CN101710116A - Method for determining trace terramycin by utilizing molecularly imprinted immunosensor - Google Patents
Method for determining trace terramycin by utilizing molecularly imprinted immunosensor Download PDFInfo
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- CN101710116A CN101710116A CN200910114541A CN200910114541A CN101710116A CN 101710116 A CN101710116 A CN 101710116A CN 200910114541 A CN200910114541 A CN 200910114541A CN 200910114541 A CN200910114541 A CN 200910114541A CN 101710116 A CN101710116 A CN 101710116A
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Abstract
The invention discloses a method for determining trace terramycin by utilizing a molecularly imprinted immunosensor. When an terramycin molecularly imprinted membrane is formed on the surface of a gold electrode and acts with terramycin molecules, an electrochemical signal of hydroquinone on the gold electrode changes; and when hydrogen peroxide and horseradish peroxidase exist in a detection system, the electrochemical signal has more remarkable change. When terramycin in solution to be measured is utilized to competitively replace original horseradish peroxidase labeled terramycin on the molecularly imprinted membrane, the electrochemical signal can be linearly reduced along with the increase of the concentration of unlabeled terramycin so as to establish an electrochemical analysis method for determining the trace terramycin. A differential pulse voltammetry is used to scan the solution to be measured, the scanning voltage is between -0.6V and 0.8V, and the terramycin is in good linear relation with a peak current value i within the concentration range of between 0 and 8*10-8mol/L. The method overcomes the disadvantages of high complexity and the like existing in the prior art; and the detection limit of the terramycin reaches 10-9mol/L.
Description
Technical field
The present invention relates to a kind of method of utilizing the electrochemical immunosensor fast measuring trace terramycin of molecular imprinting and immunological technique coupling.
Background technology
Terramycin is a TCs class medicine, is to produce and one of clinical practice microbiotic at most, and its chemical constitution belongs to hydrogenation aphthacene ring derivatives, and oral absorption is good, and is widely distributed in vivo, can enrichment in hepatic tissue, cause hepatic injury.Terramycin often is added in the animal feed, with growth and the control various diseases that promotes animal, if can not strictly control withdrawal time, be easy to cause in animal flesh and tissue residual, bring harm to health, therefore all there is a strict requirement of limiting the quantity of countries in the world to the telracycline family in the livestock and poultry meat is residual, therefore research terramycin is had a high selectivity detection method highly significant.Molecular engram is that a kind of that development in recent years is got up has optionally synthetic method of polymers to microsphere.The trace sensor of having reported is used for the detection of tens kinds of agricultural chemicals such as atrazine, desmetryn, glyphosate, parathion, chloromycetin, does not appear in the newspapers but the terramycin molecularly imprinted immunosensor that utilizes immunological technique to combine with molecular engram detects the research of trace terramycin.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing the electrochemical immunosensor fast measuring trace terramycin of molecular imprinting and immunological technique coupling.
Conceive as follows: we find that p-dihydroxy-benzene can be used as ion probe, when the terramycin molecular engram film forms and the terramycin molecular action time in gold electrode surfaces, its electrochemical signals on gold electrode changes, when detection architecture the inside exist hydrogen peroxide with horseradish peroxidase the time, electrochemical signals changes more remarkable.When utilizing the terramycin of original horseradish peroxidase mark on the terramycin competition replacement molecular engram film in the solution to be measured, electrochemical signals can linearity reduces along with unlabelled terramycin concentration increase, so can be used for detecting the content of terramycin.
The present invention relates to molecular imprinting immuno-enzymatic mark enhanced sensitivity technology.When the competition replacement took place the terramycin of horseradish peroxidase mark on testing molecule terramycin and the terramycin molecular engram film, the electrochemical signals on the p-dihydroxy-benzene gold electrode changed, and the concentration of peak current and terramycin to be measured is in 0mol/L～8 * 10
-8Linear in the mol/L scope.
Concrete steps are:
One, the processing of gold electrode:
Successively with the polishing of the alumina powder of 1.0 μ m, 0.3 μ m and 0.05 μ m, is 1: 1 nitric acid, absolute ethyl alcohol and pure water bubble wash in volume ratio successively with gold electrode, takes out back supersound washing 5min;
Two, the preparation of terramycin molecularly imprinted immunosensor:
Measure 0.5mL 2 * 10 respectively
-4Mol/L～8 * 10
-4The catechol of mol/L and 2.5mL 8 * 10
-5Mol/L～3.5 * 10
-4The terramycin solution of mol/L joins in the acetic acid that 7mL pH=5.2 concentration is 0.1mol/L-sodium acetate buffer solution, fully between 0 to 0.8V, use cyclic voltammetry continuous sweep 30 to 60 to enclose behind the stirring and evenly mixing, can make the terramycin molecularly imprinted immunosensor with the speed of 50mV/s;
Three, detection method:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mol/L～8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 1 * 10
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the terramycin solution of mol/L, inserts detection architecture, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L; With the differential pulse voltammetry in the electrochemical workstation liquid to be measured is scanned, scanning voltage-0.6V is to 0.8V;
Four, the drafting of standard working curve:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mol/L～to 8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 1 * 10 one by one
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the mol/L terramycin solution, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L, differential pulse voltammetry scanning, scanning voltage-0.6V is to 0.8V; Terramycin is in 0mol/L～8 * 10
-8Be good linear relationship, linear equation with peak point current i in the mol/L concentration range: i=24.07921-2.27396 * C, coefficient R is 0.99973;
Five, terramycin Determination on content to be measured:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mol/L～to 8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 1 * 10 one by one
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the mol/L terramycin solution, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L, differential pulse voltammetry scanning, scanning voltage-0.6V is to 0.8V; Read peak point current i; Calculate C according to calibration curve; The i.e. amount of contained terramycin in this 10mL solution to be measured as can be known.
The present invention has overcome prior art and had too many shortcomings such as complexity, and is highly sensitive, for concentration 10
-8The detection of the terramycin that mol/L is above is easy to robotization.
Description of drawings
Fig. 1 be on the embodiment of the invention gold electrode terramycin molecular engram film 5 * 10
-3Cyclic voltammogram in the mol/L potassium ferricyanide solution.
Mark among the figure: the cyclic voltammogram of the naked gold electrode of a-; The cyclic voltammogram of the molecularly imprinted immunosensor of wash-out not after the b-polymerization; The cyclic voltammogram of the molecularly imprinted immunosensor behind the c-wash-out.
Fig. 2 is the graph of a relation of embodiment of the invention terramycin content and differential pulse voltammetry peak current.
Embodiment
Embodiment:
One, the processing of gold electrode:
Successively with the polishing of the alumina powder of 1.0 μ m, 0.3 μ m and 0.05 μ m, is 1: 1 nitric acid, absolute ethyl alcohol, pure water bubble wash in volume ratio successively with gold electrode, takes out back supersound washing 5min.
Two, the preparation of terramycin molecularly imprinted immunosensor
Measure 0.5mL 7 * 10 respectively
-4The catechol of mol/L, 2.5mL 3 * 10
-4The terramycin solution of mol/L and 7mL pH=5.2 concentration are acetic acid-sodium acetate buffer solution of 0.1mol/L, fully between 0 to 0.8V, use cyclic voltammetry continuous sweep 30 to enclose behind the stirring and evenly mixing, can make the terramycin molecularly imprinted immunosensor with the speed of 50mV/s.
Three, detection method
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 5 * 10
-4The phosphate buffered solution of mol/L p-dihydroxy-benzene.Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 3 * 10
-5Competitive Adsorption is 5 minutes in the terramycin solution of mol/L, inserts detection architecture, drips 20 μ L 1 * 10
-3The hydrogen peroxide of mol/L.Select for use the differential pulse voltammetry in the CHI660 type electrochemical workstation that liquid to be measured is scanned, scanning voltage-0.6V is to 0.8V.
Four, the drafting of standard working curve:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 5 * 10
-4The phosphate buffered solution of mol/L p-dihydroxy-benzene.Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 3 * 10 one by one
-5Competitive Adsorption is 5 minutes in the mol/L terramycin solution, inserts detection architecture, drips 20 μ L 1 * 10
-3The hydrogen peroxide of mol/L.Select for use the differential pulse voltammetry in the CHI660 type electrochemical workstation that liquid to be measured is scanned, scanning voltage-0.6V is to 0.8V.Terramycin content and peak current relation such as Fig. 2, terramycin is in 0mol/L～8 * 10
-8Be good linear relationship, linear equation with peak point current i in the mol/L concentration range: i=24.07921-2.27396 * C, coefficient R is 0.99973.
Five, terramycin Determination on content in the sample
Get milk sample and measure, but do not detect terramycin, reclaim experiment so adopt calibration curve method to carry out mark-on.Get the 15mL small beaker, add 10mL 0.1mol/L and contain 5 * 10
-4The phosphate buffered solution of mol/L p-dihydroxy-benzene.With in horseradish peroxidase mark terramycin solution fully the terramycin molecularly imprinted immunosensor after the absorption immersed in the 10mL terramycin solution competitive Adsorption 5 minutes, insert detection architecture, drip 20 μ L 1 * 10
-3The hydrogen peroxide of mol/L.Select for use the differential pulse voltammetry in the CHI660 type electrochemical workstation that liquid to be measured is scanned, scanning voltage-0.6V is to 0.8V.Read peak point current i.Calculate C according to calibration curve.Replicate determination three times, calculate recovery rate, the result is as shown in table 1.
Table 1 mark-on recovery test data
Claims (1)
1. method of utilizing molecularly imprinted immunosensor to measure trace terramycin is characterized in that concrete steps are:
One, the processing of gold electrode:
Successively with the polishing of the alumina powder of 1.0 μ m, 0.3 μ m and 0.05 μ m, is 1: 1 nitric acid, absolute ethyl alcohol and pure water bubble wash in volume ratio successively with gold electrode, takes out back supersound washing 5min;
Two, the preparation of terramycin molecularly imprinted immunosensor:
Measure 0.5mL 2 * 10 respectively
-4Mol/L～8 * 10
-4The catechol of mol/L and 2.5mL 8 * 10
-5Mol/L～3.5 * 10
-4The terramycin solution of mol/L joins in the acetic acid that 7mL pH=5.2 concentration is 0.1mol/L-sodium acetate buffer solution, fully between 0 to 0.8V, use cyclic voltammetry continuous sweep 30 to 60 to enclose behind the stirring and evenly mixing, can make the terramycin molecularly imprinted immunosensor with the speed of 50mV/s;
Three, detection method:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mo l/L～8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL1 * 10
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the terramycin solution of mol/L, inserts detection architecture, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L; With the differential pulse voltammetry in the electrochemical workstation liquid to be measured is scanned, scanning voltage-0.6V is to 0.8V;
Four, the drafting of standard working curve:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mol/L～to 8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 1 * 10 one by one
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the mol/L terramycin solution, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L, differential pulse voltammetry scanning, scanning voltage-0.6V is to 0.8V; Terramycin is in 0mol/L～8 * 10
-8Be good linear relationship, linear equation with peak point current i in the mol/L concentration range: i=24.07921-2.27396 * C, coefficient R is 0.99973;
Five, terramycin Determination on content to be measured:
Get the 15mL small beaker, add 10mL 0.1mol/L and contain 3 * 10
-6Mol/L～to 8 * 10
-6The phosphate buffered solution of mol/L p-dihydroxy-benzene; Terramycin molecularly imprinted immunosensor after fully adsorbing in horseradish peroxidase mark terramycin solution is immersed 10mL 1 * 10 one by one
-5Mol/L～3 * 10
-3Competitive Adsorption is 3 to 10 minutes in the mol/L terramycin solution, drips 20 μ L 5 * 10
-4Mol/L～3 * 10
-3The hydrogen peroxide of mol/L, differential pulse voltammetry scanning, scanning voltage-0.6V is to 0.8V; Read peak point current i; Calculate C according to calibration curve; The i.e. amount of contained terramycin in this 10mL solution to be measured as can be known.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621216A (en) * | 2012-04-11 | 2012-08-01 | 桂林理工大学 | Method for detecting trace terramycin with double-amplification-effect molecular imprinting electrochemical sensor |
| CN103293299A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Method and kit for broadening double-antibody sandwich immunodetection concentration range |
| CN108226240A (en) * | 2017-12-11 | 2018-06-29 | 江苏奔拓电气科技有限公司 | A kind of preparation method for the marking sensor for detecting terramycin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101130580A (en) * | 2007-08-10 | 2008-02-27 | 浙江省疾病预防控制中心 | Tsiklomitsin molecular engram polyalcohol and uses of the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293299A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Method and kit for broadening double-antibody sandwich immunodetection concentration range |
| CN102621216A (en) * | 2012-04-11 | 2012-08-01 | 桂林理工大学 | Method for detecting trace terramycin with double-amplification-effect molecular imprinting electrochemical sensor |
| CN108226240A (en) * | 2017-12-11 | 2018-06-29 | 江苏奔拓电气科技有限公司 | A kind of preparation method for the marking sensor for detecting terramycin |
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Effective date of registration: 20210324 Address after: No. 99-321, Taizhong Road, cross strait science and Technology Industrial Park, Nanjing, Jiangsu, 211800 Patentee after: Nanjing Pukou Kechuang investor group Co.,Ltd. Address before: 541004 the Guangxi Zhuang Autonomous Region Guilin Construction Road No. 12 Patentee before: GUILIN University OF TECHNOLOGY |
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