CN105241932B - The preparation method of a kind of fumonisin sensor based on ceria hollow ball structure and application - Google Patents
The preparation method of a kind of fumonisin sensor based on ceria hollow ball structure and application Download PDFInfo
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Abstract
The present invention relates to the preparation method of a kind of fumonisin sensor based on ceria hollow ball structure and application, belong to new function material, bio-sensing detection technique field。Based on the absorption property that ceria hollow ball is good, significantly improve the sensitivity of biosensor, the detection of fumonisin in grain is had great importance。
Description
Technical field
The preparation method of the fumonisin sensor that a kind of ceria hollow ball of the present invention builds and application。Specifically adopt and there is the ceria hollow ball of good adsorption properties, prepare and a kind of detect the sensor of fumonisin in grain, belong to new function material and bio-sensing detection technique field。
Background technology
The fungus producing fumonisin in nature is mainly fusarium moniliforme, next to that many births Fusarium spp.。Both funguses are widely present in various grain and goods thereof, and the fumonisins that therefore they produce easily pollutes grain agricultural products, especially Semen Maydis。Research finds, even if in dry warm environment, fusarium moniliforme be Semen Maydis occurs one of most frequent strain it is reported that, people, animal are not only a kind of short cancer thing by fumonisins, and are entirely a kind of carcinogen。Animal experiment and epidemiologic data it has been shown that fumonisins major determinant hepatic and renal function, can cause horse brain substantia alba malacia and Pulmonis Sus domestica edema etc., and the esophageal carcinoma occurred frequently with China and some areas, South Africa is relevant, has now caused worldwide extensive attention。
Current biosensor is widely used for the detection of various mycotoxin because biosensor have highly sensitive, selectivity good, simple in construction, easy and simple to handle, be prone to miniaturization, can the series of advantages such as detection analysis continuous, rapid automatized。Wherein, owing to unmarked type sensor is used directly for the identification process of detection antigen-antibody and avoids the interference that label brings, obtains and pay close attention to more widely。
Ceria hollow ball-methylene blue-chitosan-ionic liquid water is modified glassy carbon electrode surface by the present invention, builds unmarked type sensor。First, ceria hollow ball has good absorption property, it is possible to a large amount of methylene blue of load effectively, it is provided that electrochemical signals；Second, chitosan can effectively be coated with ceria hollow ball surface, to prevent the leakage of methylene blue；3rd, ionic liquid can improve electronics electron transport rate in chitosan, accelerates the electron transfer of methylene blue and electrode surface。The method creates good electrochemical signals in detection process, can be used for the analysis of fumonisin。The method has that cost is low, highly sensitive, specificity is good, detect the advantages such as quick, and preparation process is relatively simple, at present effectively detection fumonisin provide new way。
Summary of the invention
An object of the present invention is based on ceria hollow ball and builds unmarked type biosensor。
The two of the purpose of the present invention are in highly sensitive, the specific detection of fumonisin by this unmarked type biosensor application。
Technical scheme is as follows
1.A kind of preparation method of the fumonisin sensor built based on ceria hollow ball
(1) being polished with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, ultrasonic cleaning in ultra-pure water and ethanol respectively, nitrogen dries up；
(2) drip, at electrode surface, ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution that 6 μ L concentration are 1 ~ 2mg/mL, dry；
(3) continue the fumonisin antibody-solutions that 6 μ L concentration are 5 ~ 20 μ g/mL is added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1h, clean up；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5 ~ 20mg/mL, in 4 DEG C of refrigerators, hatch 1h, clean up；
(5) the fumonisin antigen of a series of variable concentrations that 6 μ L concentration are 0.0001 ~ 10ng/mL is used for the specific recognition with antibody, incubated at room temperature 1h, cleans up, store for future use in 4 DEG C of refrigerators。
The preparation of ceria hollow ball-methylene blue-chitosan-ionic liquid
0.05 ~ 0.2g seven cerium hydrate and 0.89 ~ 0.356g polyvinylpyrrolidone are dissolved in 19mL water, it is subsequently added the Methanamide of 0.5 ~ 2mL and the hydrogen peroxide of 0.05 ~ 0.2mL, after stirring 30min, above-mentioned mixed solution is transferred in reactor, 24h is reacted at 180 DEG C, dry after centrifuge washing, obtain ceria hollow ball；
1 ~ 4mg/mL methylene blue solution of 1 ~ 4mg/mL ceria hollow ball aqueous solution of 1mL and 1mL is mixed, it is centrifuged after concussion 12h, add the chitosan solution that concentration is 10mg/mL of the ionic liquid containing 0.4 ~ 1.6mL, continue concussion 1h, again it is dispersed in the water of 1mL after centrifugal, obtains ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution；
Described chitosan is carboxymethyl chitosan, and described ionic liquid is 1-butyl-pyridinium tetrafluoroborate。
The detection method of fumonisin
(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in the phosphate buffered solution that pH value is 6.8 of 10mL；
(2) selecting differential pulse voltammetry that fumonisin antigen is detected, select to be scanned under-0.6 ~ 0V voltage, record current changes；
(3) testing sample solution replace fumonisin antigen standard solution detect。
The useful achievement of the present invention
(1) present invention adopts ceria hollow ball to have good absorption property, it is possible to the substantial amounts of methylene blue of load。
(2) chitosan that the present invention adopts can effectively be coated with ceria hollow ball, it is prevented that the leakage of methylene blue。
(3) ionic liquid that the present invention adopts can improve electronics electron transport rate in chitosan, accelerates the electron transfer of methylene blue and electrode surface。
(4) the unmarked type biosensor of preparation is used for the detection of fumonisin by the present invention, and detection limit is low, range of linearity width, it is possible to achieve simple, quick, sensitive and specific detection。
Detailed description of the invention
Embodiment 1A kind of preparation method of the fumonisin sensor built based on ceria hollow ball
(1) being polished with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, ultrasonic cleaning in ultra-pure water and ethanol respectively, nitrogen dries up；
(2) drip, at electrode surface, ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution that 6 μ L concentration are 1mg/mL, dry；
(3) continue the fumonisin antibody-solutions that 6 μ L concentration are 5 μ g/mL is added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1h, clean up；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5mg/mL, in 4 DEG C of refrigerators, hatch 1h, clean up；
(5) the fumonisin antigen of a series of variable concentrations that 6 μ L concentration are 0.0001 ~ 10ng/mL is used for the specific recognition with antibody, incubated at room temperature 1h, cleans up, store for future use in 4 DEG C of refrigerators。
Embodiment 2A kind of preparation method of the fumonisin sensor built based on ceria hollow ball
(1) being polished with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, ultrasonic cleaning in ultra-pure water and ethanol respectively, nitrogen dries up；
(2) drip, at electrode surface, ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution that 6 μ L concentration are 1.5mg/mL, dry；
(3) continue the fumonisin antibody-solutions that 6 μ L concentration are 10 μ g/mL is added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1h, clean up；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 10mg/mL, in 4 DEG C of refrigerators, hatch 1h, clean up；
(5) the fumonisin antigen of a series of variable concentrations that 6 μ L concentration are 0.0001 ~ 10ng/mL is used for the specific recognition with antibody, incubated at room temperature 1h, cleans up, store for future use in 4 DEG C of refrigerators。
Embodiment 3A kind of preparation method of the fumonisin sensor built based on ceria hollow ball
(1) being polished with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, ultrasonic cleaning in ultra-pure water and ethanol respectively, nitrogen dries up；
(2) drip, at electrode surface, ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution that 6 μ L concentration are 2mg/mL, dry；
(3) continue the fumonisin antibody-solutions that 6 μ L concentration are 20 μ g/mL is added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1h, clean up；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 20mg/mL, in 4 DEG C of refrigerators, hatch 1h, clean up；
(5) the fumonisin antigen of a series of variable concentrations that 6 μ L concentration are 0.0001 ~ 10ng/mL is used for the specific recognition with antibody, incubated at room temperature 1h, cleans up, store for future use in 4 DEG C of refrigerators。
Embodiment 4The preparation of ceria hollow ball-methylene blue-chitosan-ionic liquid
0.05g seven cerium hydrate and 0.89g polyvinylpyrrolidone are dissolved in 19mL water, it is subsequently added the Methanamide of 0.5mL and the hydrogen peroxide of 0.05mL, after stirring 30min, above-mentioned mixed solution is transferred in reactor, 24h is reacted at 180 DEG C, dry after centrifuge washing, obtain ceria hollow ball；
The 1mg/mL methylene blue solution of the 1mg/mL ceria hollow ball aqueous solution of 1mL and 1mL is mixed, it is centrifuged after concussion 12h, add the chitosan solution that concentration is 10mg/mL of the ionic liquid containing 0.4mL, continue concussion 1h, again it is dispersed in the water of 1mL after centrifugal, obtains ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution；
Described chitosan is carboxymethyl chitosan, and described ionic liquid is 1-butyl-pyridinium tetrafluoroborate。
Embodiment 5The preparation of ceria hollow ball-methylene blue-chitosan-ionic liquid
0.1g seven cerium hydrate and 0.178g polyvinylpyrrolidone are dissolved in 19mL water, it is subsequently added the Methanamide of 1mL and the hydrogen peroxide of 0.1mL, after stirring 30min, above-mentioned mixed solution is transferred in reactor, 24h is reacted at 180 DEG C, dry after centrifuge washing, obtain ceria hollow ball；
The 2mg/mL methylene blue solution of the 2mg/mL ceria hollow ball aqueous solution of 1mL and 1mL is mixed, it is centrifuged after concussion 12h, add the chitosan solution that concentration is 10mg/mL of the ionic liquid containing 0.8mL, continue concussion 1h, again it is dispersed in the water of 1mL after centrifugal, obtains ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution；
Described chitosan is carboxymethyl chitosan, and described ionic liquid is 1-butyl-pyridinium tetrafluoroborate。
Embodiment 6The preparation of ceria hollow ball-methylene blue-chitosan-ionic liquid
0.2g seven cerium hydrate and 0.356g polyvinylpyrrolidone are dissolved in 19mL water, it is subsequently added the Methanamide of 2mL and the hydrogen peroxide of 0.2mL, after stirring 30min, above-mentioned mixed solution is transferred in reactor, 24h is reacted at 180 DEG C, dry after centrifuge washing, obtain ceria hollow ball；
The 4mg/mL methylene blue solution of the 4mg/mL ceria hollow ball aqueous solution of 1mL and 1mL is mixed, it is centrifuged after concussion 12h, add the chitosan solution that concentration is 10mg/mL of the ionic liquid containing 1.6mL, continue concussion 1h, again it is dispersed in the water of 1mL after centrifugal, obtains ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution；
Described chitosan is carboxymethyl chitosan, and described ionic liquid is 1-butyl-pyridinium tetrafluoroborate。
Embodiment 7The detection method of fumonisin
(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in the phosphate buffered solution that pH value is 6.8 of 10mL；
(2) selecting differential pulse voltammetry that fumonisin antigen is detected, select to be scanned under-0.6 ~ 0V voltage, record current changes；
(3) testing sample solution replace fumonisin antigen standard solution detect；
(4) the fumonisin Detection of antigen range of linearity is 0.0001 ~ 10ng/mL by this biosensor, detection limit 0.05pg/mL。
Claims (2)
1. the preparation method of the fumonisin sensor built based on ceria hollow ball, it is characterised in that step is as follows:
(1) preparation of ceria hollow ball
0.05 ~ 0.2g seven cerium hydrate and 0.89 ~ 0.356g polyvinylpyrrolidone are dissolved in 19mL water, it is subsequently added the Methanamide of 0.5 ~ 2mL and the hydrogen peroxide of 0.05 ~ 0.2mL, after stirring 30min, above-mentioned mixed solution is transferred in reactor, 24h is reacted at 180 DEG C, dry after centrifuge washing, obtain ceria hollow ball；
(2) preparation of ceria hollow ball-methylene blue-chitosan-ionic liquid
1 ~ 4mg/mL methylene blue solution of 1 ~ 4mg/mL ceria hollow ball aqueous solution of 1mL and 1mL is mixed, it is centrifuged after concussion 12h, add the chitosan solution that concentration is 10mg/mL of the ionic liquid containing 0.4 ~ 1.6mL, continue concussion 1h, again it is dispersed in the water of 1mL after centrifugal, obtains ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution；
Described chitosan is carboxymethyl chitosan, and described ionic liquid is 1-butyl-pyridinium tetrafluoroborate；
(3) preparation of sensor
1) being polished with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, ultrasonic cleaning in ultra-pure water and ethanol respectively, nitrogen dries up；
2) drip, at electrode surface, ceria hollow ball-methylene blue-chitosan-ionic liquid aqueous solution that 6 μ L concentration are 1 ~ 2mg/mL, dry；
3) continue the fumonisin antibody-solutions that 6 μ L concentration are 5 ~ 20 μ g/mL is added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1h, clean up；
4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5 ~ 20mg/mL, in 4 DEG C of refrigerators, hatch 1h, clean up；
5) the fumonisin antigen of a series of variable concentrations that 6 μ L concentration are 0.0001 ~ 10ng/mL is used for the specific recognition with antibody, incubated at room temperature 1h, cleans up, store for future use in 4 DEG C of refrigerators。
2. a kind of fumonisin sensor built based on ceria hollow ball that prepared by the preparation method as claimed in claim 1 detection method to fumonisin, it is characterised in that step is as follows:
(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in the phosphate buffered solution that pH value is 6.8 of 10mL；
(2) selecting differential pulse voltammetry that fumonisin antigen is detected, select to be scanned under-0.6 ~ 0V voltage, record current changes；
(3) testing sample solution replace fumonisin antigen standard solution detect。
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| CN102226779A (en) * | 2011-03-28 | 2011-10-26 | 中国人民解放军第三军医大学第三附属医院 | Electrochemical immunodetection method |
| CN102778453A (en) * | 2012-08-08 | 2012-11-14 | 济南大学 | Manufacture method and application of silver hybridization SBA-15 electrochemical luminescence immunosensor |
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