CN103981268A - Preparation method of biochip based on compound nano material and application of biochip - Google Patents
Preparation method of biochip based on compound nano material and application of biochip Download PDFInfo
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Abstract
The invention relates to a preparation method of a biochip based on a compound nano material and application of the biochip and relates to preparation and application of biochips. The preparation method of the biochip based on the compound nano material comprises the following steps: carrying out oil phase-water phase transformation on a compound type nano material; treating a gold membrane with an activating solution to remove organic matters on the surface of the gold membrane by utilizing strong oxidizing property of the activating solution; introducing the compound type nano material modified with a sulfydryl into the surface of the gold membrane for assembling; sequentially dipping the obtained gold membrane in a phosphate buffer solution, ultrapure water and mercaptoethanol for fixing the gold membrane; intruding DNA or protein with amino as a biological probe into the obtained gold membrane, so that the biochip is obtained. The preparation method provided by the invention has the advantages that multiple proteins and DNAs can be easily assembled on a substrate to serve as the biochip, and highly sensitive biological detection can be carried out.
Description
Technical field
The invention belongs to nano material manufacturing technology field, relate to preparation and the application of biochip.
Background technology
Biochip (biochip or bioarray) is according to special interactional principle between biomolecules, by biochemical analysis process integration in chip surface, thereby realize the high-throughput rapid detection to DNA, RNA, polypeptide, protein and other biological composition.The biochip concept of narrow sense refers to by different methods biomolecules (oligonucleotide, cDNA, genomic DNA, polypeptide, antibody, antigen etc.) is bonded to the biomolecules dot matrix forming on the solid phase mediators such as sheet glass, sheet glass (pearl), plastic sheet (pearl), gel, nylon membrane.Therefore biochip technology claims again micro-display (microarray) technology, and the solid-phase matrix that contains large number of biological information is called microarray, claims again biochip.
Nano particle refers to the particle of size between 1～100 nm, and it is by the few atom of number or molecular former subgroup or molecular group, and the field in the middle of macroscopic material and microcosmic atom and molecule, is that a kind of typical Jie sees system.Its special structure causes it to have the character of many uniquenesses, as surface effects, small-size effect, quantum size effect, macro quanta tunnel effect etc.
Present biochip is all directly integral body to be carried out to the assembling of protein or DNA take after situation that silicon or glass is substrate plates layer of gold nano thin-film mostly, although this way can be saved the Production Time of biochip, but owing in making processes, gold nanometer film not being carried out to the modification of any functional group, thereby can cause the adhesive rate of the biochip protein produced or DNA lower.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method based on composite nano materials biochip.
Another object of the present invention is to disclose this biochip in the application of medical science context of detection.
it is one, provided by the invention a kind of based on composite nano granular biological chip,first prepare the needed composite nano particle of biochip, first the method for utilizing oil phase synthesis method to prepare Au-Cu
2s is as kernel, and shell is ZnS.Then apply mercapto-polyglycol resulting composite nano material is carried out to the conversion between oil-water, and make material surface carry sulfydryl and be then passed in the substrate of glass that is attached with golden film and assemble, finally apply mercaptoethanol and be fixed with the surface active of quantum dot in order to the follow-up assembling of carrying out protein and DNA.
We select a kind of novel composite nano materials A u-Cu in the present invention
2s/ZnS makes our biochip probe as carrier.Its reason is with respect to single Cu
2s quantum dot nanometer Au is easier to and various functional groups, and Cu
2the stability of S is better than again nanometer Au, and when having guaranteed stability, we have more strengthened the bio-compatibility of composite nano material monolithic like this, and then we are again at Au-Cu
2s finishing ZnS reduces the bio-toxicity of composite nano material with this.The conversion of again it being carried out to oil-water makes it carry the sulfydryl that is easy to bio-compatible afterwards, thereby biochip can easierly be assembled with protein or DNA.
A kind of making method based on composite nano granular biological chip provided by the invention is as follows:
(1) first prepare and make the needed composite nano material of biochip, the method utilizes oil phase synthesis method to prepare Au-Cu
2s is as kernel, interior nuclear diameter is 10 ± 0.6nm, shell is ZnS, shell thickness is 2 ± 0.6nm, then the mercapto-polyglycol of 30 ~ 50mg is dissolved in to central the stirring 3 ~ 5 minutes of chloroform of 3 ~ 5ml, and be the ratio mix and blend of 1:1 3 ~ 8 minutes in molar ratio by the mixing solutions of the composite nano particle making and chloroform and mercapto-polyglycol, finally solution to be put into Rotary Evaporators and gone out chloroform and obtain the composite nano material of water, nano-material surface has now been modified sulfydryl;
(2) by the ratio that the vitriol oil and hydrogen peroxide are 3:1 according to volume ratio, make activation solution, and by activation solution with the speed of 50-150ul/s at the uniform velocity by the thick gold nanometer film surface of 40-80nm 8 ~ 15 minutes, utilize the strong oxidizing property of activation solution remove the organism on golden film surface and other impurity and expand its surface tension and reduce electrostatic force, then with ultrapure water, rinse, pass into nitrogen and carry out drying treatment;
(3) the composite nano material that is modified with sulfydryl is at the uniform velocity passed through to golden film with the speed of 50-150ul/s equally, carry out at normal temperatures for time 2 ~ 4 hours circulate, owing to having expanded the surface tension of golden film in step (2), making composite nano material with sulfydryl more easily be attached to golden film surface assembles, composite nano material is fixed on to golden film surface, finally passes into PH and be 7 phosphate buffered saline buffer (PBS) and clean 10 ~ 15 minutes and then clean with ultrapure water;
(4) to the golden film with composite nano material, the speed with 50-150ul/s circulates the mercaptoethanol solution that is finally 1mmol/L by concentration, make composite nano material be fixed on golden film surface, then with ultrapure water, clean to wash off the residual mercaptoethanol solution of biochip surface;
(5) protein with amino or DNA are at the uniform velocity passed through to golden film surface 2 ~ 4 hours at normal temperatures, carry out the last assembling of biochip, then with ultrapure water, clean, make biochip.
Wherein, the optimum flow rate that flows through golden film surface in step 2,3 and 4 is 100ul/s.
The optimum thickness of gold nanometer film is 50nm.
three, the application based on composite nano material biochip of the present invention:of the present inventionly based on composite nano granular biological chip, can be applicable to that gene is differentiated and diagnosing tumor
(1) usage of differentiating for gene based on composite nano granular biological chip of the present invention is as follows: due to the strong compatibility of composite nano particle, the method that applied chemistry is modified, first the corresponding RNA of gene that needs are differentiated modifies with amino, then cdna solution being passed into composite nano material surface assembles, the gene recombination liquid of finally needs being differentiated passes into biochip, and the PBS solution with different concns after 2 hours rinses.By colorimetry or electrochemical process, biochip is detected, show that testing sample is whether with the gene of needs.
2) of the present invention as follows for diagnosing tumor usage based on composite nano granular biological chip: the method that applied chemistry is modified, first the distinctive DNA that tumour cell is entrained modifies with amino, then cdna solution passes into composite nano material surface and assembles, the tumour cell solution of finally needs being diagnosed passes into biochip and hybridizes the PBS solution with different concns after 2 hours and rinse, by colorimetry and electrochemical process, biochip is detected again, show whether testing sample suffers from this kind of tumour illness.
Biochip technology of the present invention is basis, usings composite nano material as bioprobe, is applied on biochip technology.The composite nano materials A u-Cu that the present invention is selected
2s/ZnS is a kind of hypotoxicity nano material.The prepared biochip size of the present invention is less, is easy to carry.
The present invention applies afterwards mercaptoethanol and carries out between the two fixing owing to being fixed on golden film surface at composite nano material, also utilizes mercaptoethanol that quantum dot surface is necessarily activated simultaneously, is easier to carry out combination with protein or DNA with amino.
Structure design of the present invention is ingenious, simply applicable.The material that the present invention can be applicable to biology, chemistry and medical field detects.
Accompanying drawing explanation
Fig. 1 is the composite nano material after oil-water transforms;
Fig. 2 is the golden film having adhered to after composite nano material;
Fig. 3 is the made biochip that is attached with composite nano material.
embodiment:
embodiment 1
(1) first prepare and make the needed composite nano material of biochip, the method utilizes oil phase synthesis method to prepare Au-Cu
2s is as kernel, interior nuclear diameter is 10nm, shell is ZnS, shell thickness is 2nm, then in the middle of the mercapto-polyglycol of 40mg being dissolved in to the chloroform of 4ml, stir 4 minutes, and be the ratio mix and blend of 1:1 5 minutes in molar ratio by the mixing solutions of the composite nano particle making and chloroform and mercapto-polyglycol, finally solution to be put into Rotary Evaporators and gone out chloroform and obtain the composite nano material of water, nano-material surface has now been modified sulfydryl.
(2) by the ratio that the vitriol oil and hydrogen peroxide are 3:1 according to volume ratio, make activation solution, and by activation solution with the speed of 100ul/s at the uniform velocity by the thick gold nanometer film surface of 50nm 10 minutes, utilize the strong oxidizing property of activation solution remove the organism on golden film surface and other impurity and expand its surface tension and reduce electrostatic force, then with ultrapure water, rinse, pass into nitrogen and carry out drying treatment.
(3) the composite nano material that is modified with sulfydryl is at the uniform velocity passed through to golden film with the speed of 100ul/s equally, carry out at normal temperatures for time 3 hours circulate, owing to having expanded the surface tension of golden film in step (2), make composite nano material with sulfydryl more easily be attached to golden film surface and assemble, composite nano material is fixed on to golden film surface.Finally passing into PH and be 7 phosphate buffered saline buffer (PBS) cleans 12 minutes and then cleans with ultrapure water.
(4) to the golden film with composite nano material, the speed with 100ul/s circulates the mercaptoethanol solution that is finally 1mmol/L by concentration, make composite nano material be fixed on golden film surface, then with ultrapure water, clean to wash off the residual mercaptoethanol solution of biochip surface.
(5) protein with amino or DNA are at the uniform velocity passed through to golden film surface 3 hours at normal temperatures, carry out the last assembling of biochip, then with ultrapure water, clean, make biochip.
embodiment 2
(1) first prepare and make the needed composite nano material of biochip, the method utilizes oil phase synthesis method to prepare Au-Cu
2s is as kernel, interior nuclear diameter is 10.6nm, shell is ZnS, shell thickness is 1.6nm, then in the middle of the mercapto-polyglycol of 30mg being dissolved in to the chloroform of 5ml, stir 5 minutes, and be the ratio mix and blend of 1:1 3 minutes in molar ratio by the mixing solutions of the composite nano particle making and chloroform and mercapto-polyglycol, finally solution to be put into Rotary Evaporators and gone out chloroform and obtain the composite nano material of water, nano-material surface has now been modified sulfydryl;
(2) by the ratio that the vitriol oil and hydrogen peroxide are 3:1 according to volume ratio, make activation solution, and by activation solution with the speed of 50ul/s at the uniform velocity by the thick gold nanometer film surface of 40nm 8 minutes, utilize the strong oxidizing property of activation solution remove the organism on golden film surface and other impurity and expand its surface tension and reduce electrostatic force, then with ultrapure water, rinse, pass into nitrogen and carry out drying treatment;
(3) the composite nano material that is modified with sulfydryl is at the uniform velocity passed through to golden film with the speed of 50ul/s equally, carry out at normal temperatures for time 2 hours circulate, owing to having expanded the surface tension of golden film in step (2), make composite nano material with sulfydryl more easily be attached to golden film surface and assemble, composite nano material is fixed on to golden film surface.Finally passing into PH and be 7 phosphate buffered saline buffer (PBS) cleans 15 minutes and then cleans with ultrapure water;
(4) to the golden film with composite nano material, the speed with 50ul/s circulates the mercaptoethanol solution that is finally 1mmol/L by concentration, make composite nano material be fixed on golden film surface, then with ultrapure water, clean to wash off the residual mercaptoethanol solution of biochip surface.
(5) protein with amino or DNA are at the uniform velocity passed through to golden film surface 2 hours at normal temperatures, carry out the last assembling of biochip, then with ultrapure water, clean, make biochip.
embodiment 3
(1) first prepare and make the needed composite nano material of biochip, the method utilizes oil phase synthesis method to prepare Au-Cu
2s is as kernel, interior nuclear diameter is 9.4nm, shell is ZnS, shell thickness is 1.6nm, then in the middle of the mercapto-polyglycol of 50mg being dissolved in to the chloroform of 3ml, stir 3 minutes, and be the ratio mix and blend of 1:1 8 minutes in molar ratio by the mixing solutions of the composite nano particle making and chloroform and mercapto-polyglycol, finally solution to be put into Rotary Evaporators and gone out chloroform and obtain the composite nano material of water, nano-material surface has now been modified sulfydryl;
(2) by the ratio that the vitriol oil and hydrogen peroxide are 3:1 according to volume ratio, make activation solution, and by activation solution with the speed of 150ul/s at the uniform velocity by the thick gold nanometer film surface of 50nm 15 minutes, utilize the strong oxidizing property of activation solution remove the organism on golden film surface and other impurity and expand its surface tension and reduce electrostatic force, then with ultrapure water, rinse, pass into nitrogen and carry out drying treatment;
(3) the composite nano material that is modified with sulfydryl is at the uniform velocity passed through to golden film with the speed of 150ul/s equally, carry out at normal temperatures for time 4 hours circulate, owing to having expanded the surface tension of golden film in step (2), make composite nano material with sulfydryl more easily be attached to golden film surface and assemble, composite nano material is fixed on to golden film surface.Finally passing into PH and be 7 phosphate buffered saline buffer (PBS) cleans 8 minutes and then cleans with ultrapure water;
(4) to the golden film with composite nano material, the speed with 150ul/s circulates the mercaptoethanol solution that is finally 1mmol/L by concentration, make composite nano material be fixed on golden film surface, then with ultrapure water, clean to wash off the residual mercaptoethanol solution of biochip surface.
(5) protein with amino or DNA are at the uniform velocity passed through to golden film surface 4 hours at normal temperatures, carry out the last assembling of biochip, then with ultrapure water, clean, make biochip.
the usage of differentiating for gene based on composite nano granular biological chip:
Strong compatibility due to composite nano particle, the method that applied chemistry is modified, first the corresponding RNA of gene that needs are differentiated modifies with amino, then cdna solution being passed into composite nano material surface assembles, the gene recombination liquid of finally needs being differentiated passes into biochip, and the PBS solution that is 7 with pH value after 3 hours rinses.By colorimetry or electrochemical process, biochip is detected, show that testing sample is whether with the gene of needs.
based on composite nano granular biological chip, be used for diagnosing tumor usage:
The method that applied chemistry is modified, first the distinctive DNA that tumour cell is entrained modifies with amino, then cdna solution passes into composite nano material surface and assembles, the tumour cell solution of finally needs being diagnosed passes into the biochip PBS solution that to hybridize after 4 hours be 7 with pH value and rinses, by colorimetry and electrochemical process, biochip is detected again, show whether testing sample suffers from this kind of tumour illness.
Claims (4)
1. in a preparation method for composite nano materials biochip, it is characterized in that being completed by following steps:
(1) utilize oil phase synthesis method to prepare Au-Cu
2s is as kernel, interior nuclear diameter is 10 ± 0.6nm, shell is ZnS, shell thickness is 2 ± 0.6nm, then the mercapto-polyglycol of 30 ~ 50mg is dissolved in to central the stirring 3 ~ 5 minutes of chloroform of 3 ~ 5ml, and be the ratio mix and blend of 1:1 3 ~ 8 minutes in molar ratio by the mixing solutions of the composite nano particle making and chloroform and mercapto-polyglycol, solution is put into Rotary Evaporators and go out chloroform and obtain the composite nano material of water;
(2) by the ratio that the vitriol oil and hydrogen peroxide are 3:1 according to volume ratio, make activation solution, and by activation solution with the speed of 50-150ul/s at the uniform velocity by the thick gold nanometer film surface of 40-80nm 8 ~ 15 minutes, then with ultrapure water, rinse, pass into nitrogen and carry out drying treatment;
(3) the composite nano material that is modified with sulfydryl is at the uniform velocity passed through to gold nanometer film with the speed of 50-150ul/s equally, carry out at normal temperatures for time 2 ~ 4 hours circulate, composite nano material is fixed on to golden film surface, then pass into PH and be 7 phosphate buffered saline buffer and clean 10 ~ 15 minutes, then clean with ultrapure water;
(4) to the gold nanometer film with composite nano material, the speed with 50-150ul/s circulates the mercaptoethanol solution that is 1mmol/L by concentration, make composite nano material be fixed on golden film surface, then with ultrapure water, clean to wash off the residual mercaptoethanol solution of biochip surface;
(5) protein with amino or DNA are at the uniform velocity passed through to golden film surface 2 ~ 4 hours at normal temperatures, carry out the last assembling of biochip, then with ultrapure water, clean, make biochip.
2. preparation method according to claim 1, is characterized in that: the optimum flow rate that flows through gold nanometer film surface is 100ul/s.
3. preparation method according to claim 1, is characterized in that: the optimum thickness of gold nanometer film is 50nm.
4. the application in gene resolution and diagnosing tumor based on composite nano materials biochip obtaining according to preparation method claimed in claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105714288A (en) * | 2014-12-01 | 2016-06-29 | 国家纳米科学中心 | Preparation method of quantum dot self-assembled film |
| CN109777400A (en) * | 2019-01-28 | 2019-05-21 | 山东师范大学 | A kind of nontoxic Cu2S/ZnS core shell quantum dots and preparation method thereof |
| CN114371159A (en) * | 2021-05-19 | 2022-04-19 | 南京医科大学第二附属医院 | RNA biochip and preparation method and application thereof |
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| CN101046472A (en) * | 2006-03-31 | 2007-10-03 | 成都夸常医学工业有限公司 | Biochip and its prepn process and the rit therewith |
| CN103642491A (en) * | 2013-11-22 | 2014-03-19 | 长春理工大学 | Strong signal and low toxicity composite nanometer material and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101046472A (en) * | 2006-03-31 | 2007-10-03 | 成都夸常医学工业有限公司 | Biochip and its prepn process and the rit therewith |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105714288A (en) * | 2014-12-01 | 2016-06-29 | 国家纳米科学中心 | Preparation method of quantum dot self-assembled film |
| CN105714288B (en) * | 2014-12-01 | 2018-11-06 | 国家纳米科学中心 | A method of preparing quantum dot self-assembled film |
| CN109777400A (en) * | 2019-01-28 | 2019-05-21 | 山东师范大学 | A kind of nontoxic Cu2S/ZnS core shell quantum dots and preparation method thereof |
| CN109777400B (en) * | 2019-01-28 | 2022-02-08 | 山东师范大学 | Non-toxic Cu2S/ZnS core/shell quantum dot and preparation method thereof |
| CN114371159A (en) * | 2021-05-19 | 2022-04-19 | 南京医科大学第二附属医院 | RNA biochip and preparation method and application thereof |
| CN114371159B (en) * | 2021-05-19 | 2023-11-07 | 南京医科大学第二附属医院 | RNA biochip, preparation method and application thereof |
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