CN103163124A - Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor - Google Patents
Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor Download PDFInfo
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- CN103163124A CN103163124A CN2013100858770A CN201310085877A CN103163124A CN 103163124 A CN103163124 A CN 103163124A CN 2013100858770 A CN2013100858770 A CN 2013100858770A CN 201310085877 A CN201310085877 A CN 201310085877A CN 103163124 A CN103163124 A CN 103163124A
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Abstract
The invention discloses a method for detecting a trace amount of gibberellin A3 by a molecular imprinting electrochemical luminescence sensor. According to the method, when the molecular gibberellin A3 to be detected and the rhodamine B marked gibberellin A3 on a molecularly imprinted membrane of the surface of an electrode are subjected to competitive replacement, rhodamine B is oxidized under a certain voltage to form an oxidation state intermediate product which can greatly induce and amplify weak electrochemical luminescence signals of luminal, thus resulting in reduction of electrochemical luminescence of a luminol base solution, the electrochemical luminescence intensity changes on a gold electrode of the rhodamine B and the luminol in the base solution have a good linear relationship with the concentration of the gibberellin A3 in the concentration range of 10*10<-11>-3.0*10<-9>mol/L, and the detection limit is 3.45*10<-12>mol/L. The method has the advantages of overcoming the detects of too complex detection and the like of the prior art, improving the sensitivity and selectivity and being easy in automation of detection of the gibberellin A3 with low concentration.
Description
Technical field
The present invention relates to a kind of method of utilizing molecular imprinting, rhodamine B enlarge-effect and electrochemical luminous sensor coupling Fast Measurement trace gibberellin.
Background technology
Gibberellin (Gibberellin, GA) is a very important plant hormone of class, is widely used in agricultural production.Gibberellin plays regulating action to the stages of plant growth, growth, and vegetable and fruit etc. is had significant effect of increasing production.Different gibberellin biologically actives is different, and the activity of gibberellin A 3 (GA3) is the highest.But, there are some researches show, gibberellin may accumulate in human body in enrichment after long-term the use, causes slow poisoning, and even canceration causes potential safety hazard to human health.At present, some developed countries have made strict regulations to the gibberellin maximum residue limit(MRL) in fruit, vegetables and beer.Therefore, the method for the detection trace gibberellin of a kind of quick and easy, highly sensitive, low detection limit of searching is significant.The molecular specificity recognition technology of molecular engram rising in recent years.And the electrochemiluminescence detection means has the advantage that many galvanochemistry do not possess, molecular imprinting and electrochemiluminescence technology are combined, can obtain the biology sensor of high sensitivity, high selectivity, relevant report has been arranged at present, but the detection that is applied to micro-gibberellin there is not correlative study temporarily.
Summary of the invention
The purpose of this invention is to provide a kind of high sensitivity, high selectivity and be different from the rhodamine B mark enlarge-effect that enzyme amplifies, and the molecular imprinting electrochemical luminescence sensor that can measure micro-gibberellin.
Conceive as follows: under the condition that rhodamine B exists, under certain voltage, all oxidation reaction can occur due to luminol and rhodamine B, generate the intermediate product of oxidation state.Under certain voltage and in medium, the dissolved oxygen DO of the intermediate oxidation attitude product of rhodamine B in can reducing solution generates super oxygen base negative ion (O
2 -), the luminous precursor of luminol is surpassed oxygen base negative ion (O
2 -) be further oxidized to the 3-aminophthalic acid radical ion (ATP of excited state
-)
*, i.e. luminophor, luminophor discharges photon in returning to the relaxation process of ground state, produce stronger chemiluminescence.So rhodamine B can greatly strengthen the faint electrochemical luminescence signals of luminol excited state intermediate product in system.In addition, introduced the rhodamine B mark and amplified, replaced the higher enzyme of cost, well improved sensitivity and reduced testing cost.The change of electrochemical luminescence signals is namely to compete by the gibberellin A 3 in liquid to be measured the gibberellin A 3 that replaces the rhodamine B mark on having hatched on molecular engram film to reach.Gibberellin A 3 concentration in liquid to be measured is larger, competitiveness is stronger, make the gibberellin A 3 amount of the rhodamine B mark of electrode surface reduce, the reacting dose that is luminol in rhodamine B and end liquid reduces, the luminescent intermediates that produces reduces, weaken thereby electrochemical luminescence signals detected, can reach the purpose that indirectly detects gibberellin A 3 like this.
The present invention relates to the molecular imprinting electrochemiluminescence enhanced sensitivity technology of rhodamine B mark enlarge-effect.When the gibberellin A 3 of rhodamine B mark on the molecular engram film of testing molecule gibberellin A 3 and electrode surface is at war with replacement, the electrochemical luminescence signals of luminol on gold electrode in rhodamine B and end liquid changes, and the concentration of electrochemiluminescence Strength Changes and gibberellin A 3 to be measured is 1 * 10
-11~ 3 * 10
-9Be good linear relationship in the mol/L scope.
Concrete steps are as follows:
(1) processing of gold electrode:
After using successively 1.0,0.3 and 0.05 μ m alumina powder with the gold electrode polishing, each supersound washing 5 minutes in absolute ethyl alcohol and water respectively is then at 0.5 mol/L H
2SO
4In carry out cyclic voltammetric (CV) electrochemical treatment in-0.2 ~ 1.0 V, until obtain stable CV response.
(2) preparation of gibberellin A 3 molecular imprinting electrochemical luminescence sensor:
First with the acetate buffer solution dissolving of 0.009 ~ 0.036g o-phenylenediamine with 10 mLpH=5.2, then add 2 * 10
-4~ 8 * 10
-4The gibberellin A 3 of mol/L (GA3) solution fully mixes, and with cyclic voltammetry scanning 20 ~ 40 circles, sweep limit-0.2 ~ 1.0V sweeps fast 50 mV/s; After polymerization is completed, gold electrode is taken out, after rinsing well with redistilled water, put into volume ratio and be the absolute methanol of 7 ~ 9:1-glacial acetic acid system, under agitation wash the polymeric membrane 5 ~ 15 minutes of gold electrode surfaces, remove the gibberellin A 3 in polymer film and be adsorbed on other adsorbate on molecular engram film, make the gibberellin A 3 molecular imprinting electrochemical luminescence sensor that remains with template molecule configuration hole;
(3) detection method:
At first the gibberellin A 3 molecular imprinting electrochemical luminescence sensor that step (2) is made is placed in the solution hatching 18 minutes of the gibberellin A 3 of rhodamine B mark, then takes out gibberellin A 3 molecular imprinting electrochemical luminescence sensor and rinses the surface; To hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and put into respectively 0,1 * 10
-11, 3 * 10
-11, 6 * 10
-11, 12 * 10
-11, 25 * 10
-11, 50 * 10
-11, 80 * 10
-11, 120 * 10
-11, 160 * 10
-11, 200 * 10
-11, 250 * 10
-11, 300 * 10
-11 Absorption 10 ~ 20 minutes is at war with in the gibberellin A 3 standard solution of mol/L; Access at last detection system, detection system is: 10 mL contain 1.2 * 10
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; System carries out electrogenerated chemiluminescence scanning with MPI-E type Electrochemiluminescprocess process, scanning voltage-0.3 ~ 0.8 V, and sweeping speed is 100 mV/s, photomultiplier high pressure 900 V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s;
(4) drafting of standard working curve:
Add 10 mL to contain 1.2 * 10 in 15 mL small beakers
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; Will be hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 standard solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution, carry out electrochemiluminescdetection detection; Gibberellin A 3 is 1.0 * 10
-11~ 3.0 * 10
-9In the mol/L concentration range with electrochemiluminescence intensity minimizing value
I F Be good linear relationship, linear equation is respectively:
I F =11.51
C(10
-11Mol/L)+134.44, linearly dependent coefficient
r=0.999;
(5) mensuration of gibberellin A 3 content in sample:
Add 10 mL to contain 1.2 * 10 in 15 mL small beakers
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; To hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution; Utilize the Electrochemiluminescprocess process system to carry out electrogenerated chemiluminescence scanning to liquid to be measured, scanning voltage scanning voltage-0.3 ~ 0.8 V, sweeping speed is 100 mV/s, photomultiplier high pressure 900 V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s obtain electrochemiluminescence intensity
I F Calculate the concentration of gibberellin A 3 according to calibration curve
C
The present invention has overcome prior art and has had when detecting that too method is loaded down with trivial details, and the complicated shortcomings that waits of step has improved sensitivity and selectivity, is easy to robotization for the detection of the gibberellin A 3 of low concentration.
Description of drawings
Fig. 1 is the electrochemiluminescence figure of gibberellin A 3 molecular engram film under different conditions on embodiment of the present invention gold electrode.
The naked gold electrode of a. wherein; B. remove the gibberellin A 3 molecular imprinting electrochemical luminescence sensor of template molecule in liquid at the bottom of luminol; C. again adsorb gibberellin A 3 molecular imprinting electrochemical luminescence sensor after gibberellin A 3 in liquid at the bottom of luminol; D. the gibberellin A 3 molecular imprinting electrochemical luminescence sensor of removing template molecule is at the bottom of containing rhodamine B in liquid; E. the gibberellin A 3 molecular imprinting electrochemical luminescence sensor after having competed in the gibberellin A 3 standard solution is not in containing the end liquid of luminol; F. the gibberellin A 3 molecular imprinting electrochemical luminescence sensor after competing in the gibberellin A 3 standard solution is in liquid at the bottom of luminol.
Fig. 2 is the graph of a relation of embodiment of the present invention gibberellin A 3 content and electrochemiluminescence intensity.
Embodiment
Embodiment:
(1) processing of gold electrode:
After using successively 1.0,0.3 and 0.05 μ m alumina powder with the gold electrode polishing, each supersound washing 5 minutes in absolute ethyl alcohol and water respectively is then at 0.5 mol/L H
2SO
4In carry out cyclic voltammetric (CV) electrochemical treatment in-0.2 ~ 1.0 V, until obtain stable CV response.
(2) preparation of gibberellin A 3 molecular imprinting electrochemical luminescence sensor:
First 0.018 g o-phenylenediamine is dissolved with 10 mLpH=5.2 acetate buffer solutions, then add 5 * 10
-4The GA3 solution of mol/L fully mixes, and with cyclic voltammetry scanning 30 circles, sweep limit-0.2V～1.0V sweeps fast 50 mV/s; After polymerization is completed, gold electrode is taken out, after rinsing well with redistilled water, put into volume ratio and be the absolute methanol of 8:1-glacial acetic acid system, under agitation wash the polymeric membrane 10 minutes of gold electrode surfaces, remove the gibberellin A 3 in polymer film and be adsorbed on other adsorbate on molecular engram film, make the gibberellin A 3 molecular imprinting electrochemical luminescence sensor that remains with template molecule configuration hole;
(3) detection method:
At first the gibberellin A 3 molecular imprinting electrochemical sensor that step (2) is made is placed in the solution hatching 18 minutes of the gibberellin A 3 of rhodamine B mark, then takes out gibberellin A 3 molecular imprinting electrochemical luminescence sensor and rinses the surface; Then it is put into respectively and contain 0,1 * 10
-11, 3 * 10
-11, 6 * 10
-11, 12 * 10
-11, 25 * 10
-11, 50 * 10
-11, 80 * 10
-11, 120 * 10
-11, 160 * 10
-11, 200 * 10
-11, 250 * 10
-11, 300 * 10
-11Be at war with in the gibberellin A 3 standard solution of mol/L and adsorbed 15 minutes, access at last detection system; Detection system is: 10 mL contain 1.2 * 10
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; Utilize MPI-E type Electrochemiluminescprocess process system to carry out electrogenerated chemiluminescence scanning to liquid to be measured, scanning voltage scanning voltage-0.3 V is to 0.8 V, and sweeping speed is 100 mV/s, photomultiplier high pressure 900V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s;
(4) drafting of standard working curve:
Add 10 mL to contain 1.2 * 10 in 15 mL small beakers
-30.05 mol/LTris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; Will be hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 standard solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution, carry out electrochemiluminescence scanning; Gibberellin A 3 is 1 * 10
-11Mol/L～3 * 10
-9In the mol/L concentration range with electrochemiluminescence intensity minimizing value
I F Be good linear relationship, linear equation is:
I F =11.51
C(10
-11Mol/L)+134.44, linearly dependent coefficient
r=0.999;
(5) mensuration of gibberellin A 3 content in beer sample: the beer that is used for sample detection is purchased from the supermarket, uses simultaneously recovery testu.Add in 15 mL small beakers and contain 1.2 * 10
-30.05 mol/L Tris-HCl buffering molten (pH=7.8) of mol/L luminol; To hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution; Utilize the Electrochemiluminescprocess process system to carry out electrogenerated chemiluminescence scanning to liquid to be measured, scanning voltage scanning voltage-0.3 ~ 0.8 V, sweeping speed is 100 mV/s, photomultiplier high pressure 900 V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s obtain electrochemiluminescence intensity
I F Calculate the concentration of gibberellin A 3 according to calibration curve
CAnd the recovery, result such as table 1:
Claims (1)
1. method that detects micro-gibberellin A 3 with the molecular imprinting electrochemical luminescence sensor is characterized in that concrete steps are:
(1) processing of gold electrode:
After using successively 1.0,0.3 and 0.05 μ m alumina powder with the gold electrode polishing, each supersound washing 5 minutes in absolute ethyl alcohol and water respectively is then at 0.5 mol/L H
2SO
4In to carry out cyclic voltammetric in-0.2 ~ 1.0 V be the CV electrochemical treatment, until obtain stable CV response;
(2) preparation of gibberellin A 3 molecular imprinting electrochemical luminescence sensor:
First with the acetate buffer solution dissolving of 0.009 ~ 0.036g o-phenylenediamine with 10 mLpH=5.2, then add 2 * 10
-4~ 8 * 10
-4The gibberellin A 3 solution of mol/L fully mixes, and with cyclic voltammetry scanning 20 ~ 40 circles, sweep limit-0.2 ~ 1.0V sweeps fast 50 mV/s; After polymerization is completed, gold electrode is taken out, after rinsing well with redistilled water, put into volume ratio and be the absolute methanol of 7 ~ 9:1-glacial acetic acid system, under agitation wash the polymeric membrane 5 ~ 15 minutes of gold electrode surfaces, remove the gibberellin A 3 in polymer film and be adsorbed on other adsorbate on molecular engram film, make the gibberellin A 3 molecular imprinting electrochemical luminescence sensor that remains with template molecule configuration hole;
(3) detection method:
At first the gibberellin A 3 molecular imprinting electrochemical luminescence sensor that step (2) is made is placed in the solution hatching 18 minutes of the gibberellin A 3 of rhodamine B mark, then takes out gibberellin A 3 molecular imprinting electrochemical luminescence sensor and rinses the surface; To hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and put into respectively 0,1 * 10
-11, 3 * 10
-11, 6 * 10
-11, 12 * 10
-11, 25 * 10
-11, 50 * 10
-11, 80 * 10
-11, 120 * 10
-11, 160 * 10
-11, 200 * 10
-11, 250 * 10
-11, 300 * 10
-11Absorption 10 ~ 20 minutes is at war with in the gibberellin A 3 standard solution of mol/L; Access at last detection system, detection system is: 10 mL contain 1.2 * 10
-3The 0.05mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; System carries out electrogenerated chemiluminescence scanning with MPI-E type Electrochemiluminescprocess process, scanning voltage-0.3 ~ 0.8 V, and sweeping speed is 100 mV/s, photomultiplier high pressure 900 V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s;
(4) drafting of standard working curve:
Add 10 mL to contain 1.2 * 10 in 15 mL small beakers
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; Will be hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 standard solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution, carry out electrochemiluminescdetection detection; Gibberellin A 3 is 1.0 * 10
-11~ 3.0 * 10
-9In the mol/L concentration range with electrochemiluminescence intensity minimizing value
I F Be good linear relationship, linear equation is respectively:
I F =11.51
C(10
-11Mol/L)+134.44, linearly dependent coefficient
r=0.999;
(5) mensuration of gibberellin A 3 content in sample:
Add 10 mL to contain 1.2 * 10 in 15 mL small beakers
-30.05 mol/L Tris-HCl buffer solution of mol/L luminol, Tris-HCl buffer solution pH=7.8; To hatch completely gibberellin A 3 molecular imprinting electrochemical luminescence sensor and immerse in 10 mL gibberellin A 3 solution competitive Adsorption 15 minutes in rhodamine B mark gibberellin A 3 solution; Utilize the Electrochemiluminescprocess process system to carry out electrogenerated chemiluminescence scanning to liquid to be measured, scanning voltage scanning voltage-0.3 ~ 0.8 V, sweeping speed is 100 mV/s, photomultiplier high pressure 900V, sampling rate 10 T/S, enlargement factor 4, Measuring Time 90 s obtain electrochemiluminescence intensity
I F Calculate the concentration of gibberellin A 3 according to calibration curve
C
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849262A (en) * | 2015-04-09 | 2015-08-19 | 宁波大学 | Electrochemical luminescence sensor array, and preparation method and application thereof |
| CN106404868A (en) * | 2016-11-04 | 2017-02-15 | 北京农业信息技术研究中心 | Microelectrode biosensor for online detection of GAs (gibberellins) in living plant and application of microelectrode biosensor |
| CN108837813A (en) * | 2018-06-27 | 2018-11-20 | 桂林理工大学 | A kind of preparation method and application of the ion surface imprinted material of mesoporous diatom As (V) |
| CN109254047A (en) * | 2018-11-05 | 2019-01-22 | 济南大学 | A kind of preparation method and application of chrysanthemum ester insecticide Electrochemiluminescsensor sensor |
| CN113092453A (en) * | 2021-04-13 | 2021-07-09 | 中国热带农业科学院分析测试中心 | Dipterex electrochemiluminescence molecularly imprinted sensor, and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849262A (en) * | 2015-04-09 | 2015-08-19 | 宁波大学 | Electrochemical luminescence sensor array, and preparation method and application thereof |
| CN106404868A (en) * | 2016-11-04 | 2017-02-15 | 北京农业信息技术研究中心 | Microelectrode biosensor for online detection of GAs (gibberellins) in living plant and application of microelectrode biosensor |
| CN106404868B (en) * | 2016-11-04 | 2018-07-06 | 北京农业信息技术研究中心 | A kind of microelectrode biosensor of live body on-line checking plant gibberellin and its application |
| CN108837813A (en) * | 2018-06-27 | 2018-11-20 | 桂林理工大学 | A kind of preparation method and application of the ion surface imprinted material of mesoporous diatom As (V) |
| CN109254047A (en) * | 2018-11-05 | 2019-01-22 | 济南大学 | A kind of preparation method and application of chrysanthemum ester insecticide Electrochemiluminescsensor sensor |
| CN113092453A (en) * | 2021-04-13 | 2021-07-09 | 中国热带农业科学院分析测试中心 | Dipterex electrochemiluminescence molecularly imprinted sensor, and preparation method and application thereof |
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Application publication date: 20130619 |