CN100491390C - Reversible Encapsulation Microfluidic Separation and Purification Biological Sample Processing Chip - Google Patents
Reversible Encapsulation Microfluidic Separation and Purification Biological Sample Processing Chip Download PDFInfo
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- CN100491390C CN100491390C CNB2005100766613A CN200510076661A CN100491390C CN 100491390 C CN100491390 C CN 100491390C CN B2005100766613 A CNB2005100766613 A CN B2005100766613A CN 200510076661 A CN200510076661 A CN 200510076661A CN 100491390 C CN100491390 C CN 100491390C
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Abstract
本发明可逆封装微流体分离提纯生物样品处理芯片，用于生物样品中分离提纯，采用硅片、聚二甲基硅氧烷和玻璃为芯片主体材料，在硅片上刻蚀出微沟道，在微沟道两端刻蚀出进样、出样孔或进样、出样流通池，聚二甲基硅氧烷和玻璃复合成盖板，对微沟道进行修饰，在微沟道表面制备氧化硅层或多孔氧化硅层，该氧化硅层或多孔氧化硅层作为分离提纯的固相载体。通过硅片与盖板(盖板为聚二甲基硅氧烷和玻璃)可逆封装制备封闭微沟道，无需键合工艺完成芯片的制造，提高了芯片的成品率，降低了生产成本，同时由于可逆封装有利于清洗芯片，该芯片可以重复多次使用，延长芯片的使用寿命。
The present invention reversibly encapsulates the microfluidic separation and purification biological sample processing chip, which is used for the separation and purification of biological samples. Silicon wafers, polydimethylsiloxane and glass are used as chip main materials, and microchannels are etched on the silicon wafers. At both ends of the microchannel, the sample inlet and outlet holes or the sample inlet and outlet flow cells are etched, polydimethylsiloxane and glass are combined to form a cover plate, and the microchannel is modified. On the surface of the microchannel A silicon oxide layer or a porous silicon oxide layer is prepared, and the silicon oxide layer or the porous silicon oxide layer is used as a solid phase carrier for separation and purification. The closed microchannel is prepared by reversible packaging of the silicon wafer and the cover plate (the cover plate is polydimethylsiloxane and glass), and the chip is manufactured without a bonding process, which improves the yield of the chip and reduces the production cost. Since the reversible packaging is beneficial to cleaning the chip, the chip can be used repeatedly to prolong the service life of the chip.
Description
Technical field
The present invention relates to a kind of reversible packaging micro fluent chip for separating and purifying biological sample, be used for biological sample and separate purification, be specifically related to from various biological samples, extract biological samples such as DNA, RNA, protein or cell.
Background technology
Microfluid biochip is based on MEMS (micro electro mechanical system) (MEMS) processing technology, binding analysis chemistry and analytical biochemistry technology, with little raceway groove network is constitutional features, can finish multinomial functions such as sample pretreatment, single composition detection, pcr amplification and dna sequencing, can realize gene, part, antigen and active somatic cell etc. are carried out the purpose of test analysis, in biomedical and clinical diagnosis, have a wide range of applications.The microfluid biochip volume is little, has significantly reduced the consumption of reagent, has shortened the time of reaction, makes high-throughput, extensive, the real-time detection become possibility simultaneously.At present, the research to microfluid biochip mainly contains DNA cloning microfluid biochip, capillary electrophoresis biochip, dielectrophoresis cell separating chips etc.
At present, microfluid biochip adopts the bonding technology of silicon-glass to form the little raceway groove of permanent closure mostly, and the advantage of this technology is can leakage; Shortcoming is that permanent little raceway groove can not be dismantled repeatedly, therefore easy cleaning chip not, and chip is prone to blockage problem.
The separating and purifying technology of biomacromolecule or cell is the gordian technique of biochemical analysis, can directly obtain highly purified biomacromolecule or cell; And highly purified biomacromolecule or cell will directly influence the success of subsequent experimental, therefore seek quick, efficient, simple and easy, separating and purifying method is biochemical analysis worker's continuous pursuit always cheaply.At present, the method for Chang Gui separation and purification has: dialysis, micro-filtration, saltout and centrifugal etc.
Dialysis method is a kind of simple separation and purification method, only needs the sample of purifying to be separated is enclosed in the dialysis tubing of being made up of semi-permeable membranes, then sack is put into the dialyzate of low ionic strength and is dialysed.Have only the interior molecular weight of film can enter in the dialyzate through film, thereby realized that macromole separates with micromolecular less than the molecule of a certain molecular weight.Though this is a kind of simple method, but maximum shortcoming is consuming time, and use range is limited, is mainly used in salt or sample concentration in the elimination biomacromolecule sample.
The micro-filtration method is to be filtration medium with the porous membrane, allows sample solution pass through membrane filtering.Be mainly used in the filtering small-particle and make muddy liquid clarification, collect pure precipitation for analysis; Also can be used for collecting bacterial cell.The disadvantage of this method also is consuming time.
Salting-out process is to make protein slough hydration shell according to salt, and simultaneously the electrostatic double layer around the compressed protein matter molecule makes protein molecule assemble mutually and precipitate, thereby realizes proteinic separation and purification.This method is mainly used in proteinic separation and purifies, and different protein can select different salt to separate purification under the condition of suitable concentration and pH value.Shortcoming is that this method not only is subjected to Temperature Influence, and selects suitable salt, regulates the way more complicated of suitable concn and pH value, and the shortcoming of maximum to be the protein of separation and Extraction also will carry out the desalination step mostly.
Centrifuging is to utilize the character that have nothing in common with each other such as the quality of variable grain, density, size and shape, and settled speed is also inequality in same fixed centrifugal field, therefore obtains mutual separation.It is one of protein, enzyme, nucleic acid and the isolating the most frequently used method of cell subfraction.But also have very big deficiency, for example high speed centrifugation may destroy the structure of some biomacromolecule.In order to reach best separating effect, adopt some special solution to carry out centrifugal mostly, for example adopt phenol and chloroform extraction DNA, though this method is applicable to most of biological specimens, but bring pollutent easily in operation steps complexity, extraction efficiency instability, the centrifugal process, and phenol, chloroform all have volatilization toxicity, life-time service serious harm HUMAN HEALTH.In addition, can also in centrifuge tube, add granulated glass sphere, rely on centrifugation simultaneously and separate purification DNA with the adsorption of granulated glass sphere surface electrostatic with DNA, its advantage is to remove most of impurity, more suitable to polluting biological specimen, but because the granulated glass sphere specific surface area is little, extraction efficiency is lower, and the experimentation needs are repeatedly centrifugal, complex steps.
This laboratory is to the existing preliminary research of microchip Solid-Phase Extraction (SPE method) purification technique, the SPE method is to utilize the surface of some solid matters, as silicon chip, glass, ion exchange resin or material surfaces such as magnetic bead through modifying, as carrier specificity absorption biomacromolecule, adopt centrifuging to separate purification then; Microchip SPE method is meant the microstructure utilized in the chip carrier as Solid-Phase Extraction, and whole separation purification process is finished in chip.But conventional SPE method is mostly because the restriction of the specific area of solid phase carrier makes extraction efficiency limited.
Summary of the invention
But the object of the invention has been to prepare a kind of reversible encapsulation microfluid biological sample process chip that the good repeated multiple times of refining effect is used of separating, and proposes preparation method and the application in the biological sample separation is purified thereof that this reversible encapsulation microfluid biological sample is handled sheet.
For achieving the above object, technical solution of the present invention provides a kind of reversible encapsulation microfluid biological sample process chip, be used for biological sample and separate purification, adopting silicon chip, polydimethylsiloxane and glass is the chip body material, on silicon chip, etch little raceway groove, etch sample introduction, sample outlet hole or sample introduction at little raceway groove two ends, go out the sample flow-through cell, sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell Open Side Down or upwards, polydimethylsiloxane film and sheet glass are combined into cover plate, the polydimethylsiloxane film is overlying on the internal surface of sheet glass, is connected with silicon chip; Simultaneously little raceway groove is modified, prepared silicon oxide layer or porous silica layer in little channel surface, this silicon oxide layer or porous silica layer are as separating the solid phase carrier of purifying;
Silicon chip and cover plate can carry out reversible encapsulation, but repeated disassembled and assembled is repeatedly used.
Described reversible encapsulation microfluid biological sample process chip, its described silicon chip and cover plate carry out reversible encapsulation, are to adopt the polydimethylsiloxane of silicon chip and sheet glass internal surface to carry out the little raceway groove of reversible encapsulation formation sealing.
Described reversible encapsulation microfluid biological sample process chip, its described silicon oxide layer or porous silica layer are on the surface of the little raceway groove of chip, and this silicon oxide layer or porous silica layer are as separating the solid phase carrier of purifying.
Described reversible encapsulation microfluid biological sample process chip, its described little raceway groove are V-arrangement, trapezoidal, U-shaped or rectangle.
Described reversible encapsulation microfluid biological sample process chip contains microtrabeculae in the little raceway groove of its described rectangle.
Described reversible encapsulation microfluid biological sample process chip, its described microtrabeculae is square column or cylinder.
Described reversible encapsulation microfluid biological sample process chip, its described silicon oxide layer or porous silica layer are on the surface and the microtrabeculae wall in little raceway groove of the little raceway groove of chip.
The preparation method of described reversible encapsulation microfluid biological sample process chip, process is:
(a) silicon nitride adopts negative glue or positive glue as photo etched mask as the mask of etching silicon chip and preparation porous silica layer;
(b) photoetching behind the whirl coating;
(c) on silicon chip, etch little raceway groove, sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell; It also is included in the microtrabeculae that etches on the silicon chip in little raceway groove and the little raceway groove in (c) step;
(d) microtrabeculae surface preparation silicon oxide layer or the porous silica layer in little raceway groove or little raceway groove and little raceway groove;
(e) the polydimethylsiloxane prepolymer is spin-coated on the internal surface of punching or nonporous glass, forms cover plate with sheet glass after the polydimethylsiloxane prepolymer cures; The polydimethylsiloxane prepolymer is spin-coated on the internal surface of glass, forms cover plate with glass after the polydimethylsiloxane prepolymer cures;
(f) encapsulation is carried out reversible encapsulation with silicon chip and cover plate, gets finished product.
The preparation method of described reversible encapsulation microfluid biological sample process chip, its described (c) is in the step, adopt each diversity corrosive fluid corrosion of silicon to form V-arrangement, U-shaped or trapezoidal little raceway groove, or the employing deep etching technology, on silicon chip, etch the microtrabeculae in little raceway groove of rectangle or the little raceway groove of rectangle and the little raceway groove of rectangle; Adopt each diversity corrosive fluid corrosion technology or deep etching technology to prepare sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell.
The preparation method of described reversible encapsulation microfluid biological sample process chip, its described (d) is in the step, adopt electrochemical erosion method to prepare porous silicon in hydrogen fluoroethanol solution, prepare the porous silica layer at low thermal oxidation then, temperature range is 400-800 ℃; Or directly high temperature oxidation forms silicon oxide layer, and temperature range is 1000-1200 ℃.
The preparation method of described reversible encapsulation microfluid biological sample process chip, its described (d) and (e) between the step also has on sheet glass corresponding silicon chip sample introduction, goes out the punching of sample flow-through cell position.
The preparation method of described reversible encapsulation microfluid biological sample process chip, its described (f) are in the step, and encapsulation is to adopt the polydimethylsiloxane of silicon chip and cover inner surface to carry out reversible encapsulation to form the little raceway groove of sealing.
The preparation method of described reversible encapsulation microfluid biological sample process chip, its described c) in the step, sample introduction, sample outlet hole are the through hole on the silicon chip, and sample introduction, to go out the sample flow-through cell be blind hole, and opening upwards.
Described reversible encapsulation microfluid biological sample process chip is characterized in that, can separate in the purification at biological sample, repeatedly uses repeatedly.
Described reversible encapsulation microfluid biological sample process chip, it can separate purification DNA from biological sample, RNA, protein or cell.
The technology of the present invention effect is: seal little raceway groove by silicon chip and polydimethylsiloxane film (PDMS), the reversible encapsulation preparation of sheet glass, need not bonding technology and finish the manufacturing of chip, improved the yield rate of chip, reduced production cost, simultaneously because reversible being packaged with is beneficial to the cleaning chip, this chip can repeat repeatedly to use, and prolongs the work-ing life of chip.
Description of drawings
Fig. 1 is the silicon chip open-type porous silica microfluid biochip structural representation of reversible packaging micro fluent chip for separating and purifying biological sample of the present invention;
Fig. 2 is the cover plate open-type porous silica microfluid biochip structural representation of reversible packaging micro fluent chip for separating and purifying biological sample of the present invention;
Fig. 3 is the porous silicon sem photograph of little channel surface electrochemical erosion method preparation of the silicon chip open-type preprocessed chip of porous silica microfluid sample of reversible packaging micro fluent chip for separating and purifying biological sample of the present invention.
Embodiment
Reversible packaging micro fluent chip for separating and purifying biological sample of the present invention, be used for biological sample and separate purification, adopt silicon chip 7, polydimethylsiloxane (PDMS) 8, glass 9 is the chip body material, on silicon chip 7, etch little raceway groove 1, etch sample introduction at little raceway groove 1 two ends, sample outlet hole 2 or sample introduction, go out sample flow-through cell 3, reversible encapsulation polydimethylsiloxane 8 and glass 9 are formed cover plate 4 on the silicon chip 7, simultaneously little raceway groove 1 is modified, at little raceway groove 1 surface preparation silicon oxide layer or porous silica layer 5, this silicon oxide layer or porous silica layer 5 are as separating the solid phase carrier of purifying.Little raceway groove 1 is V-arrangement, trapezoidal or rectangle, is shaped on array microtrabeculae 6 in the little raceway groove 1 of rectangle, and the spacing of adjacent microtrabeculae 6 is selected on demand, and microtrabeculae 6 shapes are generally cylinder or square column.Silicon oxide layer or porous silica layer 5 exist only on microtrabeculae 6 walls in the little raceway groove of chip 1 surface and the little raceway groove.Reversible encapsulation microfluid biological treatment chip, its cover plate 4 is formed with polydimethylsiloxane (PDMS) layer 8 and sheet glass 9, and polydimethylsiloxane (PDMS) layer 8 is overlying on the internal surface of sheet glass 9, is connected with silicon chip 7, and sheet glass 9 is a simple glass.Adopt silicon chip 7 and cover plate 4 to carry out reversible encapsulation, form the little raceway groove 1 of sealing.
The preparation method of porous silica microfluid biological sample process chip is: at silicon chip 7 cvd silicon nitrides, as the mask of etching silicon chip 7 and preparation porous silica layer 5; Adopt negative glue or positive glue as photo etched mask, photoetching behind the whirl coating; Adopt each diversity corrosive fluid corrosion of silicon 7 to form V-arrangement or trapezoidal little raceway groove 1, or the employing deep etching technology etch the microtrabeculae 6 in the little raceway groove 1 of rectangle or the little raceway groove 1 of rectangle and the little raceway groove 1; Adopt each diversity corrosive fluid corrosion technology or deep etching technology to prepare sample introduction, sample outlet hole 2 or sample introduction, go out sample flow-through cell 3; Adopt electrochemical erosion method to prepare porous silicon in hydrogen fluorine (HF) ethanolic soln, the low thermal oxidation legal system is equipped with porous silica layer 5 then, and temperature range is 400-800 ℃; Or directly high temperature oxidation forms silicon oxide layer 5, and temperature range is 1000-1200 ℃; The unnecessary silicon nitride of etching; On sheet glass 9 corresponding silicon chip 7 sample introductions, go out sample flow-through cell 3 positions punchings, polydimethylsiloxane 8 prepolymers are spin-coated on the internal surface of punching or nonporous glass 9, form cover plates 4 with sheet glass 9 after polydimethylsiloxane 8 prepolymer cures; Adopt silicon chip 7 and cover plate 4 to carry out reversible encapsulation and form the little raceway groove 1 of sealing.
Among the present invention, the cover plate 4 that polydimethylsiloxane (PDMS) 8 and glass 9 form encapsulates with silicon chip 7 and is meant: cover plate 4 carefully is placed on the silicon chip 7, because the effect of hydrogen bond, Van der Waals force, electrostatic force etc., polydimethylsiloxane on the cover plate 4 (PDMS) 8 bonds together with silicon chip 7, and then has formed little raceway groove 1 of sealing.Owing to the less cause of reactive force of hydrogen bond, Van der Waals force, electrostatic force, cover plate 4 can thoroughly clean little raceway groove 1 by carefully stripping down with utensils such as thin blades simultaneously.Reversible encapsulation is meant: because cover plate 4 and the silicon chip 7 peeled away remain intact, cover plate 4 and the silicon chip 7 peeled away after thoroughly cleaning all can be bonding once more, repeat repeatedly to use.Because traditional die adopts glass 9 and silicon chip 7 anode linkages, formed firm chemical bond between glass 9 and the silicon chip 7, in case bonding is just finished can't be with glass 9 and silicon chip 7 intact peeling away, simultaneously because little channel dimensions is little, easy cleaning not after carrying out Bioexperiment.And the present invention adopts glass 9 and polydimethylsiloxane (PDMS) 8 formation cover plates 4 to overcome above-mentioned shortcoming by the reversible encapsulation of cover plate 4 and silicon chip 7.
Characteristics of the present invention are that silicon chip 7 and cover plate 4 carry out reversible encapsulation, but repeated disassembled and assembled is repeatedly used.Utilize MEMS (micro electro mechanical system) (MEMS) processing technology in little raceway groove 1 of silicon 7, to prepare silicon oxide layer or porous silica layer 5 are arranged; this silicon oxide layer or porous silica layer 5 are as separating the solid phase carrier of purifying; and on the microchip in the width of little raceway groove 1, length and the degree of depth and the little raceway groove diameter, height and the gap of microtrabeculae 6 can change to some extent as the case may be; little in addition raceway groove 1 can be designed to whole little pond shape, and the Any shape and the size of little raceway groove 1 and microtrabeculae 6 all drop in protection scope of the present invention.
Describe the preferred scheme of the present invention below in conjunction with accompanying drawing, but do not limit the scope of the invention.
Embodiment one:
Clean N type low resistance (0.011-0.014 Ω .cm) twin polishing monocrystalline silicon piece 7; Growth 2000
(silicon nitride thickness can select 1000-7000 to silicon nitride
) as the mask of etching silicon chip 7 with preparation porous silica layer 5; Get rid of negative photoresist BN303 (negative glue), develop in the exposure back on Karlsuss MA4 type lithography machine; Erode away sample introduction, sample outlet hole 2 with each diversity corrosive fluid of K0H, sample introduction, sample outlet hole 2 are the through hole on the monocrystalline silicon piece 7, and eroding away the degree of depth simultaneously is that 140 μ m (degree of depth may be selected to be 80-150 μ m), width are the little raceway grooves 1 of 200 μ m; 1050 ℃ of high-temperature thermal oxidation growing silicon oxides 5; On the plasma etching machine, use SF
6Destroy silicon nitride; Silicon chip 7 and cover plate 4 (cover plate 4 is composited for PDMS8 and simple glass 9) carry out reversible encapsulation, preparation chip as shown in Figure 1.Biological sample (PCR reaction product, whole blood etc.) and reagent import porous silica microfluid biological sample process chip by peristaltic pump, can be at 40 minutes with interior extraction DNA.This reversible encapsulation microfluid biological sample process chip has been avoided bonding technology, improved yield rate greatly and shortened preparation time, can produce in batches, can be repeatedly used, avoid biological sample obstruction chip in the use, thereby prolonged the work-ing life of process chip.
Embodiment two:
Clean N type low resistance (0.011-0.014 Ω .cm) twin polishing monocrystalline silicon piece 7; Growth 2000
(silicon nitride thickness can select 1000-7000 to silicon nitride
) as the mask of etching silicon chip 7 with preparation porous silica layer 5; Get rid of positive photoresist AZ1500 and AZ4620 (positive glue), develop in the exposure back on Karlsuss MA4 type lithography machine; It is 80 μ m sample introductions that deep etching technology etches the degree of depth, go out sample flow-through cell 3, sample introduction, go out sample flow-through cell 3 and be blind hole, opening upwards, simultaneously etching the degree of depth deeply is 80 μ m (degree of depth may be selected to be 80-200 μ m), width be in little raceway groove 1 of 200 μ m and the little raceway groove 1 microtrabeculae 6 (microtrabeculae 6 gaps are 10 μ m (gap may be selected to be 5-50 μ m), microtrabeculae 6 highly is 80 μ m (highly may be selected to be 50-200 μ m), the diameter of circular microtrabeculae 6 is 10 μ m (diameter may be selected to be 5-50 μ m), or the length of side of square microtrabeculae 6 is 20 μ m (length of side may be selected to be 5-50 μ m)); In hydrofluoric acid (HF) ethanolic soln, adopt electrochemical erosion method to prepare porous silicon (porous silicon is a dendroid or spongy); 570 ℃ of low thermal oxidation growth porous silica layers 5; On the plasma etching machine, use SF
6Destroy silicon nitride; Silicon chip 7 and cover plate 4 (cover plate 4 is for PDMS8 and simple glass 9 are composited, on the cover plate 4 with the chip sample introduction, go out on sample flow-through cell 3 opposite positions to punch) carry out reversible encapsulation, prepare chip as shown in Figure 2.Biological sample (PCR reaction product, whole blood etc.) and reagent import porous silica microfluid biological sample process chip by peristaltic pump, can be at 60 minutes with the interior separation and Extraction of finishing DNA.
Claims (12)
1. reversible encapsulation microfluid biological sample process chip, be used for biological sample and separate purification, adopting silicon chip, polydimethylsiloxane and glass is the chip body material, on silicon chip, etch little raceway groove, etch sample introduction, sample outlet hole or sample introduction at little raceway groove two ends, go out the sample flow-through cell, sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell Open Side Down or upwards, the compound composition cover plate of polydimethylsiloxane film and sheet glass, polydimethylsiloxane is overlying on the internal surface of sheet glass, is connected with silicon chip; It is characterized in that, little raceway groove is modified, prepare silicon oxide layer or porous silica layer in little channel surface, this silicon oxide layer or porous silica layer are as separating the solid phase carrier of purifying; Silicon chip and cover plate can carry out reversible encapsulation, but repeated disassembled and assembled is repeatedly used.
2. reversible encapsulation microfluid biological sample process chip as claimed in claim 1 is characterized in that, described little raceway groove is V-arrangement, trapezoidal, U-shaped or rectangle.
3. reversible encapsulation microfluid biological sample process chip as claimed in claim 1 is characterized in that, contains microtrabeculae in the little raceway groove of described rectangle.
4. reversible encapsulation microfluid biological sample process chip as claimed in claim 3 is characterized in that described microtrabeculae is square column or cylinder.
5. reversible encapsulation microfluid biological sample process chip as claimed in claim 1 is characterized in that described silicon oxide layer or porous silica layer are on the surface and the microtrabeculae wall in little raceway groove of the little raceway groove of chip.
6. the preparation method of reversible encapsulation microfluid biological sample process chip as claimed in claim 1, process is:
(a) silicon nitride adopts negative glue or positive glue as photo etched mask as the mask of etching silicon chip and preparation porous silica layer;
(b) photoetching behind the whirl coating;
(c) etching or erode away little raceway groove, sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell on silicon chip; It is characterized in that,
(d) prepare silicon oxide layer or porous silica layer in little channel surface;
(e) the polydimethylsiloxane prepolymer is spin-coated on the internal surface of punching or nonporous glass, forms cover plate with sheet glass after the polydimethylsiloxane prepolymer cures;
(f) encapsulation is carried out reversible encapsulation with silicon chip and cover plate, makes finished product.
7. the preparation method of reversible encapsulation microfluid biological sample process chip as claimed in claim 6, it is characterized in that, in described (c) step, adopt each diversity corrosive fluid corrosion of silicon to form V-arrangement, U-shaped or trapezoidal little raceway groove, or the employing deep etching technology, on silicon chip, etch in little raceway groove of rectangle or the little raceway groove of rectangle and the little raceway groove of rectangle and be shaped on the array microtrabeculae; Adopt each diversity corrosive fluid corrosion technology or deep etching technology to prepare sample introduction, sample outlet hole or sample introduction, go out the sample flow-through cell.
8. the preparation method of reversible encapsulation microfluid biological sample process chip as claimed in claim 6, it is characterized in that, described (d) step, adopt electrochemical erosion method in the hydrofluoric acid ethanolic soln, to prepare porous silicon, prepare the porous silica layer at low thermal oxidation then, temperature range is 400-800 ℃; Or directly high temperature oxidation forms silicon oxide layer, and temperature range is 1000-1200 ℃.
9. the preparation method of reversible encapsulation microfluid biological sample process chip as claimed in claim 6 is characterized in that, described (d) and (e) between the step also has on sheet glass corresponding silicon chip sample introduction, goes out the punching of sample flow-through cell position.
10. the preparation method of reversible encapsulation microfluid biological sample process chip as claimed in claim 6, it is characterized in that, in described (f) step, encapsulation is to adopt the polydimethylsiloxane of silicon chip and cover inner surface to carry out reversible encapsulation to form the little raceway groove of sealing, wherein contain sample introduction, outlet silicon chip and the encapsulation of atresia cover plate, contain sample introduction, go out sample flow-through cell silicon chip and the encapsulation of punching cover plate.
11. the preparation method as claim 6 or 7 described reversible encapsulation microfluid biological sample process chip is characterized in that described c) in the step, sample introduction, sample outlet hole are the through hole on the silicon chip, and sample introduction, to go out the sample flow-through cell be blind hole, and opening is upwards.
12. reversible encapsulation microfluid biological sample process chip as claimed in claim 1 is characterized in that, separates purification DNA, RNA, protein or cell from biological sample.
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