WO2000071746A1 - Novel method for producing microarray chip of chemical compound and the microarray chip of chemical compound produced thereby - Google Patents

Abstract

Method for producing microarray chip of chemical compound, in particular the one that comprises multi steps of micro reaction chamber fixed point synthesis, also the microarray chip produced thereby.

Description

 New method for preparing compound microarray chips

 And compound microarray chip prepared by the method

 The present invention relates to a new method for preparing a compound microarray chip, and in particular, to a compound microarray chip prepared by a microreverse method. '' Compound microarray chip refers to the preparation of a group of compound arrays composed of different molecular microunits on the surface of a solid substrate. Compounds mainly refer to biological macromolecular substances including nucleic acids such as DNA, RNA, oligonucleotides, etc., peptides and proteins such as enzymes, antibodies, antigens, etc., and other artificially synthesized biologically active substances such as PNA (peptide nucleic acid) and the like. When a compound has the properties of a nucleic acid molecule, a compound microarray chip is also called a gene chip.

Background of the invention

Compound microarray chips are of great significance in biological detection, medical testing, drug screening, gene sequence analysis, and compound library synthesis. In biology, for example, with the continuous development of molecular biology, especially since the implementation of the world-renowned Human Genome Project, data on nucleic acid, protein sequence and structure have grown exponentially. The most challenging task in the next century is how to analyze a large amount of biomolecular information after the completion of the Human Genome Project, that is, in the post-gene era, to find out the laws and make biology rise from experiments to theories. To understand life phenomena and revolutionize medical treatment. Modern medicine is moving from the second-stage medicine at the system, organ, tissue, and cell level to DNA → RNA → protein at the molecular level, the interaction between proteins and nucleic acids, and their third-stage medicine at the level of interaction with the environment "Transformation. This kind of genetic diagnosis and gene therapy at the molecular level will fundamentally understand the root causes of diseases, and will hopefully fundamentally understand and treat major diseases, including cancer. These biological and medical changes, A fundamental premise is the rapid determination and analysis of a large number of gene sequences. The ability to efficiently and quickly perform a large number of gene sequencing and analysis affects the implementation of the human genome project, and further the further development of biology and medicine. The method includes a series of complicated steps such as chemical reaction and gel electrophoresis. This method takes a long time and requires complicated operations. It is time-consuming in large-scale sequencing, and it is not suitable for portable and rapid sequencing. In traditional gene sequencing Method for improving process to gene chip Representative biochip technology emerged. Biochip is a plurality of discrete analysis process involved in the life sciences, such as sample preparation, testing and analysis of chemical reactions and the like by employing Microelectronics, micromechanics and other processes are integrated into the chip to make it continuous, integrated and miniaturized. The maturity and application of this technology will bring a revolution in the life science-related fields such as disease diagnosis and treatment, new drug development, forensic identification, biomedical research, food and the environment in the next century, and provide a powerful tool for the acquisition and analysis of biological information. Powerful means.

 Compound microarray chips are very important and necessary for life science research. The sequence of biological substances (such as proteins, nucleic acids, etc.) is detected or sequenced by the interaction between the array of known compound molecules on the chip and the biomolecules being measured. Taking nucleic acid detection as an example, it includes firstly preparing an oligonucleotide molecule probe array on a solid-phase plant, that is, a compound microarray, and then hybridizing the gene to be tested with the oligonucleotide molecule probe array, The hybridization results are analyzed to obtain the information of the gene sequence to be tested. The key point in the preparation of gene chips is the preparation of oligonucleotide molecular probe arrays.

It is expected that the probe array of the chip has high spatial resolution, small synthesis workload, fast speed, simple method, and low cost. Currently, there are two methods for preparing oligonucleotide probe arrays. One is to use a conventional solid-phase synthesis technique to synthesize the required single probe molecules respectively, and then use spray or printing techniques to combine different probe molecules at different positions on the substrate to form a probe array. It is difficult to achieve high spatial resolution by preparing probe arrays by spraying or printing methods, and the probe molecules are synthesized one by one during the preparation of the probe molecules. The synthesis workload is large and time-consuming. The integration of the chip is small and the cost is high. Conducive to mass production. The other method is to use a template localized photochemical reaction proposed by Affymetrix in the United States to synthesize a probe array on a substrate. Using this method to prepare a probe array can achieve a high spatial resolution (40 x 0 μm ² ), and it is a parallel synthesis during chip synthesis, and the synthesis speed is fast. However, due to the low yield of the photochemical reaction and the large number of side-effect reaction products in the reaction, the accuracy of the synthetic probe sequence is not high, and reagents with special protective groups are needed. The cost is high. Therefore, the preparation of compound microarray chips There still needs to be a better way.

To this end, Southeast University has proposed a method for preparing chemical microarrays by multiple imprinting fixed-point synthesis methods. The method has high synthetic yield, can use conventional DN Α or PN ΑΑ synthesis reagents, has the advantages of low preparation cost, simple equipment, and the like, and has successfully prepared high-density DNA array chips with high sequence accuracy. On the basis of the multiple-imprint fixed-point synthesis method, the present invention also proposes an on-chip localized solid-phase DNA synthesis method based on a microreactor cell master. This method is basically consistent with the current universal DNA synthesis methods, which can further improve the yield and accuracy of synthesis, reduce costs, and accurately control the position of in situ synthesis of compounds on the chip surface, and can carry out high-density large-size DNA chips. Synthesis. Object of the invention

 The purpose of the present invention is to provide a simple, reliable, high spatial resolution, high accuracy, and low cost compound sign array chip preparation method, that is, a micro-reaction cell mother board is used for multiple step-by-step positioning synthesis to prepare compounds. Microarray chip.

 After long and extensive research, the inventors have found a new method for preparing compound microarray chips, which method includes:

 First, prepare a microreactor cell mother board with a microreaction cell structure on the surface according to the microarray design of the desired compound, and then press the microreactor cell master plate on a solid substrate according to the previous design, and make the two tightly combined To form a microfluidic system. The reaction solution is injected into the microfluidic system to generate a chemical reaction on the surface of the solid substrate in contact with the microreaction cell, and a suitable chemical gene is connected to a specific position on the substrate surface. By replacing the micro-reaction cell mother plate pressed on the solid substrate, or by controlling the micro-fluid valve on the micro-reaction cell mother plate, the chemical reaction can be controlled to proceed at a specified position on the surface of the solid substrate, and the required Chemical groups to obtain compound microarray chips. The present invention has been completed based on the above steps. In the above method, the position of the microreaction cell (including the groove) formed by the microstructure on the microreaction cell mother board, and / or by changing the flow of the microfluid in the reaction cell, and / or changing its chemical reaction conditions Control the chemical reaction on the solid substrate in contact with the microreactor. Thus, a specific chemical gene is covalently coupled to a substrate surface molecule, and a desired compound microarray chip is finally formed on the substrate.

Detailed description of the invention

 The first aspect of the present invention relates to a new method for preparing a compound microarray chip, and in particular, the invention in another aspect relates to a compound microarray chip prepared by the method of the present invention, especially a high-density DNA microarray chip and Density PNA microarray chip.

According to the present invention, the method of the present invention is characterized by: (a) designing and preparing a micro-reaction cell mother board having a structure such as a micro-reaction cell on the surface according to the required compound micro-array chip; (b) combining the micro The mother plate of the reaction cell is pressed on the surface of the solid substrate of the compound microarray to be prepared, and forms a microfluidic system with the surface of the solid substrate. (C) introducing the reactant and catalyst solution, cleaning solution, and chemical treatment solution into the microfluid system composed of the mother board and the solid substrate step by step, and controlling the appropriate reaction conditions to contact the solution in the microreaction cell The surface of the solid substrate is chemically reacted to connect with specific chemical groups. (D) According to the order of design, the microreactor cell master plates with specific microreactor cell distributions prepared in different (a) are pressed on the same solid substrate surface by positioning devices, and the reaction cells are changed by Chemical reaction conditions, and / Or control the liquid in the reaction cell through the microfluidic valve on the microreaction cell mother board prepared in (a), controllably synthesize the required compound microunit array on the surface of the solid substrate, and prepare the corresponding core f

 According to the method of the present invention, it is further characterized in that: (a) the micro-reaction cell mother board is processed into a template with a designed concave-convex pattern on a substrate such as a silicon wafer by means of photolithography and etching, and then the liquid polymer is Raw materials are introduced into the template, and after the polymerization is cured, the cured polymer is peeled off from the template, or (a) the micro-reaction cell mother board is prepared by directly etching the silicon wafer and other substrates by methods such as photolithography and etching. The chip is prepared by processing on the chip to have the designed microstructure, or (a) the micro-reaction cell mother board is directly processed on the substrate by using a laser beam, a particle beam, or a micro-tool according to the designed pattern to prepare owned.

 According to the method of the present invention, the micro-reaction cell mother board (a) may further include micro-electromechanical structures such as micro-valves, micro-pumps, and in / out micro-fluid channels. Through external energy such as electricity, magnetism, light, sound, heat, etc., Control the state of the micro-pump and micro-valve, so that the flow of liquid in the micro-reaction cell can be controlled.

 According to the method of the present invention, the material used to prepare the mother plate of the microreaction cell in (a) may be a single material, such as a polymer material such as silicon, glass, rubber, etc., or it may be covered with a layer on the surface and the solid to be prepared. The surface of the microchip substrate has good contact properties, has a certain elasticity, and is not easy to cause a leakage of the reaction solution at the interface of the substrate.

 According to the method of the present invention, in step (c), a microfluidic system formed by the microreaction cell mother plate obtained in (b) and the surface of the solid substrate may also be introduced to promote the microfluidic solution in step (c). The catalyst or biological enzyme is attached to the substrate.

 According to the method of the present invention, in step (d), different micro-reaction cell mother boards are replaced on the same substrate multiple times, and a set of different mother boards are accurately positioned on the same substrate by an accurate mechanical pressing device. On the chip, the levitation reaction cell of the micro-reaction cell mother board coincides with different positions on the surface of the substrate. During multiple pressings of different micro-reaction cell mother boards, the position of the micro-reaction cell array and the surface of the chip substrate may be overlapped or non-overlapping.

 According to the method of the present invention, the reaction cell in the micro-reaction cell mother board in (d) may include small components such as electrochemical electrodes, temperature control, and the like, by controlling the reaction conditions (such as electricity, heat, light, magnetism, machinery, and other energy) In order to control the chemical reaction between the trace solution in the reaction cell and the surface of the solid substrate.

According to the method of the present invention, wherein step (b)-(d) is performed in a vacuum or a gas having no adverse effect on steps (b) and (d), such as nitrogen, argon can be selected as the gas. According to the present invention, more specifically, a method for preparing a microreactor cell mother board is to use a photolithographic etching method, for example, to process a template with a designed concave-convex pattern on a substrate such as a silicon wafer, and then pour the liquid polymer into the template After the polymer is cured, the cured polymer is removed from the template, and the convex W pattern on the original template is copied on the surface of the cured polymer to form the micro-reaction cell mother board of the present invention. .

 According to the present invention, another method for preparing the microreactor cell mother board of the present invention is to directly process the microreactor with a designed microstructure on a silicon wafer substrate, such as a photolithographic etching method, to become the microreactor cell master according to the invention board.

 According to the present invention, the third method for preparing the micro-reaction cell mother board is to use a laser beam, a particle beam (including an electron beam or an ion beam), or a micro-tool under the control of a computer under a designed pattern directly on silicon or glass. And polymer materials, etc., to process the micro-reaction cell mother board according to the present invention.

 According to the present invention, the fourth method for preparing a microreactor cell mother board is to first use laser beam, particle beam (including electron beam or ion beam), or micro-tools to prepare a set of different Through-hole distribution membranes and a microfluidic cover plate corresponding to these membranes. The required micro-reaction cell mother plate is formed by tightly combining membranes with different micro-pore distributions and microfluidic cover plates. At this time, different micro-reaction cell mother boards can be obtained by replacing different membranes with the combination of the micro fluid cover plate.

 According to the present invention, the micro-reaction cell mother board may include grooves and reaction cells necessary for collecting liquid flow.

 According to the present invention, the method for introducing the reactant solution into the micro-reaction mother board may be to form a closed fluid system together with the solid substrate by adding a sealing cover. The reaction solution was injected through the injection port. It is also possible to directly spray the reactants through the micro-reaction cell mother board on the surface of the solid substrate by spraying (spraying) to perform a chemical reaction. The chip of different compound microarrays can be prepared by changing the mother board with different reaction cell distribution.

 According to the present invention, the micro-reaction cell mother board may also include a valve and a pump micro-reaction cell mother board. By controlling these micro-valves and pumps, the liquid flow in the micro-groove and the micro-reaction cell is controlled, thereby realizing the connection with the surface of the chip substrate. Position-controlled in-situ synthesis.

 According to the present invention, a set of micro-electric plate arrays can be prepared in the micro-reaction cell mother board. By adding a specific potential distribution to the micro-electrode array, the electric capillary phenomenon and electric phenomenon of the fluid are used to control the solution in the micro-reaction cell mother board. The motion in the plate controls the chemical reaction in each microreaction cell.

According to the present invention, the chemical reaction positions positioned multiple times on the same substrate may be overlapping That is, multi-step chemical reactions are performed at the same location, and the order of the multi-step chemical reactions can be controlled to avoid mutual or cross-chemical reactions. It is also possible to perform fixed-point chemical reactions without overlapping. After each pressing and positioning chemical reaction, the substrate can be cleaned and chemically treated to meet the requirements of the subsequent positioning chemical reaction.

 According to the present invention, during the localization chemical reaction of the microreaction cell, a (super) sound field, light energy, thermal energy, electric field, magnetic field, photoacoustic surface wave, surface exciton ( (Resonance) and other physical action energy to control or accelerate the chemical reaction at the solid surface position corresponding to the micro-reaction cell, or a chemical reactant solution injected in a micro-fluid system composed of the micro-reaction cell mother board and the surface of the solid chip substrate. A catalyst or a biological enzyme is added to the chemical reaction to accelerate the chemical reaction at the substrate position corresponding to the microreaction cell by a chemical method. The entire preparation process of the microarray chip must be performed in a vacuum or inert gas such as nitrogen or argon.

 The substrates used to prepare compound microarray chips can be silicon, glass, ceramics, metals, polymers, and other inorganic or organic solid materials, as well as molecular films modified or assembled on the surface of these materials. The surface can be dense, or Can be porous.

According to the present invention, the present invention also relates to a compound microarray chip prepared by the method of the present invention, wherein the compound microarray chip prepared by the method of the present invention has high spatial resolution, for example, the spatial resolution of the DNA chip prepared by the method of the present invention is 30 0μιη ² , the degree of integration of the array is high, such as the number of arrays of DNA chips prepared by the method of the present invention can reach 6. 5536 XI 0 ⁴ (square centimeter), high accuracy rate, the accuracy rate of each step synthesis is 99.8% or more The total accuracy of the 20-mer oligonucleotide is above 95%. With the improvement of the precision of the micro-reaction cell mother board and the corresponding pressing mechanism: the above indexes can also be greatly improved.

Brief description of the drawings:

 FIG. 1 is a schematic diagram of a microreactor mother board in the present invention.

 FIG. 2 is a micro reaction cell mother board proposed by the present invention. FIG. 2 (a) It includes a through-hole micro reaction cell with a specific distribution. Its bottom surface is in close contact with a solid substrate, and the other surface is provided with inflow and flow. The outlet sealing cover Fig2 (b) is connected to form a microfluidic system.

 Fig. 3 is a schematic diagram of a microfluidic system composed of a microreactor cell substrate 4 and a solid substrate 7 in the present invention. Molecules A can be covalently coupled to the surface of the solid substrate in contact with the microreactor.

FIG. 4 is a micro-flow system using a micro-fluid system mother plate and a solid substrate according to the present invention. By replacing micro-reaction cell mother plates with different micro-reaction cell distributions, the present invention will contain four chemical reactants A, B, and C. D. Schematic diagram of compound microarray prepared by the chemical reaction of the solution being introduced into the microfluidic system on the substrate surface in multiple step-by-step positioning. FIG. 5 is a No. 1 microreaction cell mother board for preparing a dinucleotide full array.

 Figure 6 shows the No. 2 microreactor master plate for the preparation of a full array of dinucleotides.

 Figure 7 shows the No. 3 microreactor mother plate for the preparation of a full array of dinucleotides.

 Fig. 8 shows the No. 4 microreaction cell mother plate for preparing a dinucleotide full array.

 Figure 9 shows the No. 5 microreactor master plate for the preparation of a full array of dinucleotides.

 Fig. 10 shows the No. 6 microreaction cell mother board for preparing a dinucleotide full array.

 Fig. 11 is a master plate of microreactor No. 7 for preparing a dinucleotide full array.

 Fig. 12 shows the No. 7 microreactor mother plate for preparing a full array of dinucleotides.

 Figure 13 is a schematic diagram of a full array of dinucleotides on a substrate

 The following implementations are further explanations of the present invention, but they are not meant to limit the scope of the present invention in any way.

 Example 1. Preparation of high-density DNA microarray chip.

 A- Preparation of micro-reaction cell master. Apply a layer of photolithography with a thickness of about 20 μm on a clean silicon wafer using a glue coater, and place the adhesive under the designed lithographic mask for exposure and deep etching. Thereby, a micro-reaction cell mother board composed of a set of transparent micro-reaction cells is formed. The size of the micro-reaction cell is 30μιπ χ30μιπ> 300μιη. Cover the bottom raised surface of the silicon wafer with a layer of 5 μm thick silicone rubber (PDMS) material. At this time, the micro-reaction cell mother board is made.

 B. Preparation of oligonucleotide probe arrays on a substrate. After washing and drying the slides, they were respectively placed in a benzene solution of APTES (aminopropyltriethoxysilane) for 2 hours, rinsed in benzene, and then reacted in a stupid solution of succinic acid for 1 hour. The surface of the glass sheet forms hydroxyl groups. The entire preparation process of the gene chip is performed under the protection of argon.

The microreaction cell mother plate is pressed and fixed on the substrate surface by a high-precision alignment device, and then another cover plate with a liquid sealing group is pressed and placed on the microreaction cell mother plate to form a closed microfluidic system. An anhydrous acetic acid solution whose 5, -0Η nucleotides, such as dA-NB ₂ and tetrazole (catalyst), which have been protected with di-trimethoxytrityl (DMT), is injected into the enclosure cover, and from the enclosure The lumen flows into the micro-reaction cell on the motherboard. Through the action of tetrazole, 3, -0H of deoxyadenosine triphosphate is covalently coupled to the substrate. A piezoelectric ultrasonic vibration source is introduced on the substrate, and the speed of the chemical reaction is accelerated by the action of ultrasonic waves. After the nucleotide reacts with the active gene on the solid substrate, it is covalently attached to the surface of the substrate, and the acetonitrile solution is discharged through the micro-fluid channel between the mother plate and the substrate and is discharged from the fluid collection system. Then, the protective agent DMT on the nucleotide 5, -0H on the substrate was removed with an acetonitrile solution of thiophenol (or trichloroacetic acid), and the 5, -OH was exposed. Collect the stripped DMT solution, adjust it to a certain volume, and use DMT-C1 monomer as the standard solution to detect DMT and light absorption value (0D value) at 495nm. The ratio of adjacent quadratic OD values can obtain the synthetic yield of this layer.

Take out the first mother board, press the second mother board on the surface of the substrate with a high-precision alignment device in a certain order, and repeat the above process to place different single nucleotides (such as dC-NB ₂ , dG-N-iBu, T). Repeat the above process at different positions on the substrate to form a single nucleotide array.

Repeating the above embossing process, the second, third, ... layers of nucleotide molecules can be bonded. After synthesizing to twenty layers (that is, a 20-base oligonucleotide), the substrate was treated with 30% ammonia water to remove the protective groups on the base and phosphoric acid. Rinse the chip with water and store it in a dry package. At this point, the preparation of the gene chip is complete. The size of each gene probe unit of the chip is 30 × 30μπι ² and there are 6. 5536 XI 0 ⁴ different gene probes on the surface of lcm ² ; according to the 0D measurement method of DMT, the synthesis efficiency of each layer of the chip is 99. Above 8%, the accuracy of the synthetic probe is above 95%; the preparation time of each layer is about 20 minutes, and the synthesis time of the entire chip is about 6 hours.

Example 2. Preparation of high-density DNA microarray chip.

 A. Preparation of the micro-reactor mother board. A layer of about 20 μm thick lithography is coated on a clean 0.3 mm stainless steel sheet, and the glue is placed under the designed lithographic mask, and exposed and deeply etched to form a set of distributed according to design requirements. Through-holes. If the length of the nucleic acid to be synthesized is 20, 80 stainless steel membranes with different through-hole distributions are required. The surface of the stainless steel membranes is covered with a 5 μm thick silicone rubber (PDMS) membrane.

Β- A microfluidic cover plate corresponding to the stainless steel membrane prepared in A above is prepared by photolithography, and the required micro-reaction cell mother plate is formed by tightly combining the stainless steel membrane and the microfluidic cover plate. The size of the micro-reaction cell is 100 μm ^χ 1 0 Ομιπ><300 μιη. At this time, the micro-reaction cell mother board is made.

 C. Chemical modification of the substrate surface. After washing and drying the slides, they were respectively placed in a benzene solution of APTES (aminopropyltriethoxysilane) for 2 hours, rinsed in benzene, and then reacted in a benzene solution of succinic acid for 1 hour. The surface of the glass sheet forms hydroxyl groups. The entire gene chip preparation process is performed under the protection of argon.

D. Preparation of an array of oligonucleotide probes on a substrate. The micro-reaction cell mother plate is pressed and fixed on the surface of the substrate by a high-precision alignment device to form a closed micro-fluidic system. Inject its 5, -0Η nucleotides protected with di-p-methoxytrityl (DMT), such as dA-N-B ₂ and tetrazolium (catalyst), into the inlet of the microfluidic motherboard. Into the micro-reaction cell on the motherboard. Through the action of tetrazolium, 3, -0Η of deoxytriphosphate is covalently coupled to the substrate. A piezoelectric ultrasonic vibration source is introduced on the substrate, and the speed of the chemical reaction is accelerated by the action of ultrasonic waves. when After the nucleotide reacts with the active gene on the solid substrate, it is covalently attached to the surface of the substrate, and the acetonitrile solution is discharged through the micro-liquid channel between the motherboard and the substrate and is discharged from the fluid collection system. Then, the protective agent DMT on the nucleotide 5, -0H on the substrate was removed with an acetonitrile solution of thiophenol (or trichloroacetic acid), and the 5, -0H was exposed. Collect the stripped DMT solution, adjust it to a certain volume, use DMT-C1 monomer as the standard solution to detect DMT and light absorption value (0D value) at 495nm. According to the ratio of adjacent secondary 0D values, the layer can be obtained. Synthetic yield.

Take out the first stainless steel diaphragm, and press the high-precision alignment device to fix and fix the second stainless steel diaphragm on the surface of the substrate in a certain order. Repeat the above process to separate different single nucleotides (such as dC -NB ₂ , dG-N-iBu, T). Repeat the above process at different positions on the substrate to form a single nucleotide array.

Repeating the above embossing process, the second, third, ... layers of nucleotide molecules can be bonded. After synthesizing to twenty layers (that is, a 20-base oligonucleotide), the substrate was treated with 30% ammonia water to remove the protective groups on the base and phosphoric acid. Rinse the chip with water and store it in a dry package. At this point, the preparation of the gene chip is complete. The size of each gene probe unit of the chip is 100 X ΙΟΟμπι ² and there are 10 ⁴ different gene probes on the surface of lcm ² ; according to the 0D measurement method of DMT, the synthesis efficiency of each layer of the chip is 99.8% or more. The accuracy of the synthetic probe is more than 95%; the preparation time of each layer is about 20 minutes, and the synthesis time of the entire chip is about 6 hours. Example 3: Preparation of a high-density PNA microarray chip.

 Peptide nucleic acid (PNA) is an oligomeric N-2 (2-aminoethyl glycine) with a reduced group, and is a peptide analogue with nucleotide properties. PNA can be combined with DNA, RNA, and PNA hybrids with complementary sequences, and the hybrids have high thermal stability and high sensitivity to mismatches. Under certain conditions, single-base mismatches can be recognized. Therefore, using PNA sequences to prepare high The density gene array chip can greatly improve the hybridization accuracy and sensitivity of the gene chip, and has very important application prospects.

 The high-density PNA microarray chip is prepared as follows:

 A. Preparation of micro-reaction cell mother board (same as A in Example 1)

B. Preparation of four monomers containing four bases of thymine, cytosine, adenine, and guanine, which can be used for P-sequence synthesis, namely N-2 菽 butoxycarbonylaminoethyl-N-thymine-1- Acetylglycine (Gly-T), N-2 菽 butoxycarbonylethyl-N-cytosine-1-acetylglycine (Gly-C), N-2 菽 butoxycarbonylethyl-N-adenine-1- Acetylglycine (Gly- A) and N- 2 菽 Butoxycarbonylethyl-N-guanine-1-acetamidine glycine (Gly-G).

 C. Prepare a PNA probe array on the substrate. After washing and drying the slides, they were placed in APTES solution for 2 hours to form amino groups on the surface of the slides. The entire preparation process of the gene chip is performed under the protection of nitrogen. According to the set procedure, the first microreaction cell mother board is firmly pressed against the surface of the substrate with a precision-aligned mechanical device, and then the outer cover plate with the liquid sealing group is pressed against the microreaction cell mother board. To form a closed microfluidic system. A base-containing PNA synthesis monomer, such as Gly-A, and a solution of pentafluorophenyl vinegar, was used to activate the chemical groups on the substrate through pentafluorophenylhydrazone to chemically bond the C-terminus of Gly-A to the substrate. A piezoelectric ultrasonic vibration source is introduced on the substrate, and the speed of the chemical reaction is accelerated by the action of ultrasonic waves. Then replace the different micro-reaction cell motherboards and inject PNA monomers with different bases such as Gly-T, Gly-G, Gly-C and pentafluorophenyl ester mixed solutions, and repeat the above process to form a single layer of single base For the PNA array, after the synthesis of the first layer is completed, the coupling ratio of the first layer is measured by the hydrazine method. Repeating the above embossing process, the second, third, ... layer of PNA monomer molecules can be bonded. After synthesis to twenty layers (ie, oligopeptide chains of twenty bases in length), the substrate was treated with a 30% NaOH aqueous solution to remove the protective groups on the bases and phosphoric acid. Rinse the chip with water and store it in a dry package. At this point, the preparation of the PNA gene chip is complete.

The size of each gene probe unit of the chip is 30 × 30μηι ² , and there are 6. 5536 different gene probes on the surface of 1 cm ^2. According to the ninhydrin method, the synthesis efficiency of each layer of the chip is more than 99.9%, The accuracy of synthetic probes is over 98%. The preparation time of each layer is about 1 hour, and the complete chip synthesis time is about 24 hours.

Claims

Rights request

1. A method for preparing a compound microarray chip, characterized by (a) designing and manufacturing a specific microchemical reaction cell distribution motherboard according to the required compound microarray chip, (b) preparing (a) The mother plate of the microchemical reaction cell is pressed on a substrate by a positioning device to form a fixed-point microchemical reaction system. (C) A solution containing a corresponding chemical reactant is introduced into the fixed-point microchemical reaction system formed in (b). A chemical reaction occurs on the surface of the substrate in contact with the microreaction cell. (D) By changing the mother board or by controlling the liquid flow in the microchemical reaction cell of the mother board, the fixed-point control of the chemical reaction on the surface of the substrate is achieved and formed on the substrate. The required microarray chip containing different chemical microunits.

 2. The method according to claim 1, characterized in that: (a) the micro-reaction cell mother board is processed on a substrate such as a silicon wafer by a method such as photolithography or etching into a template having a designed concave-convex pattern, and then Liquid polymer material was poured onto the template. After the polymerization is cured, the cured polymer is prepared by peeling off the template.

 3. The method according to claim 1, characterized in that: (a) the preparation of the mother substrate of the micro-reaction cell is directly processed on a substrate such as a silicon wafer by a method such as photolithography and etching to have a designed unevenness or transparency. template.

 4. The method according to claim 1, characterized in that: (a) the mother plate of the micro-reaction cell uses a laser beam, a particle beam, or a micro-tool to directly process the material surface according to the designed pattern to obtain of.

 5. The method according to claim 1, characterized in that: (a) the micro-reaction cell mother board is firstly used a laser beam, a particle beam (including an electron beam or an ion beam), or a micro-tool, etc. According to the design requirements, a set of membranes with different through-hole distributions and a microfluidic cover plate corresponding to these membranes are prepared. The required combination is formed by tightly combining the membranes with different micro-hole distributions and microfluidic cover plates Micro-reaction cell mother board. At this time, different micro-reaction cell mother boards can be obtained by replacing different membranes and microfluid cover plates.

 6. The method according to claim 1 or 2 or 3 or 4, characterized in that: the micro-reaction cell mother board for preparing (a) may contain micro-mechanical and electric mechanisms such as micro-valves, electrodes, etc. to control the fluid Flow in each microreactor.

7. The method according to any one of claims 1-5, wherein in the method (c) of claim 1, a microreactor cell system prepared in (a) can also be added to promote the method (c) of claim 1 The compound is attached to a catalyst or bio-intoxicator on the substrate.

8. The method according to any one of claims 1 to 6, characterized in that: in claim 1 (d), the chemical reaction position where the microchemical reaction cell is pressed on the same substrate multiple times may be overlapped It is also possible not to overlap each other.

 9. The method according to claim 1 or 2 or 3 or 4, wherein in the micro-reaction cell of claim 1 (a), by introducing sound, light, heat, electricity to a substrate or a mother board of the micro-reaction cell Or (and) magnetic and other energy, which can control the chemical reaction at the position of the reaction cell.

 10. A method according to any one of the preceding claims, wherein steps (b)-(d) of claim 1 are performed in a vacuum or in a gas that does not adversely affect steps (b) and (c) and (d).

 11. A compound microarray chip obtained according to the method of any preceding claim.

 12. The compound microarray chip of claim 10 is a DNA microarray chip.

 13. The compound microarray chip of claim 10 is a PNA microarray chip.

 14. The compound microarray chip of claim 10 is a polypeptide microarray chip.