CN106191260A - A kind of biochip substrate - Google Patents
A kind of biochip substrate Download PDFInfo
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- CN106191260A CN106191260A CN201610564676.2A CN201610564676A CN106191260A CN 106191260 A CN106191260 A CN 106191260A CN 201610564676 A CN201610564676 A CN 201610564676A CN 106191260 A CN106191260 A CN 106191260A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- C03—GLASS; MINERAL OR SLAG WOOL
- C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
- C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
- C03C17/006—Surface treatment of glass, not in the form of fibres or filaments, by coating with materials of composite character
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- C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
- C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
- C03C17/34—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
- C03C17/42—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions at least one coating of an organic material and at least one non-metal coating
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- C03—GLASS; MINERAL OR SLAG WOOL
- C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
- C03C2217/00—Coatings on glass
- C03C2217/40—Coatings comprising at least one inhomogeneous layer
- C03C2217/42—Coatings comprising at least one inhomogeneous layer consisting of particles only
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Abstract
A kind of biochip substrate, including substrate, by the self-assembled modified substrate of fluorine containing silane, described fluorine containing silane forms the nano-particle modified layer on substrate with the interaction of amination nano-particle.The chip base decorative layer good stability of the present invention, can effectively tolerate the nano-particle modified biochip substrate of supersound washing operation, and can effectively increase the probe density of reaction site.
Description
Technical field
The present invention relates to biochip field, be specifically related to biochip substrate, be related specifically to be suitable for fabricated in situ
Biochip substrate.
Technical background
The biochip technology research in fields such as medical diagnosis on disease, drug development, transgenic analysis, toxicity tests achieves
The biggest progress.Gene chip is typically obtained by two kinds of methods, and a kind of is that pre-synthesis probe chain is passed through deposition techniques
It is fixed on solid substrate to form.Point sample method need not use complicated equipment, but requires to synthesize substantial amounts of probe, experimental cost ratio
Higher, that the homogeneity of simultaneous reactions site probe is also difficult to solve problem.Another is then by parallel fabricated in situ
Prepared by technology, in-situ synthesis can not only overcome these shortcomings of point sample method and be avoided that a large amount of of needs time prepared by cDNA chip
The problem of clone.It is the Typical Representative in superchip preparation that the light remove-insurance of Affymetrix company is defended the doctrine, for biological specimen
High throughput analysis provides important means.
DNA concentration in biological specimen typically ratio is relatively low, before implementing hybridization check, needs the DNA target in sample
Sequence carries out PCR amplification and amplifies, and takes time and effort, is difficulty with the quick diagnosis of sample；Need to use valuable PCR amplification instrument,
Increase testing cost；Meanwhile, the failure of amplification or the amplification of by-product may cause the appearance of false cloudy false positive results.Therefore,
Highly sensitive chip detection technology is always the goal in research of people.The raising of target molecules capture ability, is to improve detection
Sensitivity, simplifies one of important channel of detection of biological samples, and micro & nano technology serves critically important positive role in this respect.
The introducing of nano-particle substrate, not only increases the probe density of reaction site, can also modify further simultaneously, carry for probe
A hybridization environment closer to solution, the beneficially raising of detection sensitivity are supplied.Existing sensitivity amplifying technique is main
It is by deposition techniques, the probe of pre-synthesis to be fixed on micro-nano structure surface to prepare chip.If utilizing micro & nano technology to prepare
Be suitable to in-situ synthesized reaction and prepare the substrate of gene chip, it is achieved probe is fixed in the high density of reaction site, will improve energetically
The sensitivity of chip detection, the advantage highlighting fabricated in situ further.The composition of existing micro-nano structure has two ways a kind of
Being directly to immerse in TiO2 or SiO2 colloidal sol by slide, at the uniform velocity obtaining rough surface after lifting drying and roasting has the painting of projection
Membrane structure, structure comparison is stably firm, but the lifting to specific surface area is inadequate, in order to solve this problem, also has proposition directly
Nano-particle is covalently bound to planar substrates (such as CN201410224306), but inventor studies and finds this kind of of prior art
The operations such as the stability that method prepares substrate is the highest, the resistance to supersound washing can't stand in biochip reaction in-situ modification.
Summary of the invention
Object of the present invention is to provide a kind of probe density that not only can effectively increase reaction site, and substrate is repaiied
Decorations layer good stability, can effectively tolerate the nano-particle modified biochip substrate of supersound washing operation.
In order to realize the purpose of the present invention, inventor has made repeatedly to attempt, after failure many times, it is not intended to middle discovery
The techniques below scheme of the present invention can well realize the purpose of the present invention.
A kind of biochip substrate, including substrate, the substrate self-assembled modified by fluorine containing silane, fluorine containing silane and amination
Nano-particle effect forms the nano-particle modified layer on substrate.
Inventor is found by research
Long link layer contains a large amount of C-F key, and each-F can form hydrogen bond with amino；But also form one simultaneously and be similar to " tapetum "
Structure, every fine hair has the tentacle that much can catch amination nano silicon spheres, can be securely amination nano silicon spheres
Fix, stability can well be improved.
The carbon number of fluorine containing silane is not less than 3, preferably 6-18.
The material of described substrate can be smooth glass, pottery, silicon chip, metal or polymer.
Described fluoroalkylation silane can be perfluoro capryl triethoxysilane, perfluoro capryl trimethoxy silane, complete
Fluorine decyl triethoxysilane, perfluoro decyl trimethoxy silane perfluoro decyl dimethyl dichlorosilane (DMCS), perfluoro capryl first dichloro silicon
Alkane, trifluoro propyl trimethoxy silane, trifluoro propyl triethoxysilane, perfluor heptyl propyl trimethoxy silicane, perfluor heptan
One in base propyl-triethoxysilicane, perfluoro butyl propyl trimethoxy silicane, perfluoro butyl propyl-triethoxysilicane
Or it is several.
Described nano-particle can be chemically inert pottery, metal, metal-oxide, nonmetal oxide or polymerization
Thing nano-particle.
Described chemical inertness is acid, alkali and/or the organic solvent etc. being resistant in biochip preparation process use
Corrode.
Described nano-particle the most a diameter of 10～500nm.
The preparation method is that: first pass through chemical oxidation or plasma treatment, smooth substrate derives
Amino or hydroxyl；Then process amino or hydroxylated substrate with fluorine containing silane solution, obtain fluorine containing silane self-assembled modified
Substrate；Subsequently described substrate is immersed in amidized nanoparticles solution reaction, by gained amination nano-particle
The substrate modified through supersound process, removes the nano-particle of physical absorption, obtain intensive stable have nano-particle modified
The biochip substrate of layer.
The biochip fabricated in situ substrate of the present invention, is become by assembling functionalized nano grain fabric on a planarizing substrate.
As it is shown in figure 1, amination nanoparticle deposition is at substrate surface, by the interaction of amino Yu substrate surface fluoroalkyl, shape
Become intensive stable nano-particle modified film.
Using the chip base of the present invention, nano-particle is firmly bonded on planarizing substrate, can tolerate biochip former
The operations such as the high strength supersonic washing in the building-up process of position, good stability.The amination nano-particle closeness assembled is high, on it
Biochip prepared by fixed member probe, can be effectively improved the probe density of reaction site, improves chip probe and divides target
The capture ability of son, thus reduce the requirement to sample target content, be conducive to improving the detection sensitivity of chip.
Accompanying drawing explanation
Fig. 1 is that the amination nano-particle of the present invention combines schematic diagram in planar substrates, and small circle represents fluorine atom, arrow
Represent the hydrogen bond being likely to be formed；
Fig. 2 is the nano-particle assembling process of the present invention and building block principle and result figure；
Fig. 3 is conventional covalently bound nano-particle covalent bond schematic diagram in planar substrates, and triangle represents epoxy
Base, arrow represents the covalent bond being likely to be formed；
Fig. 4 is (a), the SEM photograph of rear (b) before amination nano silicon spheres deposition slide supersound process, in figure (a), (b)
The right and left is that fluoroalkyl modifies induction and deposition district and covalent bonding modifies crystallizing field respectively.
Fig. 5 is chip base labeled in situ fluorometric reagent rhodamine B and the flat glass chip base mark in situ of the present invention
The fluorescent scanning Comparative result of note fluorometric reagent rhodamine B.The fluorescent scanning figure of (a) amination nanoparticle deposition slide；(b)
Amination plane slide fluorescent scanning figure；(c) Fluorescence Intensity Assays Comparative result.
Detailed description of the invention
The biochip substrate of the present invention, consists of assembling amination nano-particle on smooth substrate.Such as Fig. 1 institute
Show that substrate conventional gene chip microscope slide derives fluoroalkyl decorative layer by the self-assembling reaction containing fluoroalkyl；Nano-particle is
Chemically inert amination nano SiO 2 particle；Amination nanoparticle deposition is at substrate surface, by amino and substrate
The interaction of surface fluoroalkyl, forms the intensive nano-particle modified film of stable monolayer.
Pass throughLegal system prepared silicon dioxide nano-particle, and it is modified, obtain amidized silicon dioxide
Nano-particle；The substrate of glass that perfluor silane coupler is modified is obtained by self-assembling technique；Amination silica nanometer
There is hydrogen bond action between the perfluor silane coupler that grain surface amino groups and surface of glass slide are modified, induce amidized silicon dioxide
The glass surface deposition that nano-particle is modified at perfluor silane coupler, obtains Nano microsphere functionalization chip base, modified
Journey is as shown in Figure 2
Embodiment 1:
The concrete preparation method of nanoparticle of functionalization is, adds 1.6ml deionized water, 15ml anhydrous in 50ml flask
Ethanol and 1.6ml strong aqua ammonia, stir 30min at 40-45 DEG C.Regulation magnetic stirring apparatus rotating speed is to 500 revs/min, disposably
Rapidly join 2ml tetraethyl orthosilicate, after continuing stirring 2min, turn down rotating speed to 200 revs/min.Stirring at low speed at 40-45 DEG C
4h.Supersound process 1h, stands 24h.4000 rotating speeds are centrifuged, and 50 DEG C of vacuum drying oven is dried, and obtaining particle diameter is about 200nm surface
Amidized nano silicon.
Conventional slide is by Piranha solution (concentrated sulphuric acid and the mixture of hydrogen peroxide of 30%, volume ratio is 3 to 1)
Soaking 1 hour, distilled water wash, washing with acetone, nitrogen dries up；Slide is immersed in 2% (percentage by volume) perfluoro decyl first
Reacting 20 minutes in the isooctane solution of base dichlorosilane, slide priority toluene and normal hexane respectively rinse 3 times；Obtain perfluor silicon
Alkane coupling agent self-assembled monolayer, as shown in Figure 2 b；Amidized nm-class core-and-shell particles is dispersed in toluene, by above-mentioned glass
Sheet immerses in this toluene solution, stands 20 hours, by slide as in methanol ultrasonic 3 minutes, and scanning electron microscope analysis such as Fig. 2 d institute
Show, it is seen that obtain the substrate of glass of nano-particle more uniform deposition.
Enforcement comparative example 2:
The nanoparticle of the conventional nanoparticle fixed by covalent bond on substrate of this example contrast and the present invention is fixed
The tolerance of method.By Piranha solution, (concentrated sulphuric acid and the mixture of hydrogen peroxide of 30%, volume ratio is 3 to conventional slide
Ratio 1) soak 1 hour, distilled water wash, washing with acetone, nitrogen dries up；By uniform for photoresist spin coating monoblock slide, it is placed in dry
60 degree of heat treatment 30min in case, 95 degree of heat treatment 30min；Block half slide with shadow mask plate, expose under uviol lamp
20min；It is immersed in 5min in 2.38% Tetramethylammonium hydroxide, deionized water rinsing afterwards, dries up standby.Slide is placed in
The isooctane solution of 2% (percentage by volume) perfluoro decyl dimethyl dichlorosilane (DMCS) reacts 20min, slide priority toluene and just
Hexane respectively rinses 3 times；Fall the photoresist on slide with acetone rinsing, by slide as 20min ultrasonic in acetone soln, dry up；
The chloroform of 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane that slide is placed in 5% (percentage by volume) further is molten
50 DEG C of reaction 20h overnight in liquid；Ultrasonic 10 minutes of chloroform, is dried, and obtaining half is perfluor silane coupler self assembly, and one
Half is the monofilm of 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane self assembly；Amidized nm-class core-and-shell particles is divided
It is dispersed in toluene, above-mentioned slide is immersed in this toluene solution, stand 20 hours, take out from solution, successively by toluene, first
Alcohol, distilled water and methanol washing, nitrogen dries up, and obtaining the left side is that amination nano-particle is (former in fluoroalkyl modification of surfaces deposition
Reason is as shown in Figure 1), the right is the substrate at alkoxyl modification of surfaces deposition (principle is as shown in Figure 3) of the amino nano-particle, gained
The scanning electron microscope result of nano-particle modified substrate is as shown in fig. 4 a；Further slide is placed in supersound process 3 minutes in methanol,
Scanning electron microscope result figure is as shown in Figure 4 b.
Comparison diagram 4 understands, amination nano silicon spheres slide perfluoro decyl trichlorosilane modified regions deposit than 3-(2,
3-epoxy the third oxygen) propyl trimethoxy silicane modified regions is intensive much, and in perfluoro decyl trichlorine after ultrasonic cleaning
The silicon ball in hydride modified region is still firmly bonded to surface of glass slide；And at 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane
Modified regions, silicon ball " fragmentarily " is deposited on wherein, and after ultrasonic, silicon ball all departs from.The reason of this result occurs
After being probably 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane modification hydroxyl region, the epoxy radicals site of its monolayer and nanometer
The covalent bond that silicon ball amino is formed is not enough to fix the silicon ball of more than 200nm under ultrasound environments.And perfluoro decyl trichlorosilane
Fluoroalkyl (-(CF2) 9CF3) the long link layer that modified regions is formed contains a large amount of C-F key, and each-F can form hydrogen bond with amino.
This is similar to one " tapetum ", and every fine hair has the tentacle that much can catch amination nano silicon spheres, can be securely
Amination nano silicon spheres is fixed, demonstrates the advantage than conventional covalent modification.
Enforcement comparative example 3:
This example utilizes the chip base of the present invention, by reaction in-situ, carries out fluorescence on amination nano-grain array
Labeled in situ, obtains the array of fluorescence molecule labelling, and its basic operation is consistent with fabricated in situ chip array, simply uses nanometer
Particle modification substrate replaces without nano-particle modified flat glass substrate.The present embodiment uses rhodamine B isocyanates mark
Illustrate as a example by the preparation of note array, and compare with without nano-particle modified flat glass substrate.
N-Methyl pyrrolidone (NMP) solution of the rhodamine B isothiocyanic acid of preparation 0.05M containing by spotting methods,
It is different that nano-particle modified substrate obtained in embodiment 1 and the most modified flat glass substrate point sample obtain rhodamine B
Hydrogen thiocyanate array, rinses with DMF solution after 30min, and is placed in by slide in DMF, methanol, dichloromethane
(parameter is arranged the scanning of each supersound washing 5min, GenePix 4000B biochip scanner: resolution 10um, laser intensity
100%, PMT 600, excitation wavelength 532nm, wavelength of fluorescence 635nm), obtain fluorometric result as shown in Figure 5.
As shown in Figure 5 a, totally 8 row 4 arrange amidized plane slide substrate marker fluorescent scanning result, to every a line sampling point
Interior sample analysis, sampling point mean fluorescence intensity within 3000-4500 (Fig. 5 c), ratio is more uniform, it is seen that by the method success
Carry out labeled in situ reaction.As shown in Figure 5 b, dot matrix is each for the slide marker fluorescent scanning figure that nano SiO 2 particle is modified
The swept brilliance at place relatively equalizes, and the fluorescence intensity difference in same sampling point is less.It is same to sample analysis in every a line sampling point,
The mean fluorescence intensity of sampling point be distributed in 30000-45000 in the range of (Fig. 5 c), deviation is within 4000, and relative deviation is 10%
Within, amination effects equalizer is described；It is more than 10 times of conventional plane substrate that the method modifies the fluorescence intensity of each sampling point,
Visible the method amination efficiency is high.Illustrate that, relative to amination on smooth slide, the silicon amide ball of the present invention is modified
Slide can be obviously improved the amino density of unit area of base.Simultaneously during fluorescent labeling, experienced by biochip in situ
Coupling required for synthesis and high intensity cleaning process, it is seen that the slide that amination nano silicon is modified is a kind of high-quality
Chip base.
Claims (8)
1. a biochip substrate, including substrate, by the self-assembled modified substrate of fluorine containing silane, described fluorine containing silane and amination
Nano-particle interaction forms the nano-particle modified layer on substrate.
Biochip substrate the most according to claim 1, the material of its substrate is glass, pottery, silicon chip, metal or polymerization
Thing.
Biochip substrate the most according to claim 1, the carbon number of described fluorine containing silane is not less than 3.
Biochip substrate the most according to claim 1, the carbon number of described fluorine containing silane is 6-18.
Biochip substrate the most according to claim 1, described fluorine containing silane is perfluoro capryl triethoxysilane, complete
Fluorine octyl group trimethoxy silane, perfluoro decyl triethoxysilane, perfluoro decyl trimethoxy silane perfluoro decyl methyl dichloro
Silane, perfluoro capryl first dichlorosilane, trifluoro propyl trimethoxy silane, trifluoro propyl triethoxysilane, perfluor heptyl third
Base trimethoxy silane, perfluor heptyl propyl-triethoxysilicane, perfluoro butyl propyl trimethoxy silicane or perfluoro butyl third
Ethyl triethoxy silicane alkane.
6. according to any one biochip substrate of claim 1-5, described nano-particle be chemically inert pottery, metal,
Metal-oxide, nonmetal oxide or polymer nano granules.
Biochip substrate the most according to claim 6, the chemical inertness of nano-particle is prepared for being resistant to biochip
During the erosion of acid, alkali and/or organic solvent that uses.
Biochip substrate the most according to claim 6, a diameter of the 10 of described nano-particle～500nm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110137387A (en) * | 2019-05-16 | 2019-08-16 | 京东方科技集团股份有限公司 | The patterned method of nanoparticle layers, quantum dot light emitting device and display device |
| CN114149221A (en) * | 2021-10-28 | 2022-03-08 | 乌鲁木齐汇聚路面工程有限公司 | Water-permeable high-strength asphalt concrete and preparation method thereof |
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| US20080293584A1 (en) * | 2005-12-27 | 2008-11-27 | The Furukawa Electric Co., Ltd. | Fluorescent silica nano-particle, fluorescent nano-material, and biochip and assay using the same |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110137387A (en) * | 2019-05-16 | 2019-08-16 | 京东方科技集团股份有限公司 | The patterned method of nanoparticle layers, quantum dot light emitting device and display device |
| WO2020228476A1 (en) * | 2019-05-16 | 2020-11-19 | 京东方科技集团股份有限公司 | Method for patterning nanoparticle layer, quantum dot light emitting device, and display apparatus |
| CN110137387B (en) * | 2019-05-16 | 2021-11-16 | 京东方科技集团股份有限公司 | Method for patterning nanoparticle layer, quantum dot light-emitting device and display device |
| US11795386B2 (en) | 2019-05-16 | 2023-10-24 | Boe Technology Group Co., Ltd. | Method for patterning nanoparticle layer, quantum dot light emitting device and display device all fees |
| CN114149221A (en) * | 2021-10-28 | 2022-03-08 | 乌鲁木齐汇聚路面工程有限公司 | Water-permeable high-strength asphalt concrete and preparation method thereof |
| CN114149221B (en) * | 2021-10-28 | 2023-05-26 | 乌鲁木齐汇聚路面工程有限公司 | Permeable high-strength asphalt concrete and preparation method thereof |
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