MXPA96004091A - Method and apparatus for making multiple sequential reactions on a mat - Google Patents

Abstract

A method and apparatus is provided for preparing a substrate on which are located microdropleted positions in which chemical compounds are synthesized or in which diagnostic tests are carried out. The positions are formed by applying microdroplets from a spout from which a microdrop is supplied by impulse on the surface of the substrate.

Description

MfTT.TTPT.gg ON A MATRIX CAMPO DS THE IMV? WIQN The present invention is directed to a method and apparatus for performing sequential reactions on a plurality of sites in a matrix by using non-contiguous, micro-droplet sized positions. The apparatus and method are useful for performing a test or synthesis that involves sequential steps such as .DNA sequence determination, DNA diagnosis, oligonucleotide and peptide synthesis, examination tests for target DNA, RNA or polypeptides, synthesis of various molecules, DNA separation technologies by means of which DNA binds to target molecules, preparation of polysaccharides, methods to manufacture complementary oligonucleotides and any other test, sequencing or synthetic method that uses a sequence of steps in one place. An advantage or improvement can be obtained by providing positions so that combinations of different reactions can be carried out on the same matrix. REF: 23086 BACKGROUND OF THE INVENTION Methods for performing a plurality of sequential tests or reactions at positions or places on a matrix are known by joining molecules to a solid phase. Usually, a solid phase having a free functional group such as a hydroxy group, an amino group, etc. is prepared. , and bonding groups are bound to the surface by means of covalent bonds. These linkers serve as "handles" to which molecules can be attached for sequential synthesis of such linear molecules such as for example polypeptides and polynucleotides. A disadvantage of such synthesis in the solid state is that the entire substrate or a large portion of substrate must be exposed to a single reagent, so that the reagent which is close to the molecule to be bound to the substrate, an agent of rinsing or an unprotective agent. In some cases, the positions in the substrate can be selectively treated if the reaction to be carried out is photolytic in nature, so masks must be prepared to expose selected areas to the activating radiation. However, an obvious disadvantage is that these reactions must be designed, which can be carried out by photolytic activation and different masks must be used to protect portions of substrate in which the reaction is not desired. The present invention provides a method by which reactions on non-contiguous microdrop sizes can be carried out on a substrate. Since the reagents according to the present invention are in liquid form, virtually any chemical reaction in solution or suspension can be carried out. Therefore, it is an object of the present invention to provide a method and apparatus for performing a plurality of sequential reactions on a substrate, whereby the reactions are carried out in microdropleted positions, if desired, a different sequence of Reactions can be carried out in each position. Furthermore, an object of the present invention is to provide a method and apparatus for carrying out a plurality of reactions in sequence on a matrix by the use of liquid reagents, whereby the chemical reactions can be carried out in solution or suspension. These and other objects of the invention will be apparent from the following description, the appended claims and the practice of the invention, as described herein.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method and apparatus for carrying out a plurality of chemical reactions at different sites on a substrate, wherein they can be carried out in the same places or different tests, sequenced or synthesis reactions. The invention provides a substrate having a surface which has chemical portions that are reactive with the reagents that are supplied from the microdroplet dispensing device. These reagents can be molecules that bind to the surface at the position of the microdrop to which they are supplied, as in the application of activated nucleic acid phosphoramidites, or the reagents can modify the surface at the microdrop positions for subsequent chemical reactions, such as in the deprotection of the 5 'hydroxyl group during the synthesis of oligonucleotides. In the case of delivery of reagents that bind to the surface, the invention provides a substrate having a surface to which the first reagent can be attached by supplying microdroplets of the reagent in liquid form on the substrate. The jet is displaced relative to the surface and at least one microdrop containing the same reagent or a different reagent is applied thereto. By repeating this, the use of a same or a different first reagent in liquid form, a plurality of positions on the surface can be prepared, in which the reagents are covalently bound to the microdroplet sized positions in which the limits of each position are not adjacent to any adjacent position. Subsequently, the surface can be washed to remove the unreacted reagent. If required, the entire surface can be treated, or alternatively, a selected subset of positions can be treated, with the deprotection reagents to expose the reactive sites of the molecules bound to the surface. The deprotection reagent can also be supplied from the device. Subsequently, one or more microdroplets containing a second reagent in liquid form can be delivered at selected positions on the surface of the substrate, whereby the second reagent is selected to react with molecules that are already bound to the matrix. The spout again moves in relation to the surface to apply the second reagent in different positions by using a second reagent, equal or different, which reacts with the respective joined molecules. Again, the entire surface will be washed to remove the second unreacted reagent. Subsequently, the entire surface or subgroups of selected positions can be treated with unprotective agents, and this process can be repeated. In the case of the supply of reagents that modify the reactivity of the surface, the invention provides a substrate having a surface on which a reagent is applied by supplying one or more microdroplets on the substrate. The spout is displaced in relation to the surface and one or more microdroplets are applied thereto. This process can continue until the desired group of microdropleted positions has been modified by applying the reagent. Subsequently, the surface can be washed to remove excess reagent. The entire surface or a selected subgroup of positions can be treated with a reagent that binds to the modified positions by the microdroplet delivering reagent, or alternatively, a reagent can be applied which binds to the surface except at the which has been previously modified by the microdrops supplier reagent. If the reagents that are attached to the surface contain chemical portions that can be modified by the reagent spout, the process can be repeated so that the same or different positions are modified by the microdroplet supplying reagent and subsequently reacted with a reagent or reagents that bind to the modified positions until the desired compounds have been synthesized on the substrate. It will also be recognized that a combination of the above strategies can be used in which both the reagents that bind to the surface at the droplet positions and the reagents that modify the surface at the droplet positions are supplied by the microdroplet spout device. . At the end of the desired number of sequential stages in the positions on the substrate, the compounds can be removed selectively or non-selectively, if desired, from the substrate by the use of release reagents which remove the compounds bound through linking groups to solid substrates. Release agents include enzymatic agents and other chemical agents, which can also be delivered as microdroplets at selected positions. It will be appreciated, for example, in the case of diagnostic methods, that the isolation of a final compound located at each of the points is not important, therefore the detachment of some compound from the substrate is an optional step. In some circumstances, it may be desirable to analyze the molecules directly before the release of the substrate by techniques such as mass spectrometry. In such cases, it is desirable to provide a linker (the portion through which the molecule in question binds to the substrate) which can be detached by electron beams, by laser or by another energy source so that the molecules in the position can be detached selectively from the substrate. This is particularly advantageous for analyzing the molecules by mass spectrometry, in which the laser or electron beam separates the molecules from the substrate, ionization occurs and the ions are accelerated to a mass spectrometer. The substrate can be solid, such as glass, prepared to receive binders attached to the surface. Porous substrates such as paper or synthetic filters can be used, as well as filters (such as those sold by Nucleopore®) that have straight and parallel micropores. In such a microporous substrate, the reactions can be carried out within the pores, whereby the potential signal in the position is amplified. It will also be recognized that the present invention provides a method for determining the presence of an analyte in a sample by contacting the sample with a device prepared according to the present invention having a plurality of positions sized by a microdrop of covalently linked reagents, whereby the analyte binds to at least one of the reagents. The detection of the position in which the binding is carried out can be carried out by conventional methods such as fluorescence, chemiluminescence, co-calorimetric detection, detection of radioactive labels and the like. The present invention also provides a method for delivering microdroplets to the substrate, which is based on the placement of the substrate so that the separation between the dispenser and the substrate is less than the separation required for the formation of free droplets. In this configuration, the liquid column arising from the nozzle due to the applied pressure pulses strikes the substrate before a droplet forms (i.e., a column of liquid tensions between the nozzle and the substrate). The impact on the substrate alters the flow of liquids from the nozzle so that a much smaller amount of liquid is ultimately delivered to the substrate, as compared to the case when different droplets are formed. This method allows a much smaller separation between the positions on the substrate and a greater precision in terms of position for the placement of positions.

DESCRIPTION OF THE DRAWINGS Figure IA shows a substrate having positions sized by micro droplets on the surface. Figure IB shows the cross section of a microporous substrate with parallel, straight micropores, which have a microdropleted position contained in the attached molecules. Figure 2 is a schematic side view of a droplet jet and a substrate. Figure 3 is a schematic illustration of two positions in which different peptides have been prepared. Figure 4 is a schematic illustration of a continuous, flexible substrate used with the method of the invention.

DESCRIPTION OF lAi The present invention provides a method for realizing a plurality of sections in sequence on a substrate. The substrate surface contains chemical portions that react with the reagents that are supplied from the microdroplet spout device. Reagents can be molecules that bind to the surface of the substrate at the droplet positions to which they are delivered, or reagents can modify the surface of the substrate to facilitate the formation of a covalent bond between the surface and a second reagent. In the latter case, the entire surface or a selected subset of positions can be treated with a second reagent that is covalently bound to the positions modified by the first reagent. If only a selected subgroup of positions with the second reagent is treated, this step can be repeated with a third reagent that joins another subgroup of positions modified by the first reagent. The present invention can be used to prepare, for example, molecules such as peptides. In a preferred embodiment, a linker molecule is provided as the first reagent whereby one end of the linker will bind to the surface of the substrate. The other end of the linker will be adapted to form a bond with the carboxy terminal portion of an amino acid or peptide to form, for example, an amide or ester bond. This end of the linker can be initially protected chemically by protecting groups such as t-butoxycarbonyl groups (t-BOC) or other protecting groups known in the art of peptide synthesis. By applying a second reagent on the position which removes a protecting group, such as an acid solution, the protecting group can be removed. The next reagent applied in each position subsequently would be an amino acid or polypeptide protected in its amino terminal portion or protected in its side chain, preferably an amino acid or polypeptide having an activated C-terminal group for attachment of the C-terminal portion to the end of the linker. This process can be repeated with the same or different amino acids or polypeptides in each of the microdrop positions until the substrate includes the peptides of desired sequences and lengths. Subsequently, the protecting groups are removed from part or all of the peptides, as desired. Deprotection can be carried out by the use of a common deprotection agent, which removes the protecting groups on the side chains and the amino termini, simultaneously, as is known in the peptide synthesis art. The peptides can be detached from the linker by using methods known to those ordinarily skilled in the art of peptide synthesis, methods which separate the peptides from a solid support such as, for example, those used in the Merrifield synthesis technique. . It will be understood that a particular advantage of this method is that by keeping a record of the reagents used in each of the microdropleted positions, peptides of different lengths and sequences can be manufactured concurrently on the same substrate. Such peptides can have a variety of uses including, but not limited to, screening for biological activity whereby the respective peptide sequences at each position are exposed to a labeled or unlabeled peptide receptor, such as an antibody, a cellular receptor or any other variety of receptor. The method according to the present invention can also be used to prepare oligonucleotides by sequentially delivering protected nucleic acids through the microdroplet dispenser. These can be added in sequence at each position by the use of identical or different nucleic acids or polynucleotides. Preferably, the 3 'end of the oligonucleotide will bind to the linker molecule and the oligonucleotide will be synthesized from the 3' end to the 5 'end by using known techniques for oligonucleotide synthesis. Preferably, the protecting groups are those known in the art of oligonucleotide synthesis. The oligonucleotide can be used, for example, for hybridization with an unknown oligonucleotide in order to determine the sequence of the unknown oligonucleotide. An oligonucleotide synthesized at a position can be used to synthesize its complementary oligonucleotide by the use of gDNA polymerase. Preferably, the position will comprise straight pores in a porous substrate. The complementary oligonucleotide can be removed by washing with a denaturing agent through the pores on a new substrate, resulting in a substrate (the original porous substrate) containing the oligonucleotides which are originally synthesized, and another substrate contains its complements. An array of synthesized oligonucleotides can be used to generate an array or arrangement of complementary oligonucleotides by using presynthesized oligonucleotides, which optionally contain a chemical reactive portion such as a separator with a primary amine that binds to the phosphate chain. In this embodiment, the presynthesized oligonucleotides are hybridized to the oligonucleotide array prepared by the microdroplet dispenser. Preferably located complementary oligonucleotides are removed from the array synthesized under denaturing conditions and washed on a second substrate. This second substrate is preferably a material such as nylon or a nitrocellulose membrane, or the surface with amino reactive linkers, in which the oligonucleotides are immobilized. Preferably, a flow system is used on the second substrate so that the net flow is essentially perpendicular to the original substrate so that the complementary oligonucleotides at adjacent positions do not mix. This can also be carried out by using an electric field that is perpendicular to the original substrate such as the complementary oligonucleotides subjected to electrophoresis on the second substrate. In yet another embodiment of the present invention, the substrate to which the oligonucleotides bind can be used as a tool in gene therapy by which mutations can be identified in a genome. For example, oligomeric chains complementary to fragments of the known sequences of the normal gene can be attached to the substrate. The digestion of a single strand of the gene from the subject in question and the contact with the substrate containing complementary oligomeric chain sequences can reveal oligomeric chains with which binding occurs, whereby the presence or absence of fragments in the genome of the subject. The substrate containing oligomeric chains can also be used to identify DNA in samples from an environment to detect, for example, the presence or absence of certain species, in the case in which the DNA sequences are known, or to determine the presence of DNA fragments which are aligned with the substrate in the case in which the DNA sequences are unknown. Subsequently, the oligonucleotides can be amplified by PCR amplification technology. If the substrate is a porous filter, a membrane or other material which can be cut, the substrate can be divided into portions containing a position (or a plurality of positions having identical or different molecules). These portions can be placed in microtiter wells for diagnostic or therapeutic testing, so each well is treated separately with a sample. An application of the present invention is for the preparation of an array of oligonucleotides for the determination of the DNA sequence by hybridization. The basis of this method is that a given sequence can be constructed from the knowledge of its constituent group of overlapping sequence segments, with the condition that there is a certain degree of individuality between these segments. The group of overlapping sequence segments of length n can be obtained by hybridization of unknown Jg N to a group of n-meric oligonucleotides (of n units) which represent the totality of possible sequences, 4p. The advantages of sequence determination by hybridization include faster sequence determination, lower cost, ease of automation and greater reliability (compared to a single sequence reading from a gel). For an array of oligonucleotides of length il it is possible to determine the average length of the DNA fragment from which its sequence can be determined unambiguously. Although difficulties may arise when a fragment of length n-l appears in the sequence more than once, however, statistical analysis has shown that the determination of the sequence by hybridization is a feasible method. The relationship between the length of oligonucleotides and the determinable average sequence length has also been defined. Typical quantities are shown in Table 1. For example, an array of a total of 65,536 octamers can be used in the determination of a short sequence of 100 to 200 base pair fragments.

Furthermore, it has been shown that the inclusion of a fixed length, random content free space in the oligonucleotides of the assay can be used to obtain higher sequence resolution lengths. The combination of an arrangement of the whole of 4th octamers and an arrangement of the total of 48 octamers with a random nucleotide inserted in the middle of the octamer has almost the same resolving power as an arrangement of a total of 49 nonamers, even though the arrangement of nonamers is twice as large. The preferred ink jet device used to supply the microdroplets generates directions smaller than 100 micrometers transverse, and address sizes as small as 10 micrometers can be obtained. A major advantage of using inkjet is that standard methods for oligonucleotide synthesis that have been optimized for extremely high yields can be used. By using a multiple jet device, complete array synthesis of oligonucleotides can advance four times faster and with less material compared to that obtained by performing only the addressable deprotection. The simplest design to accomplish this is a system of five jets, a jet for each of the four phosphoramidite reagents and a jet for the activating tetrazole solution. The operation of this device is directly analogous to the operation of color ink jet printers. In each coupling cycle, for each address in the array a number is assigned to indicate the correct synthon to be added. During the reagent supply process, the tray is tracked through the direction of the arrangement. Primer is applied to the substrate tetrazole. In each direction, an additional deflection motion is applied to place the correct phosphoamidite jet (A, C, G or T) in line. Subsequently one or more droplets of the phosphoramidite are dispersed. Subsequent to this, a second movement deviation is used to place the tetrazole jet in line with the direction. After the dispersion of the tetrazole reagent, the tray can advance to the next direction for a new supply cycle. The programming elements for the advanced device are very similar to the control programming elements described in the examples with a modification that a bitmap of "color" is used to represent the array. The four phosphoramidite reagents are assigned, each to a specific color. During the scan through the supply array, the color in each pixel in the bitmap is translated into the offset motion to place the correct reagent in line with the address. The tetrazole jet shoots in each direction position. The tetraethylene glycol linker is useful for unique hybridization with oligonucleotides. Low non-specific binding has been observed. Large polymers of ethylene glycol, as well as modified phosphodiesters, can be used. The phosphoramidite reagents that are commercially available and that can be polymerized in a gradual manner to provide linkers with dimethoxytrityl ends or virtually any desired length. Since this link is finally a phosphodiester with phosphates separated by alkyl chains of only a few carbons, it will have a hydrophilicity similar to that of standard .DNA. In addition, since the linker is negatively charged at neutral pH, a less non-specific binding of .DNA to the substrate is expected. To solve the question about the coupling efficiency and therefore the sequence fidelity in the synthesized arrays, the preferred method is to synthesize large arrays, in which all the directions contain the same sequence, and perform the sequence establishment of Maxam. -Gilbert directly on the substrate region that contains the array. Before starting the sequence establishment, the arrangement must be marked in an arrangement at one end with phosphate at 32P. Sequence establishment by hybridization may require larger arrays, for example, arrays of undecameros, or arrays that have been optimized for more information from a group of hybridization tests. For such large arrays, the entire group of undecameros has 4.2 million members, so small directions and regions of protection are advantageous. With directions of 100 micrometers and protection regions of 50 micrometers, parameters that are within the capacity of the examples described herein, the complete arrangement of a decamer would occupy an area of 711 cm2 (110.25 square inches). In yet another embodiment, microdroplets can be used for polysaccharides synthesized by the use of monosaccharides as building blocks. However, it will be readily apparent that many other types of polymeric materials can be manufactured according to the present invention, whereby the same or different polymers can be constructed at each position in the substrate. In a particularly advantageous use of the present invention, small molecules can be manufactured so that the molecules can be constructed in sequence by the use of reagents in a multi-step synthesis. These do not need to be polymeric molecules when it is a repetitive unit. Since different reagents can be applied at one or more positions on the substrate, there is an advantageous diversity of structures that can be obtained by the concurrent multiple synthesis technology according to the present invention. The objective compounds can be synthesized at the same time, but separately, on the substrate, to generate a composite assembly which may or may not be structurally related. Each stage of the synthesis which is carried out in each position should involve soluble reagents, and should be produced with a reasonable yield usually at room temperature, since most or all of the sites in the substrate will be essentially isothermal. For example, benzodiazepine can be prepared once from an amino acid attached to the substrate by its terminal carbon part. Treatment with a microdroplet containing 2-aminobenzophenone imine forms an imine attached to the substrate and subsequently treatment with TFA (trifluoroacetic acid) generates a benzodiazepine. By using different amino acids and different aminobenzophenones, an arrangement of different benzodiazepines can be obtained in this way. Reference will now be made to the various figures which further describe the preferred modes for practicing the invention. Referring now to the figures, Figure IA shows a substrate 20A having on a surface thereof microdrops 21 which define each position in which the chemical synthesis or the diagnostic reaction can be carried out, according to the present invention. invention. Since each drop is defined or separated and is not contiguous with adjacent microdroplets, the reactions can be carried out in each microdrop, which are independent of the reactions in other microdroplets. In FIG. IB a microporous substrate 20B having parallel, straight micropores 40 is shown. The growth chains of the molecules (41) can be bound within the pores, whereby the synthesis is amplified by the additional surface area available below the surface of the substrate. With reference to Figure 2, a schematic elevation of the substrate 20 on which the microdrops 21 are located on a surface thereof is shown. The microdropler multiple jet head jet 22 is shown schematically from which, as shown, a droplet 23 is supplied. The microdrop is supplied by a pressure pulse generator means 24., such as a piezoceramic driven pressure pulse device as is commonly known in the art of inkjet printers. The timing and amplitude of the pulse are controlled by a suitable electric controller. The placement of the spout 22 can be suitably controlled by a computer controlled mechanical grid or arm by which the precise movements of the spout can be controlled in different positions on the surface 20, by means of a control means 26. The source 28 of The reagent can serve as a reservoir for a particular reagent which is supplied, and the reagent flow is controlled by a flow controller 27. Alternatively, the spout 22 can be held stationary and the substrate 20 can be moved by appropriate controllers in a precise manner to place the microdrops on the surface 20 of the substrate. As part of controlling the position of the dispenser 22, the controlling means 26 will also contain a memory for recording the identity of each reagent and the sequence in which it was added to each microdrop position. With reference to Figure 3, an elevation view of the substrate 20 and a schematic view of the elements which may be present in two of the droplet positions are shown. In each position there is a plurality of chemical linkers 30 which are joined at one end of the substrate surface 20 and at the other end to a molecule which is synthesized at a particular position. In the figure, the letter "A" represents an amino acid. By separate treatments by droplets in one position, the peptide has the sequence (by using the conventional nomenclature for peptides, whereby the last amino acid added to the chain is the N-terminal portion), the peptide A3A2A has been manufactured! by applying, in sequence, the reagents containing the amino acids Alf A2 and A3. In another position of the microdrop, the A3A4A-L peptide was manufactured by applying, in sequence, the amino acid reagents containing A1 # A4 and A3. With reference to Figure 4, a schematic diagram of one embodiment of an apparatus using the present invention is shown. The substrate 35 is a continuous and flexible material to which covalent chemicals can be covalently attached, such as flexible polystyrene having surface groups to which chemical linkers can be attached, such as those used in the synthesis of solid phase peptides. One or more jets 36A and 36B, electromechanically controlled, are used to apply the microdroplets on the substrate 35. The movement of the substrate 35 is also electromechanically controlled in the longitudinal direction shown by the arrow. The movement of the jets 36A and 36B can be controlled along the transverse direction, as well as along the longitudinal direction. The excess reagent is removed by washing in a bath in the tank 37. The detection means 38, which is also controlled in the transverse and lateral directions, is used to observe the places for quality control or, in the case of a diagnostic use, for a signal such as fluorescence, radioactivity, polarization, chemiluminescence, and so on.

EXAMPLE 1 INK JET DEVICE A device for reagent supply is constructed consisting of two 25 mm micrometers that provide x translation and coupled to 10 V step motor, 0.5 amp per phase, of 200 stages per revolution. A single motor stage provides a trip of 2.5 μm. A 48 V power supply is incorporated with down resistors to increase the torque of the motor at high speed. A piezoelectric ink jet head is mounted vertically to a third 12.5 mm micrometer. The jet is positioned to trigger droplets upward to the underside of a microscope stage that holds the top of a platform with a spring-activated slidable support. Electric pulses are generated with electronic equipment that allows all pulse parameters to be adjusted, such as activation voltage, pulse duration and frequency. A video camera is placed, which is moved in the axes x and y with the jet, above the stage to verify the ejection of droplets by focusing on the surface of the lower stage. Alternatively, the camera can be rotated to see through the jet nozzle by providing light by a synchronized LED to allow visualization of the droplets ejected. The inkjet device is controlled by a C / C ++ ASyn program, with a built-in windows interface so that almost all functions can be performed with a mouse which can be placed inside a glove box together with the device of ink jet. ASyn provides the activation TTL level for peripheral physical equipment through a parallel multiple port and an add-in card in a PC compatible computer. The programming elements allow various modes of operation including manual movement and activation, a drawing mode that "prints" a bitmap image and a macro execution mode that can "print" various images in different positions. A bitmap is a numerical representation of a two-dimensional image consisting of an array of pixels. In the case of a black and white image, a number 1 in the bitmap produces a color, while a number 0 produces another color. Therefore, the four octets FF, 0, FF and 0, whose binary representation "in the form of bits" is 11111111, 00000000, 11111111, and 00000000 would produce alternating lines in black and white of 8 pixels in width if they will turn on a computer screen with a bitmap. The logic of the program divides the arrays into "addresses" and "protection" positions that may have variable dimensions. The decision to activate a given address is determined by the value of a pixel in the bitmap image. The activation mode in one direction can also be controlled to provide a single droplet or multiple droplets in the center of the address, as well as a pattern of single droplets to fill a square address area. In addition, a logical circuit has been incorporated in ASyn to generate the appropriate bit maps for the synthesis of oligonucleotide combinatorial arrays.

A variety of organic solvents, including dibromomethane, nitromethane, acetonitrile and dimethylformamide, are suitable for ink jet delivery. Dichloromethane is not found to be suitable for room temperature delivery, although a chilled jet assembly provides better results. A reagent consisting of 0.8 M ZnBr2 in nitromethane: isopropanol 9: 1 has been selected for the deprotection of deoxyribose protected with 5'O dimethoxytrityl during oligonucleotide chip synthesis. Although water can be supplied, the ink jet pulse parameters can be easily adjusted for the supply of single free droplets of satellites. When the jet nozzle for the preparation of stage under the microscope is greater than 100 micrometers, printing of the droplet on a silanized glycidoxypropyl plate can vary from -150 to -250 micrometers based on the activation voltage. When the separation of the nozzle to the stage is less than 60 micrometers, the footprint that is observed decreases between 60 and 80 micrometers. In this case, the trace is relatively independent of the activation voltage. The activation pulse for the ink jet is optimized by setting the video camera so that it can be seen through the jet nozzle with the stroboscopic LED at the bottom. The activation voltage and the delay parameters are adjusted while a direct stream of droplets is released. It has been found that the deprotection reagent requires an activation voltage that is about one third of that required for water. A high degree of control over the size of the droplets can be exerted when the deprotection reagent is activated by adjustment of the activation assembly. In the case of launching single droplets on a stage, the size of the "fingerprint" of the droplet as it disperses on the surface of the stage can vary from less than 100 μm to more than 250 μm, by varying the activation. A combination of suitable activation voltage in close placement has provided the supply of deprotection agent droplets with a fingerprint in the order of 60 micrometers.

EXAMPLE 2 SYNTHESIS OF OLIGCNUCLEOTIDES The synthesis of oligonucleotides is carried out by using the ink jet to supply the deprotection reagent. A standard microscope coverslip is coated with glycidoxypropyl silane and reacted with tetraethylene glycol. A standard phosphoramidite synthesis cycle is used. The complete synthesis is carried out in a glovebox filled with dry nitrogen. Prior to the first coupling reaction, the slide is moistened with acetonitrile (MeCN, distilled from calcium hydride) and dichloromethane (DCM) and dried under vacuum for one minute. The phosphoramidite monomers are dissolved at 0.1 M in acetonitrile. Tetrazole is dissolved at 0.5 M in MeCN. The coupling is carried out by adding 80 μl of each of tetrazole and phosphoramidite to an aluminum reaction vessel. The glass coverslip is placed in the container which causes the liquid to spread evenly over the surface of the coverslip. The reaction is allowed to take place for three minutes. Subsequently the coverslip is moistened with MeCN and the coupling procedure is repeated. After coupling, the coverslip is immersed for two minutes in a Teflon and glass chamber containing an oxidant solution of iodine / lutidine / MeCN / water obtained from Pharmacia (250 μl each of Oxidation 1 and Oxidation 2). The coverslip is then moistened twice with MeCN and DCM, and dried under vacuum.

After drying, the coverslip is placed on the ink jet platform to disperse the appropriate pattern of the deprotection reagent. The slide is allowed to stand for a period of five minutes after the last droplet is delivered. Subsequently, the slide is moistened twice with MeCN and with DCM, and dried under vacuum in preparation for the next coupling cycle. At the end of the synthesis, the slide is removed from the glove box and immersed overnight in a 30% ammonia bath at room temperature. A simple oligonucleotide synthesis test is carried out to generate 4 x 5 poly-T arrays. In this study, 17 coupling cycles were carried out using a single dispersion pattern that deposits 15 droplets in all directions. The addresses are placed in centers of 2 mm. At the end of the synthesis, the oligonucleotides are unblocked and subjected to hybridization with a 15-unit oligonucleotide labeled at one end of poly-A using 6x SSC / 0.5% SDS and 400 ng of a probe labeled at the end. The synthesis of poly-T arrangements is successful. It will be appreciated that what is described in the foregoing is considered as illustrative and not as restrictive, and that many modalities will be apparent to those skilled in the art upon review of the foregoing description and the claims that follow. Therefore, the scope of the invention is determined not with reference to the foregoing description, but, instead, is determined with reference to the appended claims together with the full scope of the equivalents to which such claims relate.

Table 1. Length of possible sequence determination versus the length of oligonucleotides used for hybridization.

Length of the oligonucleotide sequence length Identify * 7 80 8 180 9 260 10 560 11 1300 12 2450 * These numbers represent the length for which the construction of the sequence will be possible in 95% of all cases. 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. Having described the invention as above, property is claimed as contained in the following:

Claims (47)

RET7INDICACIQNES

1. A method of synthesis, step by step, of an arrangement or arrangement of different chemical compounds in positions sized by microdroplets, wherein each compound is covalently bound to, or below the surface of a substrate, the method is characterized in that it comprises the steps of: (a) applying through a single unit of a multiple reagent dispenser at least one microdrop of a first reagent, in liquid form, to the surface, wherein the substrate is chemically prepared to react with the first reagent for covalently attaching the reagent to the substrate; (b) moving the multiple reagent jet in relation to the surface, or the surface with respect to the multiple reagent jet, and applying at least one microdrop containing either the first reagent or a second reagent from a different unit dispenser, to the surface, wherein the substrate is chemically prepared to react with the reagent to covalently bind the reagent to the substrate; (c) optionally reacting the stage (b) at least once by using an equal or different reagent in liquid form, from the different dispensing units, wherein each of the reagents is covalently bound to the substrate to form covalently bound compounds; (d) washing the substrate to remove unbound reagents; (e) modifying the bound reagents; (f) repeating steps (a) to (e) with the same or different reagents at various positions on the substrate; and (g) optionally, selectively or non-selectively, remove bound compounds at the substrate positions.

2. A method for the step-by-step synthesis of an arrangement or arrangement of different chemical compounds in positions sized by microdroplets, wherein each compound is covalently bound to or below the surface of a substrate, the method is characterized in that it comprises steps of: (a) applying, through a single unit of a multiple reagent dispenser, at least one microdrop of a first reagent, in liquid form, to the surface, in the first position; (b) displacing the multiple reagent jet in relation to the surface and applying a second reagent to the first or first position through a second unit of a multiple reagent jet to form a mixture of reagents in the first position, wherein the substrate is chemically prepared to react with the mixture to covalently bind one or more of the reactants to the substrate in the first position; (c) optionally repeating steps (a) and (b) in additional positions sized by droplets of the surface, where mixtures of the same or different reagents are formed in each position; (d) washing the substrate to remove excess reagents; (e) modifying the bound reagents; (f) repeating steps (a) to (e) with the same or different reagents at various positions on the substrate; and (g) optionally, selectively or non-selectively, remove the bound compounds at the substrate positions.

3. The method according to claim 1 or 2, characterized in that the substrate comprises a solid, non-porous material.

4. The method according to claim 1 or 2, characterized in that the substrate comprises a porous material.

5. The method according to claim 4, characterized in that the porous material comprises paper.

6. The method according to claim 4, characterized in that the porous material comprises a sheet having essentially parallel, straight, internal pores.

7. The method for preparing a substrate according to claim 1 or 2, characterized in that the reagents in the microdroplets are constituted of protected or unprotected amino acids, whereby joined polypeptides are formed in the positions.

8. The method for preparing a substrate according to claim 1 or 2, characterized in that the reagents in the microdroplets comprise protected or unprotected nucleic acids, whereby oligonucleotides bound at the positions are formed.

9. The method for preparing a substrate according to claim 1 or 2, characterized in that the reagents in the microdroplets comprise protected or unprotected sugars, whereby oligosaccharides are formed attached in the positions.

10. The method according to claim 8, characterized in that it additionally comprises the step of amplifying the oligonucleotides by amplification by polymerase chain reaction.

11. The method according to claim 8, characterized in that it additionally comprises the steps of forming the oligonucleotide complements of the bound oligonucleotides by the use of polymerase and nucleic acids, and washing the complements of the substrate with a denaturing agent.

12. The method according to claim 8, characterized in that it additionally comprises the steps of hybridizing the oligonucleotide complements to the bound oligonucleotides, and washing the complements of the substrate with a denaturing agent.

13. The method according to claim 12, characterized in that the substrate comprises a porous membrane having straight, essentially parallel pores, and the complements are removed through the pores on a second substrate, wherein the relative positions of the complements in the second substrate correspond to the relative positions of their respective oligonucleotides on the substrate from which they were removed.

14. The method according to claim 1 or 2, characterized in that the reagents in the microdroplets comprise chemical portions for gradual synthesis of target compounds, whereby the target compounds bound in the positions are formed.

15. The method according to claim 1 or 2, characterized in that the compounds bound in the positions are detachable from the substrate by exposure to laser or electron beam.

16. The method according to claim 1 or 2, characterized in that the compounds bound in the positions are detachable from the substrate by chemical reagents.

17. A substrate characterized in that it is prepared according to the method of claims 1 or 2.

18. A brief characterized in that it is prepared in accordance with the method of claim 7.

19. A substrate characterized in that it is prepared according to the method of claim 8.

20. A substrate characterized in that it is prepared according to the method of claim 9.

21. A substrate characterized in that it is prepared according to the method of claim 13.

22. A method for determining the presence of an analyte in a sample, characterized in that it comprises the steps of contacting the sample with a substrate having a surface on which are contained positions sized by microdrop of reagents covalently bound, so that the analyte binds to at least part of the reactants in the positions; and detect the positions in which the union is carried out.

23. A method for detecting active molecules in a sample, the method is characterized in that it comprises the steps of contacting the sample with a substrate having a surface on which are contained positions sized by droplets of reagents covalently bound; whereby the active molecules bind to at least part of the reactants at the positions; and detect the positions in which the union is carried out.

24. The method according to claim 22 or 23, characterized in that the substrate is divided into separate portions and the portions are contacted, separately, with a sample.

25. The method according to claim 1 or 2, characterized in that the multiple reagent jet consists of multiple piezoelectric jet heads.

26. The method according to claim 25, characterized in that the quantities of the first reagent or of subsequent reagents delivered are microdroplets having volumes from 10 to about 50 picoliters.

27. The method according to claim 1 or 2, characterized in that the quantities of the first reagent or of subsequent reagents supplied are microdroplets having volumes from about 10 to about 150 picoliters.

28. A method for step-by-step synthesis of an array of oligonucleotides in positions sized by microdroplets, wherein each oligonucleotide is covalently bound to, or below the surface of the substrate, the method is characterized in that it comprises the steps of: (a) applying, through a single unit of a multiple reagent jet, at least one microdrop of a tetrazole solution to the first position; (b) displacing the multiple reagent jet in relation to the surface and applying, through a different unit of a multiple reagent jet, at least one microdrop of a solution of a phosphoramidite nucleotide to the first position, wherein the Phosphoramidite and tetrazole reagents react with the surface to covalently attach the nucleotide to the surface through a phosphite bond; (c) optionally repeating steps (a) and (b) in additional positions sized by droplets of the surface, wherein step (b) applies the same or different phosphoramidite nucleotide, and the nucleotide is attached to covalently to the surface; (d) washing the substrate to remove reagents that have not been bound; (e) applying an oxidant reagent to the entire substrate to modify the newly formed covalent bonds to phosphodiester linkages; (f) washing the substrate to remove oxidizing agents; (g) applying a deprotection reagent to remove the protecting groups, to allow the binding of additional nucleotides; (h) washing the substrate to remove the deprotection reagent; (i) optionally repeating steps (a) to (h), wherein phosphoramidite nucleotides equal to or different from the positions of the surface are applied; (j) optionally, removing the protecting groups from the oligonucleotides; and () optionally, selectively or non-selectively remove the oligonucleotides bound at the positions from the substrate.

29. A method for preparing a substrate having a plurality of positions, dimensioned by microdroplets, non-contiguous on a surface of the substrate, wherein at each of the positions a compound is covalently bound on or below the surface, the method is characterized in that it comprises the steps of: (a) applying through a single reagent jet, to a first position on the surface, an amount of a first activating reagent liquid, wherein the reagent modifies the surface in the first position to activate the surface and form covalent bonds; (b) displacing the single reagent jet in relation to the surface and applying a quantity of the first activating reagent to the surface in a second position and, optionally, repeating the steps of displacement and application in a third position and in subsequent positions, each one of the positions, therefore, becomes a modified position; (c) washing the substrate to remove excess reagent; and (d) applying an amount of a second reagent to the entire substrate, including the positions modified by steps a, b and c, to provide the second reagent covalently bound to the surface at the modified positions.

30. The method according to claim 29, characterized in that the first activating reagent is a deprotection reagent which eliminates position protecting groups.

31. The method according to claim 29, characterized in that the first activating reagent is an activation reagent which reactivates reactive groups of the position.

32. The method according to claim 29, characterized in that step (d) is repeated with the same reagent or with a different second reagent, in which equal or different positions have been modified according to steps (a) to (c) ).

33. The method according to claim 32, characterized in that the second reagent comprises protected or unprotected amino acids, whereby polypeptides joined at the positions are formed.

34. The method according to claim 32, characterized in that the second reagent comprises protected or unprotected nucleotides, whereby oligonucleotides bound at the positions are formed.

35. The method according to claim 34, characterized in that the activating reagent is selected from the group consisting of trichloroacetic acid in aichloromethane and zinc bromide in nitromethane: isopropanol.

36. The method according to any of claims 29 to 34, characterized in that the amount of activating reagent is applied by a pulse from a single piezoelectric jet head.

37. The method according to claim 36, characterized in that the amount of activating reagent delivered is a droplet having a volume of from about 10 to about 150 picoliters.

38. The method according to claim 29, characterized in that the amount of activating reagent delivered is a droplet having a volume of from about 10 to about 150 picoliters.

39. The method according to any of claims 29 to 34, characterized in that the substrate comprises a non-porous, solid material.

40. The method according to any of claims 29 to 34, characterized in that the substrate comprises a porous material.

41. A substrate characterized in that it is prepared according to the method of any of claims 29 to 31.

42. The substrate according to claim 41, characterized in that the substrate comprises a solid non-porous material.

43. The substrate according to claim 41, characterized in that the substrate comprises a porous material.

44. The substrate prepared according to the method of claim 32, characterized in that the repeated application of the same or a different second reagent results in the formation of polymers covalently bound to the surface at the positions.

45. The substrate according to claim 44, characterized in that the polymer is a peptide.

46. The substrate according to claim 44, characterized in that the polymer is an oligonucleotide.

47. A method for preparing a substrate having a plurality of positions or locations sized by non-contiguous microdroplets on a surface of the substrate, wherein each of the positions covalently bonds a compound on or below the surface, the The method is characterized in that it comprises the steps of: (a) placing a piezoelectric jet head jet at a predetermined distance from the substrate and applying at least one microdrop of a liquid reagent; (b) displacing the dispenser in relation to the surface and applying at least one microdrop of the liquid reagent to the surface in a second, non-contiguous position, and optionally repeating the displacement and application steps in a third position and in subsequent positions not contiguous; and (c) wherein the predetermined distance is less than the separation distance required for the formation of a free droplet, whereby a smaller volume of liquid reagent is delivered to the substrate per droplet compared to the volume per microdrop when allows a droplet to form before contact with the substrate.