CN103712951B - A kind of preparation method based on three-dimensional structure nano-array biochip and application thereof - Google Patents
A kind of preparation method based on three-dimensional structure nano-array biochip and application thereof Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/0896—Nanoscaled
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L2300/00—Additional constructional details
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- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
Abstract
The invention discloses a kind of preparation method based on three-dimensional structure nano-array biochip and application thereof, the method comprises the following steps: (1) is by the solution of silane process of golden film containing sulfydryl; (2) by the golden film acid solution process after step (1) process; (3) anodic aluminum oxide film of through hole is affixed on the golden film after step (2) process; (4) by the golden film obtained through step (3) successively be immersed in silicon tetrachloride (SiCl
4), in the mixed liquor of normal hexane, normal hexane and methyl alcohol, second alcohol and water (this process is repeatedly capable of circulation); (5) the gold substrate acid solution process that will obtain through step (4).Method provided by the invention is easy to nanotube (post) array preparing three-dimensional structure in substrate, and the size of managing (post) is controlled, can be used as biochip, and can carry out high sensitivity, cold biological detection.
Description
Right of priority
This application claims the right of priority that application number is 201310754275X, name is called the Chinese patent application of " a kind of preparation method based on three-dimensional structure nano-array biochip and application thereof ".
Technical field
The present invention relates to a kind of based on three-dimensional structure nano-array biochip and preparation method thereof, particularly, the present invention relates to one on golden film, synthesize three-dimensional structure silicon dioxide nanotube array biochip and preparation method thereof, and the application of this biochip in biomolecule specific detection.
Background technology
Highly sensitive bio-sensing in a lot of fields in be widely used, be included in the fields such as biological medicine research, environmental monitoring, proteomics, genomics, pharmacy, medical diagnosis, simultaneously it also obtain and approves more and more widely.Such as, the sensitivity based on the bio-sensing of fluorescence signal detection just can be very high, even single biomolecule can be detected.But based on bio-sensing also some drawback of fluorescence method, as very consuming time by the unusual costliness of fluorescence group mark biomolecule price, and this method even may not realize in some applications.In addition, some function of original biomolecule may be affected after mark fluorescent group.So adopt cold method to go the sensor of direct-detection biomolecule to compare just seem important.
In ten or twenty year in the past, the non-marked biology sensor much based on dissimilar nanostructured is studied widely, and these sensors all have benefited from the feature of nanometer technology.Such as there is the biology sensor etc. based on nano wire, nano-pore, nanotube or nano-pillar type.Interactional response between probe molecule and target molecule simply and fast can be changed into light signal, electric signal, thermal signal or acoustical signal etc. by dissimilar cold biology sensor.Wherein for cold optical biosensor, signal method of converting is mainly divided into two classes: optical interference and surface plasma.Optical waveguide sensor based on nano thin-film structure is exactly a kind of biology sensor of typical optical interference.It has two main advantages.First, the electromagnetic field of incident light is limited in nanometer thin rete with the optical waveguide mode strengthened, and can effectively covers the biomolecule of adsorbing in thin layer.Secondly, due to nano thin-film there is the design feature of very large Adsorption for Biomolecules surface area, and then cause the enrichment of biomolecule, so the response of biology sensor can be improved further.Even so, relative to the biology sensor detected based on fluorescence signal, the sensitivity of cold biology sensor is not also very high.Accordingly, the response how improving cold biology sensor requires further study.
Summary of the invention
Invention provides a kind of biochip based on three dimensional silica nano-tube array structure and preparation method thereof, and its in biomolecule specific detection in application.
The invention provides a kind of preparation method based on three-dimensional structure nano-array biochip, the method comprises the following steps:
(1) by the solution of silane process of golden film containing sulfydryl;
(2) by the golden film acid solution process after step (1) process;
(3) the anodised aluminium film layer of through hole is affixed on the golden film after step (2) process;
(4) by the mixed liquor being immersed in silicon tetrachloride (SiCl4), normal hexane, normal hexane and methyl alcohol of the golden film order that obtains through step (3), second alcohol and water (this process is repeatedly capable of circulation);
(5) the gold substrate acid solution process that will obtain through step (4).
Present invention also offers the biochip of the three dimensional silica nano-tube array structure prepared by method described above.
Present invention also offers the application of biochip in biomolecule specific detection of the three dimensional silica nano-tube array structure prepared by method described above.
In the present invention, by the solution of silane process of golden film containing sulfydryl, use acid solution process again, hydrophilic surface can be obtained, then the anodic aluminum oxide film of through hole is affixed on golden film, the water wettability of so golden film can the connection of enhanced film and golden film, obtaining silicon dioxide tube (post) by being alternately immersed in certain number of times in the mixed liquor of silicon tetrachloride (SiCl4), normal hexane, normal hexane and methyl alcohol, second alcohol and water again, finally the gold substrate obtained being soaked in acid solution the biochip obtaining three dimensional silica nano-tube array structure.Method provided by the invention is easy to nanotube (post) array preparing three-dimensional structure in substrate, and the size of managing (post) is controlled, can be used as carrying out high sensitivity, cold biological detection in biochip.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for instructions, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the scanning electron microscope diagram (SEM) at the biochip interface of three dimensional silica nano-tube array structure prepared by the embodiment of the present invention 1;
Fig. 2 is the surface Scanning Electron microscope figure (SEM) of the anodic aluminum oxide film of the through hole used in the embodiment of the present invention 1;
Fig. 3 is the reflectivity angle spectrogram of the biochip specific detection streptavidin (streptavidin) of the three dimensional silica nano-tube array structure that the present invention is prepared according to the method for application examples 1;
Fig. 4 is the kinetic curve figure utilizing the biochip test streptavidin (streptavidin) of three dimensional silica nano-tube array structure prepared according to the method for application examples 1 and comparative example 1.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of preparation method based on three-dimensional structure nano-array biochip, the method comprises the following steps:
(1) by the solution of silane process of golden film containing sulfydryl;
(2) by the golden film acid solution process after step (1) process;
(3) anodic aluminum oxide film of through hole is affixed on the golden film after step (2) process;
(4) the golden film that will obtain through step (3) uses sol-gal process, successively in the mixed liquor being immersed in silicon tetrachloride (SiCl4), normal hexane, normal hexane and methyl alcohol of order, second alcohol and water (this process is repeatedly capable of circulation);
(5) the gold substrate acid solution process that will obtain through step (4), removing aluminum oxide film.
According to the present invention, in step (1), described golden film is the glass substrate material that surface has layer gold.In the present invention, can use magnetron sputtering vapour deposition method or hot vapour deposition method on this base material, plate the gold that one deck has 35-50nm thickness, be preferably 40-45nm.
According to the present invention, in step (1), described hydrosulphonyl silane solution is (3-mercaptopropyi) trimethoxy silane solution, and described solution concentration is 1-30mM, is preferably 18-22mM.In solution, soak time is 3-12 hour, is preferably 3 hours.In the present invention, hydrosulphonyl silane can be dissolved in organic solvent and be made into hydrosulphonyl silane organic solution, wherein particular/special requirement be there is no to the organic solvent dissolving described hydrosulphonyl silane, it can be organic solvent known in the art, such as, can be one or more in methyl alcohol, ethanol or acetone, preferably, can be methyl alcohol.
According to the present invention, in step (2), described acid solution can be aqueous hydrochloric acid solution, and concentration is 0.05-0.2mol/L, is preferably 0.1mol/L.In solution, the processing time is 1-20 hour, and be preferably 10-12 hour, temperature is room temperature.
According to the present invention, in step (3), the anodic aluminum oxide film of through hole and can impregnated in the mixed solution of acetone or acetone and water through the substrate of step (2) gained gold film.When described solution is the mixed liquor of acetone and water, acetone and water consumption volume ratio can be 1-3:1, are preferably 1:1.Aluminum oxide film covers in golden film substrate, and then in drying box, normal temperature places 24-48 hour, is preferably 24-30 hour.
According to the present invention, in step (4), the golden film substrate processed through step (3) successively order impregnated in silicon tetrachloride (SiCl
4), in the mixed liquor of normal hexane, normal hexane and methyl alcohol, second alcohol and water.Wherein in silicon tetrachloride, dip time is 1-2 minute, is 3-5 minute in other solution, is preferably 5 minutes.The volume ratio of normal hexane and methanol usage is 1:1.The once circulation that step (4) is surperficial sol-gal process.
According to the present invention, in step (5), acid solution used is phosphoric acid solution, and concentration is 5-15 quality %, is preferably 10-12 quality %.Temperature is 20-40 DEG C.
Present invention also offers the biochip of the three dimensional silica nano-tube array structure prepared by method described above.
Present invention also offers the application of biochip in biomolecule specific detection of the three dimensional silica nano-tube array structure prepared by method described above.
Below will be described the present invention by embodiment, but protection scope of the present invention is not limited in these embodiments.
In the following Examples and Comparative Examples:
3-aminopropyl triethoxysilane (3-aminopropyltriethoxysilane) is purchased from AlfaAesar company; Biotin (Sulfo-NHS-LC-LC-Biotin) is purchased from the great Bioisystech Co., Ltd in Shanghai; Streptavidin (streptavidin) is purchased from Beijing Bo Aosen Bioisystech Co., Ltd.
Embodiment 1
The present embodiment is for illustration of the biochip adopting method of the present invention to prepare three dimensional silica nano-tube array structure.
(1) preparation of golden film: K9 glass sheet is placed in the H that volume ratio is 7:3
2sO
4(98 % by weight) and H
2o
2in (30 % by weight) mixed solution, clean 1 hour, then clean with deionized water rinsing.By gold thick for the K9 glass sheet magnetron sputtering method evaporation one deck 40nm after clean, i.e. the gold substrate of the present embodiment use;
(2) the hydrosulphonyl silane solution-treated of gold substrate: with (3-mercaptopropyi) trimethoxy silane solution of methyl alcohol configuration 20mmol.The gold substrate obtained through step (1) to be immersed in above-mentioned solution solution 3 hours.
(3) the acid solution process of gold substrate: the gold substrate of hydrochloric acid solution process after step (2) using 0.1mol, the processing time is 10 hours, normal temperature;
(4) nanohole alumine film is affixed in gold substrate: the anodic aluminum oxide film of through hole and can be immersed in the mixed solution of acetone and water through step (3) gained gold substrate, and wherein the consumption volume ratio of acetone and water is 1:1.Allow aluminum oxide film be affixed in gold substrate, then in drying box, normal temperature places 24 hours.Wherein the surface Scanning Electron microscope figure (SEM) of anodic aluminum oxide film as shown in Figure 2, in figure, S4800 represents the model of scanning electron microscope, voltage added when 5.0kV represents observing samples, 9.1mm × 50.0k represents the distance of electron gun and sample and the multiple of amplification respectively, 2013-3-13 represents the date of taking pictures, 1.00 μm of expression scales.
(5) preparation of Silica Nanotube: the gold substrate that will obtain through step (4) to immerse in silicon tetrachloride 2 minutes, and then to immerse successively in the mixed liquor of normal hexane, the normal hexane of 1:1 volume ratio and methyl alcohol, second alcohol and water each 5 minutes.Such one group is the circulation of one-time surface collosol and gel.For the present embodiment, employ 6 subsurface collosol and gel circulations.
Record the biochip of three dimensional silica nano-tube array structure prepared by embodiment 1 as shown in Figure 1.In Fig. 1,10 is golden film, 20 is silicon dioxide nanotube array, S4800 represents the model of scanning electron microscope, 5.0kV voltage added when representing observing samples, 9.9mm × 50.0k represents the distance of electron gun and sample and the multiple of amplification respectively, 2013-7-3 represents the date of taking pictures, 1.00 μm of expression scales.
Application examples 1
Specific detection streptavidin
The biochip of the three dimensional silica nano-tube array structure prepared according to embodiment 1 method should be used by use-case.Pick-up unit is based on Kretchmann structure.When meeting incident light and being coupled with the waveguide mode in nanotube array layer, occur on reflectivity angular spectrum with the form of a resonance groove through biochip reflected light.The angle position of this resonance groove is relevant with the effective refractive index of nanotube array layer.Any by causing the change of the effective refractive index of nanotube array layer all will to be amplified by waveguide mode in Adsorption for Biomolecules to nanotube array layer or due to the minor alteration of solution refractive index to be measured, the movement of the groove that resonates in the present reflectivity angular spectrum of final body.In addition, if the approximate angle that incident angle of light is fixed on resonance groove is gone measurement of reflectivity, the change of the effective refractive index of this nanotube array layer also can be quantized by the change of refraction.
It is as follows that biochip connects the method for detectable biomolecule: biochip is placed in 3-aminopropyl triethoxysilane (3-aminopropyltriethoxysilane) solution 24 hours of 5 % by weight.3-aminopropyl triethoxysilane one end can connect the hydroxyl of silica surface, and the other end can react with the functional group containing-NHS.After this, biotin (Sulfo-NHS-LC-LC-Biotin) solution same sample utilizing PDMS runner flow into NHS modification changes 10mmol phosphate buffer (buffer) certain hour into after 1 hour, then the change of the intensity incident angle of reflected light is measured, as shown in Figure 3.In Fig. 3, the longitudinal axis is reflectivity (Reflectivity), transverse axis is angle of light degree (IncidentAngle), unit is degree (degree), the reflectivity angle spectrogram that the curve that data point is rhombus obtains for using this chip detection buffer solution (buffer), data point is circular curve is that the biotin solution (500 μm of olbiotin) passing into 500 μm of ol reaches the saturated later measured reflectivity angle spectrogram of absorption, data point for square curve be pass into 100nmol streptavidin (100nmolstreptavidin) reach absorption saturated rear surveyed reflectivity angle spectrogram.For specific detection streptavidin molecule, the streptavidin of 100nmol flows into sample surfaces 90 minutes by PDMS runner, then 10mmol phosphate buffer solution (buffer) is used to rinse a period of time, in this process, incident angle is fixed on resonance angle place (62.08 °), then the change of reflectivity along with the time is recorded, as shown in Figure 4.In Fig. 4, the longitudinal axis is reflectivity (Reflectivity), transverse axis is time (time), unit is second (s), the result curve of 1 expression application examples 1, the result curve of 2 expression comparative examples 1,3 represent the time point passing into 100nmol streptavidin (100nmolstreptavidin), and 4 represent the time point passing into buffer solution (buffer).Finally measure the change of the intensity incident angle of reflected light, as shown in Figure 3, the angle of resonance groove moves to 62.72 ° in conjunction with streptavidin from 62.08 after modified biological element °.
Comparative example 1
Detect streptavidin
The method that biochip connects detectable biomolecule is as follows: for detection streptavidin molecule, first flow into sample surfaces with the phosphate buffer of 10mmol by PDMS runner and set up background baseline, then the streptavidin solution of 100nmol is passed into sample surfaces, the change of the resonance FLUTE ANGLE place measurement of reflectivity recorded after passing into phosphate buffer solution in this process, as seen from Figure 4, the change of reflectivity almost can be ignored relative in application examples 1.
Application examples 1 of the present invention and comparative example 1 can absolutely prove streptavidin molecule and biotin molecule is specific combines.In addition, also can illustrate that the biochip of this three dimensional silica nano-tube array structure can be applied to the specific detection of biomolecule.
The application that the present invention relates in biomolecule modification in biomolecule specific detection is not limited to said method, the customized alkyl of other end can be used to be connected on silicon dioxide tube, and use corresponding biomolecule method of attachment.The biomolecule connected is not limited to this embodiment yet.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out combination in any between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1., based on a preparation method for three-dimensional structure nano-array biochip, it is characterized in that:
A, by the solution of silane process containing sulfydryl of golden film;
B, by the golden film acid solution process after processing of step A;
C, the anodic aluminum oxide film of through hole is affixed on the golden film after step B process;
In D, the mixed liquor golden film obtained through step C being sequentially immersed in silicon tetrachloride, normal hexane, normal hexane and methyl alcohol, these five kinds of solution of second alcohol and water, flood once or circulate repeatedly;
E, the gold substrate acid solution process will obtained through step D, removing aluminum oxide film, obtains the biochip of three dimensional silica nano-tube array structure.
2. method according to claim 1, is characterized in that: in step, and the described solution of silane containing sulfydryl is (3-mercaptopropyi) trimethoxy silane solution, and described solution concentration is 1-30mM; In solution, soak time is 3-12 hour.
3. method according to claim 1, is characterized in that: in step, and the described solution of silane containing sulfydryl hydrosulphonyl silane is dissolved in the hydrosulphonyl silane organic solution be made in organic solvent.
4. method according to claim 1, is characterized in that: in stepb, and described acid solution is hydrochloric acid solution, and concentration is 0.05-0.2mol/L; In solution, the processing time is 1-20 hour, normal temperature.
5. method according to claim 1, is characterized in that: in step C, the anodic aluminum oxide film of through hole and impregnated in the mixed solution of acetone or acetone and water through the substrate of step B gained gold film; When described solution is the mixed liquor of acetone and water, acetone and water consumption volume ratio are 1-3:1; Aluminum oxide film covers in golden film substrate, and then in drying box, normal temperature places 24-48 hour.
6. method according to claim 1, is characterized in that: in step D, and in silicon tetrachloride, dip time is 1-2 minute, is 3-5 minute in other solution; The volume ratio of normal hexane and methanol usage is 1:1.
7. method according to claim 1, is characterized in that: in step e, and acid solution used is phosphoric acid solution, and concentration is 5-15 quality %; Temperature is 20-40 DEG C.
8. method according to claim 1, is characterized in that: in step, described golden film is attached to the surface of glass substrate material, and the thickness of golden film is 40-45nm.
9. based on a three-dimensional structure nano-array biochip, it is characterized in that: according to the method preparation described in claim arbitrary in claim 1-8.
10. a biochip for cold direct-detection biomolecule, is characterized in that: make the biochip prepared according to the method described in claim arbitrary in claim 1-8, for the specific detection of biomolecule.
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| PCT/CN2014/091814 WO2015101118A1 (en) | 2013-12-31 | 2014-11-20 | Preparation method for three-dimensional structured nanoarray-based biochip and application thereof |
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| CN103712951B (en) * | 2013-12-31 | 2016-03-09 | 清华大学深圳研究生院 | A kind of preparation method based on three-dimensional structure nano-array biochip and application thereof |
| CN104991088A (en) * | 2015-06-17 | 2015-10-21 | 清华大学深圳研究生院 | Optical waveguide sensing chip, preparation method thereof, and use |
| CN105839156B (en) * | 2016-04-19 | 2018-04-17 | 清华大学深圳研究生院 | A kind of method that orderly one-dimensional nano-array is prepared in conductive substrates |
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| WO2015101118A1 (en) | 2015-07-09 |
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