.033 + 0.001 0.036 + 0.001 0.037 + 0.005 0.033 + 0.001 Coated 0.038 + 0.002 0.040 + 0.001 0.555 + 0.041 0.044 + 0.001 1.915 + 0.029 0.066 + 0.003 PVC Uncoated 0.248 + 0.049 0.176 + 0.008 0.259 + 0.049 0.128 + 0.013 0.404 + 0.100 0.118 + 0.025 Coated 0.115 + 0.027 0.090 + 0.014 0.379 + 0.028 0.091 + 0.014 1.319 + 0.027 0.101 + 0.017 PS Uncoated 0.084 + 0.013 0.089 + 0.014 0.668 + 0.047 0.085 + 0.023 1.269 + 0.034 0.106 + 0.024 Coated 0.080 + 0.006 0.081 + 0.023 0.36 +0.010 0.089 + 0.015 1.437 + 0.012 0.098 + 0.005 Example 22 Oligonucleotide Loading Densities on Microcavity Plates Coated with Random Photo PA-PolyNOS (Compound IX-A) and Random Photo PA-PolyQuat (Compound X-A) The radiolabelled tests were carried out to determine the charge densities of the oligonucleotides and to verify the results of the colorimetric assay system. In this Example, the combination coatings of Compound IX-A and Compound XA were made on the PVC cavities as described in Example 16. The 30-mer capture oligonucleotides of ID 2 and ID 5 were immobilized on the coated cavities. The radiolabeled ID 2 probe was used to determine the charge density of the capture oligonucleotides immobilized on the surface of the cavity. A radio-labeled ID 3 detection probe, which was complementary with respect to ID 2, but not with respect to ID 5, was used to measure the hybridization reactions of the immobilized capture probes. The ID 2 and ID 3 oligonucleotides were radiolabelled at the 3 'end using the terminal transferase (Boehringer Mannheim, Indianapolis, IN) a-32P-ddATP (3000 Ci / mmole, Amersham, Arlington Heights, IL) according to manufacturer's specifications. The ID 2 and ID 5 unlabeled and ID 2 capture probes labeled with 32P were incubated in the cavities coated at 50 pmol / well for 2.25 hours at room temperature. The plates were washed and blocked as described in Example 16. Cavities with the unlabeled capture probes were hybridized with the 3P labeled ID 3 detection probe in the hybridization buffer for 1 hour at 55 ° C. The cavities containing the capture probe labeled with 32P were incubated in the hybridization buffer without the ID 3 probe. After washing three times with 2X SSC containing 0.1% SDS for 5 minutes at 55 ° C and three times with PBS / 0.05% Tween, the plates were cut into individual cavities and dissolved in tetrahydrofuran. The amount of radioactivity in each cavity was measured by the scintillation count in Aquasol-2 Fluor (DuPont NEN, Boston, MA). The results in Table 7 show that both Compound IX-A and Compound X-A were required to give a good immobilization of the capture probe. Also, increasing the concentrations of compound IX-A and Compound X-A increased the amount of the immobilized capture oligonucleotide. At the highest concentrations tested, the signal to noise ratio was greater than 3000 to 1.

Table 7: Densities of the Immobilized Capture Oligonucleotide and the Oligo Detection with 32P Hybridized.

Example 23 Comparison between Random Photo-Polyntiary Amin (Compound XIII), Random Photo-PA-PolyNOS (Compound IX-A) and a Mixture of Photo PA-PolyNOS (Compound IX-A) and Random Photo-Polinatorial Amina (Compound) XIII) Compound XIII at 0.02 mg / ml in deionized water is incubated in microcavity PP plates for 10 minutes. The wells were illuminated as described in Example 16. Compound IX-A was coated on the PP cavities at 2 mg / ml deionized water as described for Compound XIII. A coating solution containing a mixture of 2 mg / ml of Compound IX-A and 0.02 mg / ml of Compound XIII in deionized water was prepared and coated as described for Compound XIII. The 30-mer ID capture oligonucleotide at 5 pmole / well is incubated in each well at 37 ° C for one hour. Hybridization was done as described in Example 16 using the complementary ID 3 detection oligonucleotide and the non-complementary ID 4 oligonucleotide. The contents of each cavity were transferred to the PS cavities after a 10 minute incubation with the peroxidase substrate. The results listed in Table 8 indicate that the combination of Compound IX-A and Compound XIII gave higher signals compared to those of the coating of Compound IX-A or Compound XIII alone.

Table 8: Hybridization Signs (A65s) of Microcavity Plates Coated with PP.

Example 24 Immobilization of the Nucleic Acids Sequence on an Amine Derived Superie A copolymer of the present invention is prepared in the following manner. The acrylamide, 5.686 g (80.0 mmol), is dissolved in 100 ml of DMSO, followed by the addition of 3.083 g (10.0 mmol) of Compound IV, prepared according to the general method described in Example 4, and 2.207 g. (10.0 mmoles) of MAPTAC, supplied as a solution of Dry DMSO prepared according to the general method described in Example 11. The TEMED, 0.134 ml (0.89 mmol), and the AIBN, 0.197 g (1.20 mmol), are added to the mixture and the system was deoxygenated with a helium spray for 5 minutes, followed by a spray of argon for an additional 5 minutes. The sealed container is heated to 55 ° C to complement the polymerization. The polymer is isolated by pouring the reaction mixture into 800 ml of diethyl ether and centrifugation to remove the solids. The product is washed with 200 ml of diethyl ether, followed by 200 ml of chloroform. The polymer is dried under vacuum to remove the remaining solvent. A polymeric surface is derived by plasma treatment using a 3: 1 mixture of methane and ammonia gases. (See, for example, the general method described in U.S. Patent No. 5,643,580). A mixture of methane (490 SCCM) and ammonia (161 SCCM) is introduced into the plasma chamber in the company of the polymeric part to be coated. The gases are maintained at a pressure of 0.2-0.3 torr and a luminous discharge of 300-500 watts is established inside the chamber. The sample is treated for a total of 3-5 minutes under these conditions. The formation of an amine-derived surface is verified by a reduction in the contact angle of the water compared to the uncoated surface. The amine-derived surface is incubated for 10 minutes at room temperature with a 10. mg / ml solution of the above polymer in a 50 mM phosphate buffer, pH 8.5. Following this reaction time interval, the coating solution is removed and the surface is thoroughly washed with deionized water and completely dried. Immobilization of the oligomer capture probe and hybridization are performed as described in Example 16.

Example 25 Immobilization and Hybridization of Oligonucleotides on Coated Glass Slides with Photo-Polymeric NOS - Comparison of Coatings with and without Photo PA PolyQuat (Compound X-A) The glass microscope slides of soda and lime (Eire Scientific, Portsmouth, New Hampshire) were treated with silane by immersion in a mixture of p-tolyl dimethylchlorosilane (T-Silane) and N-decyl dimethylchlorosilane (D-Silane, United Chemical Technologies , Bristol, Pennsylvania), 1% of each in acetone, for 1 minute. After drying with air, the slides were cured in an oven at 120 ° C for one hour. The slides were then washed with acetone followed by immersion in DI water. The slides were further dried in an oven for 5-10 minutes. Compound IX-A, IX-D, and XV at various concentrations and with or without Compound XA, were sprayed onto the silane treated slide, which was then illuminated using a Dymax lamp (25 mjoules / cm2 as measured 335 nm with a bandpass filter of 10 nm on an International Ligth radiometer) while they are moistened, washed with water, and dried. The oligonucleotides were printed on the slides using a X, Y, Z motion controller to the position of a 0.06 cm (0.006") dull needle (internal diameter) filled with the oligonucleotide solution.Two oligonucleotides were immobilized with respect to to the prepared slides, one containing an amine on the 3 'end and the fluorescent Cy3 tag (Amersham, Arlington Heights, Illinois) on the 5' end, 5 'Cy3-GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC-NH2-3' (ID 7) ( amino modifier has a C12 spacer) and the other containing an amine on the 5 'end, 5'-NH2-TTCTGTGTCTCCCGCTCCCAATACTCGGGC-3' (ID 5) (the amino modifier has a C12 spacer). printed at a concentration of 8 pmol / μl in 50 mM sodium phosphate pH 8.5 containing 10% sodium sulfate and 1 mM EDTA The slides were placed overnight on a rack in a container sealed with sodium chloride satu to maintain a relative humidity of 75%. The slides printed with (ID 7) were then washed for 5 minutes in PBS / 0.5% Tween-20, for 90 minutes in a blocking buffer (0.2 M Tris with 10 mM ethanolamine) at 50 ° C, and for 2 hours in a buffer for washing (5X SSC, 0.1% N-lauryl sarcosine, and 0.1% sodium dodecyl sulfate). The slides were washed twice with water and centrifuged in a centrifuge machine for drying. They were then scanned using a Scan-Array 3000 fluorescent scanner from General Scanning (Watertown, Massachusetts) and the average intensities of the resulting spots or spots were measured. The slides printed with (ID 5) were washed for 5 minutes in PBS / 0.05% Tween-20 and for 30 minutes in the blocking buffer solution (0.2 M Tris with 10 mM ethanolamine) at 50 ° C. The slides were finally washed with water and dried in a centrifuge machine. The fluorescently labeled complementary oligonucleotide, 5'-Cy3-CGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGA A-3 '(ID 8), was hybridized to the slides by placing 10 μl of the hybridization solution (4X SSC, 0.1% N-lauryl sarcosine, 2 mg / ml tRNA ) on the slide and placing a coverslip on top. The slides were kept at high humidity (75%) at 50 ° C to prevent complete drying of the hybridization solution. The slides were then rinsed with 4X SSC, 2X SSC preheated at 50 ° C for 2 minutes, 2X SSC for 2 minutes, and then twice in 0.1X SSC for 2 minutes each. The slides were dried by centrifugation in a centrifuge machine. They were explored using an explorer or scanner Fluorescent General Scanning. The average intensities of the resulting spots or spots and the background levels were measured. The results listed in Table 9 show that coatings without the compound X-A immobilized slightly less the oligonucleotide but hybridization of a fluorescent oligonucleotide leads to a slightly higher signal. The resulting background is lower on the coatings which do not contain the compound X-A. It is also shown that polymers containing the PVP backbone compound (ie Compound XV) are effective to immobilize the DNA and give good hybridization results.

Table 9. Immunization and Hybridization of the Oligonucleotides for the Glass Microscope Slides. 1 Laser power adjusted to 60% and photomultiplier tube adjusted to 60% 2 Laser power adjusted to 80% and multiplier tube adjusted to 80% Example 26 Hybridization of the Immobilized PCR products on the Coated Glass Slides with the Oligonucleotide Detection Probe. Comparison between Photo-PA-PolyNOS Random (Compound IX-A) and a Mixture of Photo-PA-PolyNOS at Azar (Compound IX-A) and Random Photo-PA-PolyQuat (Compound X-A) The glass slides were coated with organosilane as described in Example 25. Compound IX-A at 1.25 mg / ml in water or a mixture of 1 mg / ml Compound IX-A and 0.25 mg / ml Compound XA in water it was coated on the glass slides treated with silane as described in Example 25.

The PCR products of the β-galactosidase gene were prepared in the customary manner by ATG Laboratories, Inc. (Eden Prairie). The primer with the modification of 5'-amino on the sense strand and the unmodified primer on the anti-sense strand were used to prepare the double-stranded PCR products at 0.5 and 1 kilobases (kb) of the length of The pairs. The control DNAs without the amine were also made. The DNAs at the concentration of 0.2 μg / μl in 80 mM of the sodium phosphate buffer solution, pH 8.5, and the 8% sodium sulfate were printed on the activated slides using TeleChem microarray or microwire dial terminals International (San José, CA). HE. allowed the coupling to proceed in a sealed container with 75% humidity overnight at room temperature. To evaluate the signals of the PCR products immobilized on the microgrids or microarrays, the slides were placed in boiling water for 2 minutes to denature the double-stranded DNA and to remove the unbound strand. The slides were then incubated with 50 mM ethanolamine in the buffer solution 0.1 M, pH 9 at 50 ° C for 15 minutes to block the residual reactive groups on the surfaces. The slides were then incubated with the pre-hybridization solution under glass coverslips at 50 ° C for 15 minutes to reduce non-specific backgrounds. The pre-hybridization solution contained 5X SSC, 5X Denhardt's solution (0.1 mg / ml each of bovine serum albumin, Ficoll and PVP), 0.1 mg / ml salmon sperm DNA and 0.1% SDS. Hybridization was then carried out with 20 fmoles / μl of a fluorescent complementary detection oligo, 5 '-Cy3-ACFCCGAGTTAACGCCATCA (ID 9), in the pre-hybridization solution overnight at 45 ° C. The slides were then washed and the hybridization signals scanned as described in Example 25. The results listed in Table 10 indicate that the glass slides coated with Compound IX-A and the mixture of Compound IX-A / XA had comparable signals. The amine-containing PCR product had hybridization signals at least 30 times higher than the unmodified DNA. The low level of the signals with an unmodified DNA was probably due to the side reactions between the amines on the heterocyclic bases for the activated surfaces.

Table 10. Signs of Hybridization with 0.5 Kb Immobilized DNA and an ID 9 of the Complementary Detection Oligonucleotide on the Glass Slides Coated with Compound IX-A / Compound X-A.

Example 27 Hybridization of Immobilized PCR products on the Glass Slides Coated with the Oligonucleotide Detection Probe - Comparison between SurModics and other Commercial Slides The PCR products of the cDNA clones can be attached to the positively charged glass surfaces, such as polylysine; DeRisi, et al., (Science, 278, 680-686, 1997), and a covalent approach having aldehyde groups has been reported by Schena (Schena et al., Proc. Nati, Acad. Sci. USA, 93, 10614-10619 ). In this example the PCR products were bound to these surfaces and the hybridization signals were compared with the coatings of this invention. The glass slides from SurModics were coated with the mixture of Compound IX-A and Compound XA as described in Example 25. Silylated glass slides having aldehyde groups reactive for immobilization of amine functionalized DNA were manufactured by CEL Associates, Inc. { Houston, TX). The polylysine glass slides were purchased from Sigma. The PCR products of 1 kb length of β-galactosidase and 1.5 pmoles / μl in 50 mM sodium phosphate buffer solution, pH 8.5, 1 mM EDTA and 3% sodium sulfate were printed on the silylated slides, the polylysine slides and slides coated with SurModics using a 0.015 cm (0.006") di (internal diameter) needle as described in Example 25. The SurModics slides were then incubated in a 75% relative humidity chamber for 2 days, they are denatured by immersion in a boiling water bath for 2 minutes, and blocked with 10 mM ethanolamine, 0.2 M Tris, pH 8.5 for 30 minutes at 50 ° C. The silylated slides were incubated in a humidified incubator. for 4 hours and then reduced with sodium borohydride as suggested by the manufacturer.The polylysine slides were cross-linked with UV and then blocked with succinic anhydride as described in lit. erature 1. All processed slides were hybridized with 50 fmoles / μl of ID 9 of the complementary detection oligonucleotide in 4X SSC, 2 mg / ml tRNA, 0.1% lauryl sarcosine at 45 ° C overnight. The slides were washed and the hybridization signals were scanned as described in Example 25. The results are shown in the following Table 11. There was no difference in the signals between the amine modified DNA against the unmodified DNA on the silylated and polylysine slides. Only the SurModics coatings showed that the specific binding was due to having a 5'-amino on the PCR products. This provides evidence of the binding of the DNA endpoint of up to 1 kb with the SurModics coatings. The polylysine slides had the highest background probably due to the ionic and / or non-specific binding of the DNA on the surfaces.

Table 11. Signs of Hybridization with the 1 Kb Immobilized DNA and an ID 9 of the Complementary Detection Oligonucleotide on the Coated Glass Slides. Comparison of Coated Slides with Compound IX-A / Compound X-A and Commercial Glass Slides.

Example 28 Immobilization and Hybridization of PCR Products with the CDNA Detection Probe on Coated Glass Slides with Photo-Polymeric NOS.

Two sets of slides were prepared as described in Example 26. Three PCR product sequences (designated Fll, XEF, daf) containing an amine on both forward and reverse strands or no strand (provided by Axys Pharmaceuticals, La Jolla, California) were dissolved in the printing buffer (80 ng / μl), heated to 100 ° C, cooled on ice, and printed on the slides using a Generation II Computer (Molecular Dynamics, Sunnyvale, California). After the overnight incubation as described in Example 25, the slides were placed in a boiling water bath for 2 minutes, washed twice with PBS / 0.05% tween-20, rinsed twice with water , and placed in the blocking buffer for 30 minutes at 50 ° C. The slides were then rinsed with water and dried by centrifugation. The slides were prehybridized as described in Example 26 and hybridized to a mixture of fluorescently labeled cDNA (Cy3) (provided by Axys Pharmaceuticals) in 50% formamide, 5X SSC, 0.1% SDS, and 0.1 mg / ml DNA of salmon sperm at 42 ° C all night. This mixture contained complementary probes for the front strand of all three targets of the PCR product. The Fll probe was canceled at a mass ratio of 1 to 50,000 relative to the other two sequences. After hybridization, the slides were washed and scanned as described in Example 25. The average intensities of the spots or spots are shown in Table 12. The slides that were hybridized to a mixture of the cDNA probe which did not contained the Fll probe did not show signs in these spots or spots. The results show that both types of coating gave comparable hybridization results. The coating containing the compound X-A had a much higher background. This was especially true in the area near where the PCR product was printed.

Table 12: Immobilization of the PCR and Hybridization Products to the cDNA Fluorescently labeled on the Microscope Glass Slides. The numbers are the Fluorescent Signal1.

Coated with amine on the composite both strands strand strand none IX-A front rear strand 0.85 Kb XEF 2664.5 6125.5 759.5 3590.5 1 Kb daf 42921.5 14294 1 Kb Fll 588 1859.5 123.5 891.5 bottom = 80 coated with amine over the mixture of both strand strand strands none the compounds front back strand IX-A and X-A 0.85 Kb XEF 3001 12896 779 4119 1 Kb daf 44132.5 13269.5 1 Kb Fll 535 1687.5 133 860.5 background = varies from 100 to 2500 1 Laser power fixed at 80% and photomultiplier tube fixed at 80% Table 13: Compounds.

COMPOSITE I COMPOUND II COMPOUND III COMPOSITE IV COMPOSITE V COMPOSITE VI COMPOSITE VII twenty COMPOSITE VIII COMPOSITE IX COMPOSITE X 25 H -CH-- C C N COMPOSITE XI COMPOSITE XII COMPOSITE XI II fifteen H2C = CH-CH2- COMPOSITE XIV twenty Compound XV It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (29)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method of attaching a target molecule to a substrate, the method is characterized in that it comprises: a) providing a reactive composition comprising a polymer skeleton, and one or more reactive groups thermochemically bound to the polymer backbone, wherein the thermochemically reactive groups are configured and arranged to form covalent bonds with the functional groups on the target molecule; b) coating and immobilizing the reactive composition on the substrate to form a bound composition; c) providing a solution comprising the target molecule having one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition; d) apply one or more points or spots of small volume of the sample, discrete, from the solution to the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule to fix the target molecule to the substrate.
2. 2. The method according to claim 1, characterized in that the step of allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule to bind the target molecule to the substrate comprises allowing the thermochemically reactive groups provided by the bound composition to form the covalent bonds with the functional groups provided by the target molecule without the use of the attraction groups, attract the target molecule to the bound reagent.
3. The method according to claim 1, characterized in that the target molecule comprises the nucleic acid and the substrate comprises plastic, silicon hydride, or a glass pretreated with organosilane or a silicone slide.
4. 4. The method according to claim 3, characterized in that the nucleic acid comprises one or more functional groups selected from the group consisting of the amine or sulfhydryl groups.
5. The method according to claim 1, characterized in that the step of providing a solution comprises the target molecule having one or more functional groups reactive with the thermochemically reactive groups provided by the reactive composition, comprising providing the solution in an amount of 20 nanoliters or less.
6. 6. The method of compliance with the claim 1, characterized in that the reactive composition further comprises one or more photoreactive groups for binding the reactive composition to the substrate during the application of the energy from a suitable source.
7. 7. The method of compliance with the claim 6, characterized in that the thermochemically reactive groups and the photoreactive groups are hanging on one or more hydrophilic polymer backbones and the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
8. 8. The method of compliance with the claim 7, characterized in that the photoreactive aryl ketones are each independently selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
9. 9. The method according to claim 1, characterized in that the polymeric skeleton is selected from the group consisting of acrylics, vinyls, polyurethanes, polyurethanes, and the thermochemically reactive reactive groups are selected from the group consisting of activated esters, epoxides , azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and oc-iodoacetamide, and the backbone further comprises one or more pendant photoreactive groups selected from the group consisting of aryl ketones.
10. 10. The method according to claim 1, characterized in that the method is used to prepare one or more micro arrays or microarrays of the nucleic acids on a substrate comprising the plastic, silicon hydride, or a glass pretreated with organosilane or silicone slides, each microframe or microarray includes discrete regions of nucleic acids in amounts of 0.1 femtomoles to 10 nanomoles.
11. 11. An activated slide for attaching the target molecule to a sample, the slide is characterized in that it comprises: a) a substrate; and b) a binding composition fixed to the substrate, wherein the bound composition comprises a polymer backbone having one or more reactive groups thermochemically bound thereto, and wherein the bound composition is configured and arranged to form covalent bonds with the functional groups on the target molecule without the use of attraction groups to attract the target molecule to the bound composition.
12. 12. An activated slide according to claim 11, characterized in that the activated slide is configured and arranged to receive a sample comprising a target molecule in an amount of 20 nanoliters or less.
13. An activated slide according to claim 11, characterized in that the activated slide is adapted to manufacture a microframe or microarray, and wherein the target molecule comprises a nucleic acid and the substrate comprises plastic, silicon hydride, or a glass pretreated with organosilane or a silicone slide.
14. 14. An activated slide according to claim 13, characterized in that the nucleic acid comprises one or more functional groups selected from the group consisting of the amine and sulfhydryl groups.
15. 15. An activated slide according to claim 11, characterized in that the bound composition comprises a product of the reaction of a reactive composition with the substrate, wherein the reactive composition comprises one or more photoreactive groups for the binding of the reactive composition to the substrate during the application of energy from a suitable source, wherein the photoreactive groups immobilize the reactive composition on the substrate to form the bound composition.
16. 16. An activated slide according to claim 15, characterized in that the reactive composition comprises the thermochemically reactive groups and the photoreactive groups, and wherein the thermochemically reactive groups and the photoreactive groups are dependent on one or more hydrophilic polymeric backbones and the groups photoreactive are photo-reactive aryl ketones.
17. 17. An activated slide according to claim 16, characterized in that the photoreactive aryl ketones are each independently selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
18. 18. An activated slide according to claim 11, characterized in that the polymer backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the thermochemically hanging reactive groups are selected from the group consisting of activated esters, epoxides , azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and α-iodoacetamide, and the backbone further comprises one or more pendant photoreactive groups selected from the group consisting of the aryl ketones.
19. 19. A microgrid or microarray comprising a substrate, a bound composition placed on the substrate, and the target molecule covalently coupled to the bound composition, characterized in that the microarray or microarray is prepared by a method comprising: a) providing a composition reactive comprising a polymer backbone, and one or more reactive groups thermochemically bound to the polymer backbone; b) coating and immobilizing the reactive composition on the substrate to form a bound composition; c) providing a solution comprising the target molecule, wherein the target molecule comprises one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition; d) apply one or more points or spots of small volume of the sample, discrete, on the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule to bind the target molecule to the substrate.
20. 20. The microarray or microarray according to claim 19, characterized in that the step of allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule comprises allowing the thermochemically reactive groups of the composition bound to form covalent bonds with the functional groups of the target molecule without the use of the attraction groups to attract the target molecule to the bound composition.
21. 21. The microarray or microarray according to claim 19, characterized in that the target molecule comprises the nucleic acid and the substrate comprises the plastic, the silicon hydride, or a glass pretreated with organosilane or silicone slides.
22. 22. The microarray or microarray according to claim 21, characterized in that the nucleic acid comprises one or more functional groups selected from the group consisting of the amine and sulfhydryl groups.
23. 23. The microarray or microarray according to claim 22, characterized in that the microarray or microarray is configured and arranged to receive the solution comprising the target molecule in volumes of 20 nanoliters or less.
24. 24. The microarray or microarray according to claim 19, characterized in that the reactive composition further comprises one or more photoreactive groups for attaching the reactive composition to the surface during the application of the energy from a suitable source.
25. 25. The microarray or microarray according to claim 24, characterized in that the thermochemically reactive groups and the photoreactive groups are hanging on one or more hydrophilic polymer backbones and the photoreactive groups are photoreactive aryl ketones.
26. 26. The microarray or microarray according to claim 25, characterized in that the photoreactive aryl ketones are each independently selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
27. 27. The microarray or microarray according to claim 19, characterized in that the polymer backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the thermochemically-hanging reactive groups are selected from the group consisting of activated esters , epoxides, azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and α-iodoacetamide, and the backbone further comprises one or more pendant photoreactive groups selected from the group consisting of aryl ketones .
28. The microgrid or microarray according to claim 19, characterized in that the microgrid or microarray comprises a surface of a plastic, silicon hydride, or glass pretreated with organosilane or silicone slides, and wherein the microgrid or microarray provides at least Approximately 100 / cm2 of different nucleic acids having a length of at least 10 nucleotides, the nucleic acids are marked with spots or spots in discrete regions and defined amounts of 0.1 femtornols to 10 nanomoles.
29. 29. The microgrid or microarray according to claim 28, characterized in that the regions are generally circular in shape, having a diameter between about 10 μm and about 500 μm and separated from other regions in the network or matrix by a center space to center from 20 μm to 1000 μm. UNION OF WHITE MOLECULES TO SURFACES SUMMARY OF THE INVENTION The present invention relates to a method and reactive composition for the covalent attachment of target molecules, such as nucleic acids, on the surface of a substrate. The reactive composition includes groups capable of covalently bonding to the target molecule. Optionally, the composition may contain photoreactive groups for use in binding the reactive composition to the surface. The reactive composition can be used to provide activated slides for use in the preparation of micro arrays or microarrays of nucleic acids.