CN109161478B - Gold/luminol nanocomposite-based biochip and preparation method and application thereof - Google Patents
Gold/luminol nanocomposite-based biochip and preparation method and application thereof Download PDFInfo
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- CN109161478B CN109161478B CN201810668414.XA CN201810668414A CN109161478B CN 109161478 B CN109161478 B CN 109161478B CN 201810668414 A CN201810668414 A CN 201810668414A CN 109161478 B CN109161478 B CN 109161478B
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
Abstract
The invention discloses a gold/luminol nano-composite-based biochip, a preparation method and application thereof. The biochip provided by the invention utilizes the principle that cholesterol generates hydrogen peroxide under the decomposition action of cholesterol oxidase, and the hydrogen peroxide can rapidly excite luminol to generate a chemiluminescent signal under the catalytic action of soybean peroxidase, so that high-flux, rapid and simple detection of cholesterol is realized.
Description
Technical Field
The invention relates to a biochip, in particular to a biochip based on a gold/luminol nano-composite and a preparation method and application thereof.
Background
Cholesterol is an indispensable component in mammalian cells and plays an extremely important role in maintaining normal cell functions. Meanwhile, cholesterol is also an indispensable raw material for maintaining normal metabolism of a human body, is an important substance for resisting aging, preventing senility and prolonging life, and is also an important raw material for synthesizing various hormones in vivo, such as steroid hormone, vitamin D and bile acid. However, cholesterol is also a double sword, and if the cholesterol in the blood of a human body is too high, atherosclerosis is formed, and the atherosclerosis is a main risk factor of coronary heart disease, myocardial infarction and cerebral apoplexy. It can be said that cholesterol has important significance for the normal physiological metabolism and life processes of the human body. Therefore, the development of a biochip for high-throughput detection of cholesterol, which has the advantages of simple preparation method, simple operation, environmental protection, sensitivity and high efficiency, is very important for further research of cholesterol.
Cholesterol can be converted into cholestenone and hydrogen peroxide under the decomposition action of cholesterol oxidase, and the hydrogen peroxide can oxidize luminol to an excited state to generate a chemiluminescent signal under the catalytic action of soybean peroxidase and gold nanoparticles, so that the quantitative detection of the cholesterol is realized. Meanwhile, polydimethylsiloxane is widely applied to preparation of chips due to good plasticity and nontoxicity, and cellulose acetate films are suitable for substrate modification due to good adsorbability and biocompatibility. Based on the principle and the material characteristics, the preparation of a simple biochip can be realized, the cholesterol can be detected with high flux, simplicity and sensitivity, and the method has profound significance for further research of the cholesterol.
Disclosure of Invention
The invention aims to provide a gold/luminol nano-composite-based biochip and a preparation method thereof, and the biochip can realize high-throughput, rapid and simple cholesterol detection.
A biochip based on gold/luminol nano-composite is composed of a polydimethylsiloxane microporous template, a substrate formed by cellulose acetate film in the micropores of said template, a gold/luminol nano-composite fixed on said substrate, and soybean peroxidase and cholesterol oxidase embedded in the micropores of said template.
Further, the gold/luminol nano-composite is obtained by compounding gold nanoparticles coated with perfluorosulfonic acid polymer and luminol-polydimethyldiallyl ammonium chloride.
Further, the gold/luminol nanocomposite was prepared as follows: mixing gold nanoparticles with a perfluorinated sulfonic acid polymer, stirring for 8 hours at room temperature, and centrifuging at 14000rpm to obtain a gold-perfluorinated sulfonic acid polymer connector; stirring 0.2 mass percent of polydimethyldiallyl ammonium chloride and 4mM luminol at 60 ℃ for 6 hours, and centrifuging to obtain a connector; and mixing and stirring the two connectors according to the volume ratio of 1:1 for 12 hours, and centrifuging to obtain the gold/luminol nano-composite.
The preparation method of the biochip comprises the following steps:
step 1, mixing a polydimethylsiloxane prepolymer with a hydrogenated siloxane cross-linking agent, pouring the mixed solution into a mold, and curing and punching to prepare a polydimethylsiloxane microporous template;
step 2, adhering a polydimethylsiloxane micropore template to a glass sheet, and adding a cellulose acetate solution into micropores to form a cellulose acetate film;
and 3, fixing the gold/luminol nano-composite on a cellulose acetate film, embedding the soybean peroxidase and the cholesterol oxidase in the micropores, and sealing the micropores with bovine serum albumin to obtain the biochip.
Further, the weight ratio of the polydimethylsiloxane prepolymer to the hydrosiloxane cross-linking agent in the step 1 is 8-10: 1-3.
Furthermore, the size of the micropores of the polydimethylsiloxane micropore template is 4-6mm in diameter and 2-3mm in depth.
Further, the mass concentration of the cellulose acetate solution in the step 2 is 2-4%.
The application of the biochip in cholesterol detection. Specifically, cholesterol solution is added into the micropores of the biochip, and a chemiluminescence signal generated by detection is collected by a charge-coupled image analyzer. The exposure time is 0-4 minutes.
As shown in FIG. 1, the biochip of the present invention uses polydimethylsiloxane to prepare a microwell template, adheres to a glass plate and modifies a cellulose acetate film in the microwells; fixing the synthesized gold/luminol nano-composite on a substrate, embedding the soybean peroxidase and the cholesterol oxidase in the microporous film, and sealing with bovine serum albumin to obtain the chip. The principle of the biochip used for high-throughput detection of cholesterol is that cholesterol oxidase specifically decomposes cholesterol to generate hydrogen peroxide, luminol is oxidized to be in an excited state under the catalytic action of soybean peroxidase and gold nanoparticles, and a chemiluminescent signal is generated and collected for detection under the condition that a charge-coupled device is exposed for a certain time, so that the cholesterol is sensitively and quantitatively detected under high throughput.
Compared with the prior art, the invention has the following remarkable advantages: the gold nanoparticles are compounded with luminol serving as a luminescent agent through electrostatic adsorption by utilizing the excellent conductivity, unique optical property, good biocompatibility and superior specific surface area of the gold nanoparticles, so that enough luminescent agent is introduced; the gold/luminol compound, the soybean peroxidase and the cholesterol oxidase required by the reaction are fixed on a substrate, the reaction can be initiated to generate a chemiluminescent signal by directly adding a cholesterol reactant, and samples with multiple concentrations can be added simultaneously, so that the cholesterol can be detected simply, conveniently and quickly with high flux. Meanwhile, the preparation process of the biochip is simple and convenient, and the cost is low.
The biochip of the invention can specifically detect cholesterol in serum and cells, collects and detects generated chemiluminescence signals through the charge coupling element, has the advantages of high sensitivity, low cost, fast detection time, high selectivity, good stability, wide detection range and the like, can simultaneously detect a plurality of samples, and realizes high-flux cholesterol detection.
Drawings
FIG. 1 is a method for preparing and detecting a biochip for detecting cholesterol based on gold/luminol nanocomposite according to the present invention.
FIG. 2 is a chemiluminescent signal of the biochip according to the present invention in response to cholesterol.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
The polydimethylsiloxane used in this example was a dow corning brand kit product comprising a polydimethylsiloxane prepolymer and a hydridosiloxane crosslinker, available from suzhou midge jengheng scientific instruments ltd; perfluorosulfonic acid polymer, poly dimethyl diallyl ammonium chloride purchased from alatin reagent ltd; cellulose acetate, bovine serum albumin, chloroauric acid solutions were purchased from the national pharmaceutical group; luminol, soy peroxidase and cholesterol oxidase were purchased from Sigma-alorich, usa.
(1) Preparing a polydimethylsiloxane micropore template: uniformly mixing a polydimethylsiloxane prepolymer and a hydrogenated siloxane cross-linking agent according to a weight ratio of 9:2, forcibly stirring in the same direction to obtain a medium-viscosity mixed solution, pouring the mixed solution into a mold, vacuumizing to remove bubbles in the mixed solution, putting the mixed solution into an oven to be cured into a tough transparent elastomer, punching the transparent elastomer by using a puncher, wherein the diameter of each hole is 4-6mm, the depth of each hole is 2-3mm, and cleaning and drying the obtained template for later use.
(2) Cutting a glass sheet according to the size of a template, then ultrasonically cleaning and washing the glass sheet by using secondary water, adhering the prepared polydimethylsiloxane micropore template on the glass sheet, and adding a cellulose acetate solution with the mass concentration of 2% into micropores to form a cellulose acetate film.
(3) Fixing the gold/luminol nano-composite on a cellulose acetate film, embedding the soybean peroxidase and the cholesterol oxidase in the micropores, and sealing with bovine serum albumin to obtain the biochip.
Gold nanoparticles are prepared by a method of reducing chloroauric acid by sodium citrate. Mixing the gold nanoparticles with 3.5% of perfluorosulfonic acid polymer according to the volume ratio of 1:1, stirring for 8h at room temperature, and centrifuging for 30min at 14000rpm to obtain a gold-perfluorosulfonic acid polymer connector; mixing 0.2% of poly dimethyl diallyl ammonium chloride and 4mM of luminol according to the volume ratio of 1:1, stirring for 6 hours at 60 ℃, and centrifuging at 1000rpm to obtain a connector; mixing and stirring the two connectors according to the volume ratio of 1:1 for 12h, and centrifuging at 1000rpm to obtain the gold/luminol nano-composite.
Adding 10 mu L of gold/luminol nano-composite into the micropores of the template, and completely fixing the gold/luminol nano-composite on the cellulose acetate film substrate; this step was repeated 3 times to ensure that enough gold/luminol nanocomposite was introduced into the microwells.
Embedding 30 mu L of soybean peroxidase with the concentration of 35U/mL and 30 mu L of cholesterol oxidase with the concentration of 20U/mL into a cellulose acetate film, standing for one night at 4 ℃ in the dark, washing off redundant enzyme by using a buffer solution with the pH of 7.4, and adding 50 mu L of bovine serum albumin solution to seal the chip to obtain the gold/luminol nano-composite-based biochip for detecting cholesterol at high flux.
Example 2
The polydimethylsiloxane used in this example was a dow corning brand kit product comprising a polydimethylsiloxane prepolymer and a hydridosiloxane crosslinker, available from suzhou midge jengheng scientific instruments ltd; perfluorosulfonic acid polymer, poly dimethyl diallyl ammonium chloride purchased from alatin reagent ltd; cellulose acetate, bovine serum albumin, chloroauric acid solutions were purchased from the national pharmaceutical group; luminol, soy peroxidase and cholesterol oxidase were purchased from Sigma-alorich, usa.
(1) Preparing a polydimethylsiloxane micropore template: uniformly mixing the polydimethylsiloxane prepolymer and the hydrogenated siloxane cross-linking agent according to the weight ratio of 8:3, forcibly stirring in the same direction to obtain a mixed solution with medium viscosity, pouring the mixed solution into a mold, vacuumizing to remove bubbles in the mixed solution, putting the mixed solution into an oven to be cured into a tough transparent elastomer, and punching by using a puncher to obtain a template which is cleaned and dried for later use.
(2) Cutting a glass sheet according to the size of a template, then ultrasonically cleaning and washing the glass sheet by using secondary water, adhering the prepared polydimethylsiloxane micropore template on the glass sheet, and adding cellulose acetate with the mass concentration of 3% into micropores to form a cellulose acetate film.
(3) Fixing the gold/luminol nano-composite on a cellulose acetate film, embedding the soybean peroxidase and the cholesterol oxidase in the micropores, and sealing with bovine serum albumin to obtain the biochip.
Gold nanoparticles are prepared by a method of reducing chloroauric acid by sodium citrate. Mixing the gold nanoparticles with 3.5% of perfluorosulfonic acid polymer according to the volume ratio of 1:1, stirring for 8h at room temperature, and centrifuging for 30min at 14000rpm to obtain a gold-perfluorosulfonic acid polymer connector; mixing 0.2% of poly dimethyl diallyl ammonium chloride and 4mM of luminol according to the volume ratio of 1:1, stirring for 6 hours at 60 ℃, and centrifuging at 1000rpm to obtain a connector; mixing and stirring the two connectors according to the volume ratio of 1:1 for 12h, and centrifuging at 1000rpm to obtain the gold/luminol nano-composite.
Adding 10 mu L of gold/luminol nano-composite into the micropores of the biochip template so as to completely fix the gold/luminol nano-composite on the substrate; this step was repeated 3 times to ensure that enough gold/luminol nanocomposite was introduced into the microwells.
Embedding 30 mu L of soybean peroxidase with the concentration of 45U/mL and 30 mu L of cholesterol oxidase with the concentration of 25U/mL into a cellulose acetate film, standing for one night at 4 ℃ in the dark, washing off redundant enzyme by using a buffer solution with the pH of 7.4, and adding 50 mu L of bovine serum albumin solution to seal the chip to obtain the gold/luminol nano-composite-based biochip for detecting cholesterol at high flux.
Example 3
The polydimethylsiloxane used in this example was a dow corning brand kit product comprising a polydimethylsiloxane prepolymer and a hydridosiloxane crosslinker, available from suzhou midge jengheng scientific instruments ltd; perfluorosulfonic acid polymer, poly dimethyl diallyl ammonium chloride purchased from alatin reagent ltd; cellulose acetate, bovine serum albumin, chloroauric acid solutions were purchased from the national pharmaceutical group; luminol, soy peroxidase and cholesterol oxidase were purchased from Sigma-alorich, usa.
(1) The preparation of the polydimethylsiloxane microporous template comprises the steps of uniformly mixing a polydimethylsiloxane prepolymer and a hydrogenated siloxane cross-linking agent according to the weight ratio of 10:1, forcibly stirring in the same direction to obtain a medium-viscosity mixed solution, pouring the mixed solution into a mold, vacuumizing to remove air bubbles in the mixed solution, putting the mixed solution into an oven to be cured into a tough transparent elastomer, and punching by using a puncher to obtain the template which is cleaned and dried for later use.
(2) Cutting a glass sheet according to the size of a template, then ultrasonically cleaning and washing the glass sheet by using secondary water, adhering the prepared polydimethylsiloxane micropore template on the glass sheet, and adding cellulose acetate with the mass concentration of 3% into micropores to form a cellulose acetate film.
(3) Fixing the gold/luminol nano-composite on a cellulose acetate film, embedding the soybean peroxidase and the cholesterol oxidase in the micropores, and sealing with bovine serum albumin to obtain the biochip.
Gold nanoparticles are prepared by a method of reducing chloroauric acid by sodium citrate. Mixing the gold nanoparticles with 3.5% of perfluorosulfonic acid polymer according to the volume ratio of 1:1, stirring for 8h at room temperature, and centrifuging for 30min at 14000rpm to obtain a gold-perfluorosulfonic acid polymer connector; mixing 0.2% of poly dimethyl diallyl ammonium chloride and 4mM of luminol according to the volume ratio of 1:1, stirring for 6 hours at 60 ℃, and centrifuging at 1000rpm to obtain a connector; mixing and stirring the two connectors according to the volume ratio of 1:1 for 12h, and centrifuging at 1000rpm to obtain the gold/luminol nano-composite.
Adding 10 mu L of gold/luminol nano-composite into the micropores of the biochip template so as to completely fix the gold/luminol nano-composite on the substrate; this step was repeated 3 times to ensure that enough gold/luminol nanocomposite was introduced into the microwells.
Embedding 30 mu L of soybean peroxidase with the concentration of 45U/mL and 30 mu L of cholesterol oxidase with the concentration of 25U/mL into a cellulose acetate film, standing for one night at 4 ℃ in the dark, washing off redundant enzyme by using a buffer solution with the pH of 7.4, and adding 50 mu L of bovine serum albumin solution to seal the chip to obtain the gold/luminol nano-composite-based biochip for detecting cholesterol at high flux.
As can be seen from FIG. 2, 1mM cholesterol solution with the same concentration was added to the microwells of different substrates modified by the chips, and the chips were placed in a CCD image analyzer, and the exposure time was set to 1 minute, so that a clear chemiluminescent signal was observed and collected by a CCD.
Claims (1)
1. A biochip based on gold/luminol nano-composite is characterized by comprising a polydimethylsiloxane micropore template, wherein cellulose acetate thin films form substrates in micropores of the template, the gold/luminol nano-composite is fixed on the substrates, and soybean peroxidase and cholesterol oxidase are also embedded in the micropores of the template; the gold/luminol nano-composite is obtained by compounding gold nanoparticles coated with perfluorosulfonic acid polymer and luminol-polydimethyldiallyl ammonium chloride; the gold/luminol nanocomposite was prepared as follows: mixing gold nanoparticles with a perfluorinated sulfonic acid polymer, stirring for 8 hours at room temperature, and centrifuging at 14000rpm to obtain a gold-perfluorinated sulfonic acid polymer connector; stirring 0.2 mass percent of polydimethyldiallyl ammonium chloride and 4mM luminol at 60 ℃ for 6 hours, and centrifuging to obtain a connector; mixing and stirring the two connectors according to the volume ratio of 1:1 for 12 hours, and centrifuging to obtain a gold/luminol nano-composite;
the preparation method of the biochip comprises the following steps:
step 1, mixing a polydimethylsiloxane prepolymer with a hydrogenated siloxane cross-linking agent, pouring the mixed solution into a mold, and curing and punching to prepare a polydimethylsiloxane microporous template;
step 2, adhering a polydimethylsiloxane micropore template to a glass sheet, and adding a cellulose acetate solution into micropores to form a cellulose acetate film;
step 3, fixing the gold/luminol nano-composite on a cellulose acetate film, embedding the soybean peroxidase and the cholesterol oxidase in micropores, and sealing the micropores with bovine serum albumin to obtain a biochip;
the weight ratio of the polydimethylsiloxane prepolymer to the hydrogenated siloxane cross-linking agent in the step 1 is 8-10:1-3, the micropore size of the polydimethylsiloxane micropore template is 4-6mm in diameter and 2-3mm in depth, and the mass concentration of the cellulose acetate solution in the step 2 is 2-4%.
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| CN101073008A (en) * | 2004-12-09 | 2007-11-14 | 拜奥迪吉特实验室技术会社 | Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same |
| CN102798627A (en) * | 2012-08-01 | 2012-11-28 | 福建医科大学 | Method for determining cholesterol by using flow injection chemiluminescence with nano-copper oxide as catalyst |
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| CN101073008A (en) * | 2004-12-09 | 2007-11-14 | 拜奥迪吉特实验室技术会社 | Lab-on-a-chip for an on-the-spot analysis and signal detection methods for the same |
| CN102798627A (en) * | 2012-08-01 | 2012-11-28 | 福建医科大学 | Method for determining cholesterol by using flow injection chemiluminescence with nano-copper oxide as catalyst |
| CN104316704A (en) * | 2014-11-06 | 2015-01-28 | 东南大学 | Biological chip adopting soybean peroxidase (SBP) for marking, and preparation method thereof |
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