CN1472340A

CN1472340A - Preparation method of biochip

Info

Publication number: CN1472340A
Application number: CNA021273359A
Authority: CN
Inventors: 林郁庭; 沈昱璋; 佘怡璇
Original assignee: BenQ Corp
Current assignee: BenQ Corp
Priority date: 2002-08-02
Filing date: 2002-08-02
Publication date: 2004-02-04
Anticipated expiration: 2022-08-02
Also published as: CN1239714C

Abstract

The invention provides a preparation method of a biochip, which comprises the following steps: spraying a hydrophobic substance on a substrate in a micro-injection manner to form a hydrophobic region on the surface of the substrate, and separating a plurality of cells by the hydrophobic region; and immobilizing a probe on the compartment by microinjection.

Description

A kind of preparation method of biochip

Invention field

The present invention relates to a kind of preparation method of biochip; Particularly, relate to the preparation method who adopts the biochip that the microinjection mode makes.

Background of invention

Biochip is the breakthrough invention of twentieth century ultimogeniture thing science and technology the next item up, can match in excellence or beauty auto production line that Henry Ford then organized to the great revolution of automotive industry for the development of biotech industry, or similar semi-conductor is for the revolutionary character development of computer industry.In broad terms, biochip is meant on glass (glass), silicon chip (slica), plastics (plastic) or nitrocotton materials such as (nitrocellulose), utilize industrial technologies such as microelectronics, micromechanics, photoelectricity, automatization to make the product that is applied to biochemical analysis, its effective object can be gene, protein or cell tissue etc.The principal feature of biochip technology be analyze confidence level and tolerance range height, analysis speed is fast, employed sample and reagent are few, can handle a large amount of samples simultaneously, and obtain the experimental data of globality and parallelization.

On the whole, biochip research still belongs to the initial stage developmental stage in the world, but existing many great achievements.The kind of biochip has microarray (microarray), DNA wafer (DNAchip), protein/antibody wafer (protein/antibody chip), organizes wafer (tissue chip) and laboratory wafer (lab-on-a-chip) etc. at present, and is wherein comparatively ripe with microarray (microarray) and DNA wafer (DNA chip) development.And the purposes of biochip is very extensive, comprise application, the screening of medicine and the detecting of chemical and biological weapons etc. on gene sequencing, toxicological analysis, disease gene performance, the calibrating of single Yeast Nucleic Acid polymorphism, the medical jurisprudence, this shows that biochip must be the large flat bread that Mirae Corp. circle competitively strives food.But, how to grasp competitive edge and then depend on and prepare highdensity biochip how at lower cost.

Biochip can be divided into three kinds according to the mode difference of probe (probe) preparation, first kind of light guiding in-situ synthesis (light-directed synthesis) that the photo-engraving process (photolithography) that is Affymetrix company develops combines with chemical synthesis.Second kind is Stanford University's employed contact point sample method (pin), is to utilize in advance synthetic good DNA, RNA or protein with robotic arm fast, be fixed on the matrix to high-density.The third is microinjection (micro-injection method), utilizes microinjection (micro-injection) mode to remove to prepare complementary nucleotide as Rosetta Inpharmatics company.The interpretation mode of last data is that sample and wafer that desire detects are carried out hybridization (hybridization), again with the mark (label) on the subject matter in the sample (target), for example modes such as fluorescent, radiating material or ferment colour generation are carried out cat scanner and data analysis afterwards.

Making the common method of biochip at present may be: (a) utilize microarray point system machine that the synthetic probe solution is planted on the wafer by precise positioning mechanism cloth, or (b) utilize the technology of optical etching to carry out Nucleotide light direct in-situ synthesis method (light-directed in-situ synthesis).Wherein, carry the physical construction of probe solution can be divided into the syringe needle (pin) of contact and contactless micro-syringe (micro-injector) in the some system machine.The syringe needle of contact (pin) cost is low, but drop is bigger, array density lower (about 150～200 μ m of every bit), and destructible wafer surface; And contactless micro-syringe (micro-injector) can not destroyed wafer surface, and drop is less, array density higher (about 50～150 μ m of every bit).

For example, carry out the mode of light direct in-situ DNA synthesis method at glass surface with the technology that is derived from optical etching, can be at area 1.6cm ²The preparation kind on glass high-density micro-array of 400,000 kinds of nucleic acid probes nearly.Though be leading present technology, be subject to the too fast and light shield cost costliness of speed that the photochemical reaction rate is successively decreased according to the increase of nucleic acid probe length, so fail to popularize.Therefore, method commonly used at present is point-needle type (pin) and microinjection (micro-injection method), and wherein microinjection is divided into piezoelectric type microinjection and hot bubble type microinjection again.For example Protogene company utilizes piezoelectric type microinjection technology (piezo micro-injection, U.S.Pat.No.5985551; 6,177,558 B1) carry out DNA original position synthetic (DNA in-situ synthesis) in wafer surface, cost is lower, but density only is about 10～10 ⁴Spots/cm ²

Though above-mentioned mentioned be that photo-engraving process, the some skill of handling needles or piezoelectric method all have its shortcoming, therefore with regard to the preparation of biochip, how preparing quality better, resolution, enough to reach low-cost biochip will be the main factor of winning victory.Prepare biochip in the microinjection mode and note the pollution to each other of probe drop.In order to overcome pollution problem, United States Patent (USP) the 5th, 552, disclosed for No. 270 with polyacrylamide colloid (polyacrylamide) and made many boothes, its colloidal thickness is 30 μ m, can prevent the mutual pollution of probe solution, and can increase probe solution fixed amount, so but shortcoming to be processing procedure trouble, cost high and because of moisture easily evaporation need be stored in the nonvolatile oil, therefore, need before using to clean with chloroform (chloroform) or ethanol (ethanol) again, generally speaking use and inconvenient.In addition, also can utilize the mode of conductor etching on silicon chip (silicon), to be etched into many sulculuses chamber and prevent pollution between probe solution, but this legal system journey trouble and spend high.The shortcoming of above-mentioned prior art promptly is the problem that institute of the present invention desire solves.

Summary of the invention

The purpose of this invention is to provide a kind of preparation method of simple and cheap biochip, it comprises the following steps: in the microinjection mode one lyophobic dust to be sprayed on the base material; Form a hydrophobicity district in this substrate surface, and be separated out a plurality of boothes by this hydrophobicity is trivial; And with the microinjection mode with a probe stationary on this booth.The inventive method not only can prevent between the probe drop interference phenomenon each other, also can increase the resolution of probe density and wafer.Therefore, the inventive method can be prepared high-density, drop is little, parsing power is good and cost is low biochip.

Description of drawings

Figure 1A to Figure 1B is sprayed on synoptic diagram base material on micro-syringe with lyophobic dust for showing the present invention.Figure 1A demonstration utilizes micro-syringe vertical direction to spray lyophobic dust to substrate surface; Figure 1B demonstration utilizes micro-syringe horizontal direction to spray lyophobic dust to substrate surface.

Fig. 2 A to Fig. 2 B shows that the present invention utilizes micro-syringe to spray lyophobic dust to substrate surface, forms the possible shape of booth at substrate surface.Fig. 2 A shows the quadrate booth; Fig. 2 B shows circular booth.

Fig. 3 shows the distribution of lyophobic dust on the substrate surface of the present invention and the sectional view that the probe drop covers situation.

After Fig. 4 A to Fig. 4 D showed the booth that the present invention separates out with lyophobic dust, the fixed nucleic acid probe was to the synoptic diagram of base material.Fig. 4 A demonstration splashes into different booth (wetting ability district) with the Nucleotide solution that micro-syringe will have protecting group; Fig. 4 B demonstration is removed protecting group with acidic solution; Fig. 4 C demonstration splashes into different booth (wetting ability district) with micro-syringe with the Nucleotide solution that the 2nd floor has protecting group; And Fig. 4 D shows the biochip that preparation is finished.

Fig. 5 is the structural map of the hot bubble type micro-syringe that adopts of most preferred embodiment of the present invention.

Fig. 6 A-Fig. 6 D is the operation chart of the micro-syringe of Fig. 5, Fig. 6 A is the cross-sectional view of micro-syringe, Fig. 6 B is for forming the situation of first bubble, Fig. 6 C is for then forming second bubble, make two bubble incorporation and extrude the situation of drop, Fig. 6 D is that two bubble collapses make unnecessary liquid flow back to the situation of liquid tank.

Embodiment

The invention provides a kind of preparation method of biochip, at first one lyophobic dust is sprayed on the base material, form a hydrophobicity district, and separate out a plurality of boothes by this lyophobic dust in this substrate surface in the microinjection mode.Then, again with the microinjection mode with a probe stationary in each booth.

Usually the material that can be used as the biochip base material can be hydrophobic substrates or hydrophilic base.Hydrophobic substrates includes but not limited to following material--glass, silicon chip, plastic cement, nylon membrane, resin, quartz, pottery and metal material.Hydrophilic base then mostly is polymkeric substance, comprise as polystyrene (polystyrene), polyester (polyester), polycarbonate (polycarbonate), polyvinyl chloride (polyvinylchloride), polyethylene (polyethylene), polypropylene (polypropylene), polysulfones (polysulfone), polyurethane(s) (polyurethane) or poly-basic methyl acrylate materials such as (PMMA), but be not limited thereto the place person of enumerating.

If adopt hydrophobic substrates, then when having sprayed lyophobic dust after, the booth surface that the hydrophobicity district of lyophobic dust formation is separated out, must be processed into possess hydrophilic property functional group earlier could combine with probe.The hydrophilic functional group of indication can be amido (NH herein ₂), carboxyl (COOH), thiol (SH), epoxy group(ing) (epoxide), aldehyde radical (aldehyde) or streptavidin (Streptavidin) etc.

If the employing hydrophilic base then needs earlier surface treatment to be become hydrophobicity, could spray lyophobic dust then, to form the hydrophobicity district.Afterwards, need again booth to be processed into wetting ability, could combine with probe.The substrate surface modifying method can be with reference to following paper: Souther, Chem.abst.113; 152979r (1990) and Anal.Biochem.157; 283 (1986), do not repeat them here.

Microinjection mode of the present invention is to adopt a micro-syringe (micro-injector), with vertical, level or any direction, unidirectionally or two-way sprays back and forth, as shown in Figure 1A and Figure 1B.The micro-syringe that is adopted comprises hot bubble type micro-syringe and piezoelectric type micro-syringe.The hot bubble type micro-syringe that is adopted comprises: a groove chamber, in order to place liquid; One microinjection hole is positioned on the groove chamber, and the liquid in the groove chamber is flowed out; And a primary heater and a secondary heater, be arranged in the both sides in this microinjection hole respectively.When the filled with fluid of groove chamber, primary heater can produce first bubble, and then, secondary heater can produce second bubble, utilizes two bubbles that liquid is cut off ejection, and primary heater and secondary heater are driven by a common signal.In addition, the generation of first bubble is as a valve, and the liquid that limits in this groove chamber flows out, and hereinafter will describe in detail.

The lyophobic dust that the present invention sprays in the microinjection mode includes, but not limited to tetrafluoroethylene (Teflon), polyimide (polyimide), siliceous and fluorine-containing water-sprinkling agent and fluorine-containing and silicon-containing compound etc.After being sprayed to lyophobic dust on the base material, lyophobic dust can form hydrophobic region at substrate surface, separates out a plurality of boothes by hydrophobic region, and booth can be for square, circular or any geometricdrawing, shown in Fig. 2 A and Fig. 2 B.The booth magnitude range can be 20～200 μ m, and the thickness range of lyophobic dust can be 1～30 μ m, and width range can be 5～100 μ m.

As shown in Figure 3, the form of desiring to be fixed in probe on the substrate surface and being with drop is sprayed out by micro-syringe, covers in the booth (wetting ability district).Probe solution is housed in the micro-syringe, this probe solution can be thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA), Nucleotide (Nucleotides), oligonucleotide (Oligonucleotides), protein (Protein), antibody (Antibodies) or peptide (Peptides) etc., but is not limited to the above person of enumerating.Above-mentioned probe stationary is to reach via the mode of a functional groups in the method for booth, and the functional groups mode comprises absorption (adsorption) method, covalent attachment method (covalent binding), coating method (encapsulation), crosslinking (cross-linking) and entrapping method (entrapment) etc., but is not limited to aforesaid method.If common probe solution DNA; when RNA or Nucleotide; must be earlier make that through some chemically modifieds the functional groups on itself and the substrate surface is tightr; for example be modified into phosphonate (phosphonate); phosphonic acids hydrogen salt (hydrogen phosphonate); phosphoramide (phosphonamidite); phosphoramidite (phosphoramidite); phosphite (phosphite) or methyl phosphoramide (methylphosphonamidite) compounds; if preparation oligonucleotide microarray (oligonucleotide microarray), in 5 of Nucleotide ' or 3 ' end must connect protecting group and combine with the Nucleotide that prevents same layer.

Produce the little microarray biochip of drop, wherein the size in the surface tension of Nucleotide solution itself, viscosity and microinjection hole is the major decision factor, and usually general drop size is about 25～250 μ m.

If prepare the Nucleotide microarray, then must more consider the viscosity of choice of Solvent in the Nucleotide solution and solvent with the hot bubble type micro-syringe.In general, therefore the drop little then more easily volatilization of healing, must contain a kind of high boiling solvent in the common employed solvent at least, and for example boiling point prevents the volatilization of nucleic acid solution greater than 140 ℃ solvent.Selected solvent is comparatively suitable with polar aprotic solvent, commonly used just like two nitriles (dinitriles), mononitriles (mononitriles), glyme class (glymes), diethylene glycol diformazan ethers (diglymes), triglyme class (triglymes), trimethyl phosphate (trimethylphosphates), dimethyl formamide (dimethylformamides, DMF) and N-Methyl pyrrolidone (N-methyl pyrrolidinone, NMP) etc.

When preparation oligonucleotide microarray (oligonucleotide microarray) wafer; utilize micro-syringe that each shielded Nucleotide (protected nucleotides) is fixed on the base material in order just like the mode of piling up wood, the protecting group that connects on Nucleotide is to be used for preventing the Nucleotide of each layer to overlap each other together.After the Nucleotide solution of the first layer fixedly gets on; utilize acidic solution that the protecting group on the Nucleotide is removed; and then the fixing Nucleotide of going up the second layer, can prepare desired biochip apace by that analogy, this kind method is more flexible in the design of nucleotide probe.If when preparation DNA, RNA or protein chip, then directly will synthesize good thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA) or protein and directly be sprayed in the booth of base material.Then peptide is connected one by one during preparation peptide (peptides) wafer as the Nucleotide micro-array.

Sample and the above-mentioned biochip for preparing that desire is detected carries out hybridization (hybridization) at last, carry out differential responses by the mark (label) on the subject matter in the sample (target) more afterwards, for example modes such as fluorescent, radiating material, ferment colour generation are carried out cat scanner and data analysis.

Embodiment

Below will specify the present invention with embodiment, right category of the present invention is not limited to the scope shown in the embodiment.

1: the first kind of preparation method who carries out batch oligonucleotide microarray (oligonucleotide microarray) with the hot bubble type micro-syringe of embodiment.

1. the glass surface with micro-syringe forms booth

With glass is base material, and lyophobic dust to contain lyophobic dust, directly, regularly sprays as the hot bubble type micro-syringe of teflon (Teflon) at glass surface in elder generation, to form the square booth that lyophobic dust separates, shown in Fig. 2 A.The width range of its intermediate chamber is about 50 μ m, and the thickness that lyophobic dust sprays is 2 μ m, and width is 5 μ m.

2. carrying out wetting ability in booth handles

In the square booth that hydrophobicity separates out, to contain the micro-syringe of octyl group trichlorosilane (octyltrichlorosilane), carrying out silanization (silanization) handles, make the booth zone become the wetting ability district, its surface has thiol and (SH) is able to combine with nucleotide probe detailed method such as United States Patent (USP) the 6th, 159, No. 695, can form a nonvolatil thioether bond or the reversible cystine linkage of a chemistry selection thiol key to surface, wetting ability district.

3. oligonucleotide is incorporated on the microarray sheet

The dimethoxy tribenzyl Nucleotide phosphamic acid solution (DMTr-nucleotide phosphoramidite) that will contain tetrazolium (tetrazole) activator is sprayed in the wetting ability district that silanization has been handled with micro-syringe, and the first layer Nucleotide can (SH) combine with the thiol in the wetting ability district.Utilize acidic solution then, protecting group is removed, spray second layer Nucleotide again as Tricholroacetic Acid (trichloroacetic acid) or hydrochloric acid (HCl) solution.Connect each Nucleotide successively with said procedure, can obtain oligonucleotide microarray (oligonucleotide microarray) at last, detailed steps can be with reference to United States Patent (USP) the 6th, 184,347B1 number, the 5th, 985, No. 551, does not repeat them here.

4. carry out hybridization (hybridization) with the oligonucleotide microarray that obtains

Target nucleotide sequence (target) that desire is detected and above-mentioned oligonucleotide microarray carry out hybridization at last, then by the mark (Label) on the target Nucleotide (target), what the sequence that can learn target Nucleotide (target) is, way can be with reference to United States Patent (USP) the 5th in detail, 985, No. 551.

The preparation method of 2: the second kinds of batch oligonucleotide microarrays of embodiment.

The compartment shape of oligonucleotide microarray (oligonucleotide microarray) can also be circle, shown in Fig. 2 B.Can directly spray lyophobic dust and form circular compartment with micro-syringe.Or doing a corresponding circular slab earlier covers on the base material, sprays lyophobic dust with micro-syringe then, takes away circular slab subsequently, can obtain circular compartment at last.Wherein the circular slab material is good with hydroaropic substance, for example nylon membrane etc.Preparation process afterwards is identical with embodiment 1.

Embodiment 3: the preparation method who carries out protein chip (Protein chip) with the piezoelectric type micro-syringe.

1. form the hydrophobicity compartment with micro-syringe at glass surface

With glass is base material, earlier at glass surface to contain lyophobic dust, as polyimide (polyimide), the piezoelectric type micro-syringe spray lyophobic dust directly, regularly, in order to form square hydrophobicity compartment, shown in Fig. 2 A, the width range of its intermediate chamber is about 50 μ m, and the thickness that lyophobic dust sprays is 2 μ m, and width is 5 μ m.

2. make protein bound on the microarray sheet

With octyl group trichlorosilane (octyltrichlorosilane), make square booth finishing that lyophobic dust is separated out become to have thiol and (SH), make that protein probe can be in conjunction with thereon.The combining method of protein probe and base material can be with reference to United States Patent (USP) the 6th, 225,047 B1 number, does not repeat them here.

3. detect the proteic test of target with protein chip

After above-mentioned protein chip prepared, in the booth on protein example (sample) the adding wafer, (Cy3 Cy5) can learn to contain which kind of protein in the sample by the fluorescent subject matter on the sample.

Though the present invention is described with preferred embodiment; right its is not in order to limit the present invention; those of ordinary skill in the art is familiar with this skill person; do not breaking away under the spirit and scope of the present invention; can do various changes and modification, so protection scope of the present invention is as the criterion with the scope of claims.

For above-mentioned and other purposes of the present invention, feature and advantage can be become apparent, hereinafter, be described in detail below by preferred embodiment and in conjunction with appended diagram:

The explanation of symbol

The symbol of Fig. 1-4D:

10～micro-syringe;

12～lyophobic dust;

14～micro-syringe moving direction;

16～base material;

18～hydrophobicity district;

20～booth;

21～wetting ability district;

22～nucleic acid drop;

24～have a Nucleotide drop of protecting group;

26～protecting group;

28～acidic solution;

The symbol of Fig. 5-6D:

1～jet orifice;

2～primary heater;

3～secondary heater;

4～electrode;

5～groove chamber;

6～microchannel;

7～liquid;

8～wafer;

A～first bubble;

B～second bubble;

P～bubble direction of expansion;

F～drop emission direction.

Claims

1. A method for preparing a biochip, comprising the following steps:

(a) providing a substrate;

(b) Spraying a hydrophobic substance on the substrate by microinjection, forming a hydrophobic region on the surface of the substrate, and separating a plurality of small compartments from the hydrophobic region; and

(c) Immobilizing a probe in the compartment by microinjection.

2. The preparation method of biochip as claimed in claim 1, wherein the hydrophobic substance comprises polytetrafluoroethylene (Teflon), polyimide (polyimide), silicon-containing and fluorine-containing water repellant, and fluorine-containing or Silicon compounds.

3. The method for preparing a biochip as claimed in claim 1, wherein the microinjection method is to use a microinjector to spray vertically, horizontally and in any direction, unidirectionally or bidirectionally.

4. The method for preparing a biochip as claimed in claim 3, wherein the microinjector comprises a thermal bubble microinjector or a piezoelectric microinjector.

5. The method for preparing a biochip as claimed in claim 1, wherein the substrate is a hydrophobic substrate composed of any of the following materials: glass, silicon wafer, quartz, mica, ceramic material or metal material.

6. the preparation method of biochip as claimed in claim 5, after step (b), also comprise step (d), carry out a hydrophilic treatment in each small compartment, make each small compartment The surface has a hydrophilic functional group.

7. The preparation method of biochip as claimed in claim 6, wherein the hydrophilic functional group comprises amine group (-NH ₂ ), carboxyl group (-COOH), thiol group (-SH) or streptavidin ( Streptavidin).

8. The preparation method of biochip as claimed in claim 1, wherein this base material is a hydrophilic base material, and it is made up of following any material: polystyrene, polyester, polycarbonate, polyvinyl chloride , polyethylene, polypropylene, polysulfone, polyurethane or polymethylene maleic anhydride.

9. The preparation method of biochip as claimed in claim 8, also comprises the following steps:

(e) after step (a), performing a hydrophobic treatment on the surface of the hydrophilic substrate to make the surface of the substrate hydrophobic; and

(f) After step (b), a hydrophilic treatment is carried out in each small compartment, so that the surface of each small compartment has a hydrophilic functional group.

10. The preparation method of biochip as claimed in claim 9, wherein this hydrophilic functional group comprises amine group (-NH ₂ ), carboxyl group (-COOH), thiol group (-SH) or streptavidin ( Streptavidin).

11. The method for preparing a biochip as claimed in claim 1, wherein the shape of the small compartment comprises a square, a circle or other geometric figures.

12. the preparation method of biochip as claimed in claim 1, wherein this probe comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotide (Nucleotides), oligonucleotide (Oligonucleotides), protein ( Protein), Antibodies or Peptides.

13. The method for preparing a biochip as claimed in claim 1, wherein the probes are immobilized in the compartments through a functional group binding method.

14. The preparation method of biochip as claimed in claim 13, wherein the functional group binding method comprises adsorption (absorption) method, covalent binding method (covalent binding), coating method (encapsulation), cross-linking method (cross-linking) ) or embedding method (entrapment).

15. The preparation method of biochip as claimed in claim 1, wherein the microinjection mode is to use a hot air bubble type microinjector, and this hot air bubble type microinjector comprises:

a tank chamber for placing liquid;

a microinjection port located on the chamber to allow the liquid in the chamber to flow out; and

A first heater and a second heater are respectively arranged on both sides of the microinjection hole; when the tank chamber is filled with liquid, the first heater will generate a first bubble, and the second heater will A second bubble is generated, and the liquid is cut off and ejected by using the two bubbles.

16. The method for preparing a biochip as claimed in claim 15, wherein the first heater and the second heater are driven by a common signal.

17. The method for preparing a biochip as claimed in claim 15, wherein the generation of the first air bubble acts as a valve to restrict the outflow of the liquid in the chamber.

18. A biochip comprising:

(i) a substrate;

(ii) a small compartment separated by a hydrophobic region and a plurality of hydrophobic regions on the substrate, wherein the hydrophobic region is formed by spraying a hydrophobic substance by microinjection; and

(iii) A probe fixed on the small compartment by microinjection.

19. The biochip as claimed in claim 18, wherein the substrate is a hydrophobic substrate composed of any of the following materials: glass, silicon wafer, quartz, mica, ceramic material or metal material.

20. The biochip as claimed in claim 19, wherein the surface of the hydrophobic substrate in each compartment has a hydrophilic functional group after a hydrophilic treatment.

21. The biochip as claimed in claim 20, wherein the hydrophilic functional group comprises amine (-NH ₂ ), carboxyl (-COOH), thiol (-SH) or streptavidin.

22. The biochip as claimed in claim 21, wherein the substrate is a hydrophilic substrate and can be made up of any of the following materials: polystyrene, polyester, polycarbonate, polyvinyl chloride, polyethylene, Polypropylene, polysulfone, polyurethane or polymethylenemaleic anhydride.

23. The biochip as claimed in claim 20, wherein before forming the plurality of small compartments, a hydrophobic pre-treatment is performed on the hydrophilic substrate to make the surface of the substrate hydrophobic.

24. The biochip as claimed in claim 23, wherein after the formation of the small compartment, a hydrophilic treatment is further performed to make the surface of the small compartment have a hydrophilic functional group.

25. The biochip as claimed in claim 24, wherein the hydrophilic functional group comprises amine (-NH ₂ ), carboxyl (-COOH), thiol (-SH) or streptavidin.

26. The biochip as claimed in claim 18, wherein the hydrophobic substance comprises polytetrafluoroethylene (Teflon), polyimide (polyimide), silicon-containing and fluorine-containing water repellant, or fluorine-containing and silicon-containing compounds .

27. biochip as claimed in claim 18, wherein this probe comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotide (Nucleotides), oligonucleotide (Oligonucleotides), protein (Protein) , Antibodies or Peptides.

28. The biochip of claim 18, wherein the probe is fixed on the compartment in one of the following ways: adsorption (absorption), covalent binding (covalent binding), coating ( encapsulation), cross-linking or entrapment.