CN1238525C - Preparation method of biochip in micro array - Google Patents

Abstract

The present invention discloses a preparation method of a micro array biochip. Straight bars with built-in through holes are arrayed and fixed according to an array. According to the condition of probe materials arranged on a biochip to be prepared, solutions of the probe materials are respectively injected in holes of the straight bars with built-in through holes, and are frozen and solidified under the temperature of 0 to 60 DEG C. A membrane containing the solidification probe materials is obtained through a mechanical slice in a direction perpendicular to the straight bars with built-in through holes. A base sheet whose surface has active groups is taken, and the membrane containing the solidification probe materials is arranged on the base sheet and is naturally heated up to 5 to 35 DEG C for unfreezing. The probe materials after unfreezing are connected with the active groups on the base sheet to form a micro array biochip. Because the biochip is prepared by using a rapid array parallel sample application method, the speed for preparing the biochip is enhanced. A block is mechanically cut, which can simultaneously obtain a large amount of the same porous membranes, and thus, a large amount of the same compound chips can be prepared.

Description

The preparation method of micro-array biochip

Technical field

The present invention relates to a kind of preparation method of biochip, especially design a kind of preparation method of micro-array biochip.

Background technology

A kind of new UltraDialUp of the preparation method of micro-array biochip is crossed the new tool that the parallel point sample of array prepares biochip.Biochip mainly refers to the microarray that the biologically active substance (comprising nucleic acid, protein, cell and small tissue etc.) assembled constitutes in the solid chip, with realize to compound (comprising medicine), protein, nucleic acid, cell and other biological components accurately, fast, the screening or the detection of large information capacity.Porous check-out console (as 96 orifice plates) is the existing a kind of conventional utensil that is used for medicine, biochemistry detection and screening that generally uses.Respectively different proteins, nucleic acid probe, cell strain or biological tissue are placed or be fixed among the different holes, by adding different chemicals, or compound combination, or biochemical reagents, or detected biological sample, observe the reaction of different chemical/biological substance, carry out fast parallel biochemical analysis, Clinical Laboratory or drug screening.96 reaction tanks on it are isolated from each other, and can select the number of reaction tank arbitrarily for use according to user's needs.This is existing a kind of method that is used for Biochemical Research and exploitation of generally using.But this method still needs one by one different compounds, biomolecules etc. are assigned among each hole, preparation process is still complicated, especially the reagent cost height also is not easy to operate continuously fast simultaneously, more is not easy to simultaneously to multi-component continuous rapid detection in the sample.It is earlier the biomolecules of analyzing to be fixed on substrate material to connect that the point sample legal system is equipped with biochip.Its main drawback is that probe density is not high, but as a kind of new way and means of biological collimation analysis have flexibly, simple, characteristics such as investment is not high, thereby be a kind of means and method that various biological samples carry out scientific research in early days that be particularly suitable for.But the expense of putting foreign instrument at present is unfavorable for promoting up to hundreds of thousands of Renminbi; Though artificial point sample then has cheap outstanding of expense, also exists the sample lattice point misaligned, the sample lattice point irregular with the sample lattice point on inhomogeneous with the consistent shortcoming of bonded compound molecule quantity.

Summary of the invention

The invention provides and a kind ofly can make probe substance molecular density on the lattice point evenly and be suitable for the preparation method of the micro-array biochip of stdn, mass production chip.

Technical scheme of the present invention is as follows:

A kind of preparation method of micro-array biochip: the first step: have the linear bar of through hole to hold in both hands row and fixing in inciting somebody to action by array, second step: according to the probe substance situation of being arranged on the biochip to be prepared, the solution of these probe substance is injected the hole that the linear bar of through hole is arranged in above-mentioned respectively, and-60 ~ 0 ℃ of freezing curing down, the 3rd step: along having the vertical direction of linear bar of through hole to obtain comprising the diaphragm that solidifies probe substance by the machinery section with interior, the 4th step: get the substrate that there is active group on the surface, to comprise the diaphragm that solidifies probe substance places on the substrate, naturally be warming up to 5 ~ 35 ℃ and thaw, probe substance after thawing is connected with on-chip active group, constitutes micro-array biochip.

Compared with prior art, the present invention has following advantage:

1) owing to adopt the method for the parallel point sample of quick array to prepare biochip, improved the speed of chip preparation; 2) because block is carried out cut mechanically, many identical porous membranes can be obtained simultaneously, many identical compound chips can be prepared then.Thereby, can stdn and produce quantitatively, preparation cost is low: make the present invention can stdn and produce biochip in large quantity, preparation cost be low.3) the present invention had both had the equipment advantage simple, with low cost that manual point sample preparation method is had, and can make resulting compound molecule lattice point marshalling again, was convenient to scanning analysis.4) have competent reaction liquid to combine with on-chip active group in each fenestra because the method for the parallel point sample of array makes, compound molecule density more becomes evenly on the lattice point that obtains, and makes the accuracy of chemical array chip analysis higher; 5) the point sample instrument point sample prepares the chemical array chip and compares, and does not need expensive point sample equipment, especially is fit to the large-scale production of ripe biochip product.6) freezing solidified technical measures under-60 ~ 0 ℃ can more effectively be avoided the crossed contamination between the different probe, are particularly suitable for the preparation such as the viable cell chip.

Specific embodiments

The preparation method of 1 one kinds of micro-array biochips of embodiment:

The first step: the linear bar that through hole is arranged in inciting somebody to action is by arrayed and fixing, in have the fixing available of linear rod of through hole not fix with the material of probe molecule generation physical adsorption and chemical bonding reaction, comprise inorganic materials (as: potter's clay etc.), organic materials (as: macromolecular materials such as rubber, plastics, gel, paraffin, nylon, biomacromolecule, poly-electric polymer), or by the mixture of inorganic materials and organic materials

Second step: according to pacifying the probe substance situation of holding in both hands on the special biochip that is equipped with, the solution of these probe substance is injected the hole that the linear bar of through hole is arranged in above-mentioned respectively, and-60 ~ 0 ℃ of freezing curing down, these probe substance mainly comprise biomacromolecule and mixtures thereof such as oligonucleotide, polypeptide, polysaccharide and peptide nucleic acid(PNA), the injection of the solution of probe substance can be directly to inject by instruments such as syringes by hand, also can adopt mechanism to inject automatically

The 3rd step: the edge has the vertical direction of linear bar of through hole to obtain comprising the diaphragm that solidifies probe substance by the machinery section with interior,

The 4th step: get the substrate that there is active group on the surface, to comprise the diaphragm that solidifies probe substance places on the substrate, naturally be warming up to 5 ~ 35 ℃ and thaw, probe substance after thawing is connected with on-chip active group, constitute micro-array biochip, above-mentioned surface has the substrate of active group can be by itself having active group inorganic materials (glass, quartz etc.), organic polymer material (poly-nitrocellulose, polymeric amide, the poly-ammonia that relies, gel etc.), or has active group organic polymer material (polypropylene by physics or chemical modification rear surface, polystyrene etc.) make, active group comprises hydroxyl, amino, aldehyde radical, carboxyl etc. can with the chemical group of other compound molecule generation chemical action, for example: the glass slide surface obtains active group amino by the reaction of 3-TSL 8330, can further obtain active group aldehyde radical etc. with the glutaraldehyde reaction simultaneously.These active groups can combine with the biomolecules of modifying, and make biomolecules be fixed on substrate that (amino as carrier surface is fixed oligonucleotide with the oligonucleotide generation chemical reaction of phosphate radical modification; The aldehyde radical of carrier surface and the amino generation chemical reaction in the protein and protein molecule is fixed).

The preparation method of 2 one kinds of micro-array biochips of embodiment:

(1) preparation of compound microarray

The preparation method one of A, compound microarray

A) parallel handful has been listed as some straight line blank pipes such as small Glass tubing, metal tube, polymer tube etc. in certain space, fill or cast polymer performed polymer in the outside of these microchannels, after the complete polymerization of polymer performed polymer, these some blank pipes that are arranged in parallel just form one with high molecular polymer, do not fill in the open tube or pour into a mould the polymer performed polymer, make between the small straight line blank pipe and isolate by macromolecular material.

B) injection contains specific chemical molecular in small straight line blank pipe, the solution that comprises biomolecules, the compound molecule difference that open tube comprised of different positions, and make in the blank pipe solution-60～0 ℃ of freezing curing, these compound molecules not with macromolecular material generation physical adsorption and chemical bonding effect;

C) obtain a series of diaphragms that comprise compound molecule in the small straight line blank pipe along the vertical direction of filling compound molecule blank pipe by the Refrigerating Machinery section;

The preparation method two of B, compound microarray

A) beat the linear microchannel that series of parallel that (brill) hole obtains is held in both hands row on a solid substrate, some microchannels of these parallel handfuls row and solid substrate formation one are separated mutually by solid substrate between the microchannel.

B) in linear microchannel, inject and contain specific chemical molecular, comprise the solution of biomolecules, the compound molecule difference that microchannel comprised of different positions, and make in the microchannel solution 0～-60 ℃ of freezing curing;

C) obtain a series of diaphragms that comprise compound molecule in the small straight line open tube along the vertical direction of filling compound molecule blank pipe by the Refrigerating Machinery section;

(2) preparation of the fixed form of chemical array and chemical array chip

The chemical array diaphragm that obtains among (1) A (c) and (1) B (c) is fixed in one through on the substrate of modifying, nature intensification degree to room temperature (5～35 ℃) is thawed freezing curing solution, and make its compound molecule and on-chip hydroxyl, aldehyde radical isoreactivity radical reaction, compound molecule is connected with substrate, and the different compounds of fixed constitute the chemical array chip on the different positions.

The preparation of embodiment 3 oligonucleotide arrays film chips

The hollow fiber conduit that some intensive that be arranged in parallel, internal diameter 0.35 mm outer diameter are 0.40 millimeter is sticking and become an integral body by tackiness agent, isolates mutually by tackiness agent between tubular fibre; Fill different oligonucleotide in the different hollow fiber conduits and constitute oligonucleotide microarray, and make the freezing curing of solution in the hollow fiber conduit; Obtain comprising in a series of hollow fiber conduits the diaphragm of oligonucleotide molecules by the Refrigerating Machinery section along the vertical direction of filling oligonucleotide hollow fiber conduit; With the diaphragm subsides with on the substrate that a process is modified, heating up naturally thaws freezing curing solution, and oligonucleotide is connected with on-chip bioactive molecule; Oligonucleotide molecules is connected with substrate to be rejected diaphragms such as tackiness agent after finishing, and the different compounds of fixed just constitute the oligonucleotide arrays chip on these different positionss.

The preparation of embodiment 4 albuminous membranae chips

On a polypropylene material, get out some intensive parallel small straight line blank pipes, isolate by polypropylene material between the small straight line blank pipe; In different straight line blank pipes, fill different protein molecules, constitute protein microarray, under 0～-60 ℃, make the freezing curing of the solution of filling in the blank pipe; Obtain a series of diaphragms that comprise protein molecule in the small straight line blank pipe along filling the vertical direction of protein molecule pipe by the Refrigerating Machinery section; Have on the glass substrate of aldehyde groups through modification, surface with the diaphragm subsides with in one, naturally heating up thaws freezing curing solution, and make the amino in the protein molecule and the reaction of the aldehyde radical generation schiff bases on the glass substrate, make protein molecule and glass substrate chemical bonding: protein molecule is connected with substrate rejects diaphragms such as tackiness agent after finishing, and the fixed different proteins just constitutes protein chip on these different positionss.

Claims (1)

1, a kind of preparation method of micro-array biochip is characterized in that:

The first step: there is the linear bar of through hole also fixing in inciting somebody to action by arrayed,

Second step: according to the probe substance situation of being arranged on the biochip to be prepared, the solution of these probe substance is injected the hole that the linear bar of through hole is arranged in above-mentioned respectively, and-60～0 ℃ of freezing curing down,

The 3rd step: the edge has the vertical direction of linear bar of through hole to obtain comprising the diaphragm that solidifies probe substance by the machinery section with interior,

The 4th step: get the substrate that there is active group on the surface, will comprise the diaphragm that solidifies probe substance and place on the substrate, be warming up to 5～35 ℃ and thaw naturally, the probe substance after thawing is connected with on-chip active group, makes micro-array biochip.