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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- gibberellin
- electrochemiluminescence
- mol
- molecularly imprinted
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 title claims abstract description 151
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000004020 luminiscence type Methods 0.000 title abstract description 4
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 21
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229940043267 rhodamine b Drugs 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 19
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000010931 gold Substances 0.000 claims abstract description 16
- 229910052737 gold Inorganic materials 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 18
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 17
- 238000001179 sorption measurement Methods 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 6
- 229920006254 polymer film Polymers 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 6
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 230000002860 competitive effect Effects 0.000 claims description 5
- 238000002484 cyclic voltammetry Methods 0.000 claims description 5
- 238000001378 electrochemiluminescence detection Methods 0.000 claims description 5
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002156 adsorbate Substances 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 238000011088 calibration curve Methods 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims 1
- 239000012528 membrane Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000008859 change Effects 0.000 abstract description 3
- 239000013067 intermediate product Substances 0.000 abstract description 3
- 230000007423 decrease Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 238000006467 substitution reaction Methods 0.000 abstract description 2
- 229930191978 Gibberellin Natural products 0.000 description 10
- 239000003448 gibberellin Substances 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 235000013405 beer Nutrition 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- -1 3-aminophthalate ion Chemical class 0.000 description 1
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- ZDGGJQMSELMHLK-UHFFFAOYSA-N m-Trifluoromethylhippuric acid Chemical compound OC(=O)CNC(=O)C1=CC=CC(C(F)(F)F)=C1 ZDGGJQMSELMHLK-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种用分子印迹电化学发光传感器检测微量赤霉素A3的方法。当待测分子赤霉素A3与电极表面的分子印迹膜上罗丹明B标记的赤霉素A3进行竞争取代时，罗丹明B在一定的电压下被氧化，形成氧化态的中间产物，该中间产物可以极大地诱导放大鲁米诺微弱的电化学发光信号，导致鲁米诺底液的电化学发光降低。罗丹明B与底液中的鲁米诺在金电极上的电化学发光强度变化与赤霉素A3的浓度在1.0×10-11~3.0×10-9mol/L浓度范围内呈良好的线性关系，方法检出限为3.45×10-12mol/L。本发明克服了已有技术在检测时存在过于复杂等诸多缺点，提高了灵敏度和选择性，对于低浓度赤霉素A3的检测易于自动化。
The invention discloses a method for detecting trace amounts of gibberellin A3 by using a molecular imprinted electrochemical luminescence sensor. When the molecule gibberellin A3 to be measured competes with rhodamine B-labeled gibberellin A3 on the molecular imprinted membrane on the electrode surface for substitution, rhodamine B is oxidized under a certain voltage to form an intermediate product in an oxidized state. The product can greatly induce and amplify the weak electrochemiluminescence signal of luminol, resulting in a decrease in the electrochemiluminescence of the luminol bottom solution. The change of the electrochemiluminescent intensity of rhodamine B and luminol in the bottom solution on the gold electrode has a good linearity with the concentration of gibberellin A3 in the concentration range of 1.0×10 -11 ~3.0×10 -9 mol/L The detection limit of the method is 3.45×10 -12 mol/L. The invention overcomes many shortcomings such as too complicated detection in the prior art, improves sensitivity and selectivity, and is easy to automate the detection of low-concentration gibberellin A3.
Description
技术领域 technical field
本发明涉及一种利用分子印迹技术、罗丹明B放大效应与电化学发光传感器联用快速测定微量赤霉素的方法。 The invention relates to a method for quickly measuring trace gibberellins by using molecular imprinting technology, rhodamine B amplification effect and electrochemiluminescence sensor.
背景技术 Background technique
赤霉素（Gibberellin，GA）是一类非常重要的植物荷尔蒙，在农业生产上得到了广泛的应用。赤霉素对植物生长、发育的各个阶段起着调节作用，对蔬菜水果等有着显著的增产效果。不同的赤霉素生物活性不同，而赤霉素A3（GA3）的活性最高。但是，有研究表明，在长期使用后赤霉素可能在人体中富集积累，引起慢性中毒，甚至癌变，对人类健康造成安全隐患。目前，一些发达国家已经对水果、蔬菜以及啤酒中的赤霉素最高残留限量做出了严格规定。因此，寻找一种快捷、方便、高灵敏、低检出限的检测痕量赤霉素的方法具有重要意义。分子印迹近年来兴起的分子特异性识别技术。而电化学发光检测手段具有许多电化学不具备的优势，将分子印迹技术与电化学发光技术结合起来，可以得到高灵敏度、高选择性的生物传感器，目前已经有相关报道，但是应用于微量赤霉素的检测暂未有相关研究。 Gibberellin (Gibberellin, GA) is a very important class of plant hormones and has been widely used in agricultural production. Gibberellin regulates various stages of plant growth and development, and has a significant effect on increasing the yield of vegetables and fruits. Different gibberellins have different biological activities, and gibberellin A3 (GA3) has the highest activity. However, studies have shown that gibberellin may accumulate in the human body after long-term use, causing chronic poisoning and even canceration, which poses a safety hazard to human health. At present, some developed countries have made strict regulations on the maximum residue limits of gibberellins in fruits, vegetables and beer. Therefore, it is of great significance to find a fast, convenient, highly sensitive and low detection limit method for detecting trace gibberellins. Molecular imprinting is a molecular-specific recognition technology that has emerged in recent years. The electrochemiluminescence detection method has many advantages that electrochemistry does not have. Combining molecular imprinting technology with electrochemiluminescence technology can obtain a biosensor with high sensitivity and high selectivity. There is no relevant research on the detection of mycin.
发明内容 Contents of the invention
本发明的目的是提供一种高灵敏度、高选择性且区别于酶放大的罗丹明B标记放大效应,并可以对微量赤霉素进行测定的分子印迹电化学发光传感器。 The purpose of the present invention is to provide a molecularly imprinted electrochemiluminescent sensor with high sensitivity and high selectivity, which is different from the rhodamine B labeling amplification effect of enzyme amplification, and which can detect trace gibberellins.
构思如下：在罗丹明B存在的条件下，由于鲁米诺和罗丹明B在一定的电压下，均会发生氧化反应，生成氧化态的中间产物。在一定的电压下和介质中，罗丹明B的中间氧化态产物能够还原溶液中的溶解氧生成超氧基阴离子（O2 -），鲁米诺发光前体被超氧基阴离子（O2 -)进一步氧化为激发态的3-氨基邻苯二甲酸根离子(ATP-)*，即发光体，发光体在返回基态的弛豫过程中释放出光子，产生较强的化学发光。所以，罗丹明B在体系中能够极大地增强鲁米诺激发态中间产物的微弱电化学发光信号。此外，引入了罗丹明B标记放大，代替了成本较高的酶，很好的提高了灵敏度和降低了检测成本。电化学发光信号的改变即是通过待测液中的赤霉素A3竞争取代分子印迹膜上已孵化上的罗丹明B标记的赤霉素A3来达到的。待测液中的赤霉素A3浓度越大，竞争能力越强，使得电极表面的罗丹明B标记的赤霉素A3量减少，即罗丹明B与底液中鲁米诺的反应量减少，产生的发光中间体减少，从而检测到电化学发光信号减弱，这样即可达到间接地检测赤霉素A3的目的。 The idea is as follows: in the presence of rhodamine B, both luminol and rhodamine B will undergo oxidation reaction at a certain voltage to generate an intermediate product in an oxidized state. Under a certain voltage and medium, the intermediate oxidation state product of rhodamine B can reduce the dissolved oxygen in the solution to generate superoxide anion (O 2 - ), and the luminescent precursor of luminol is replaced by superoxide anion (O 2 - ) is further oxidized to 3-aminophthalate ion (ATP - ) * in an excited state, that is, the luminophore, which releases photons during the relaxation process back to the ground state, resulting in strong chemiluminescence. Therefore, rhodamine B in the system can greatly enhance the weak electrochemiluminescence signal of the excited-state intermediate product of luminol. In addition, rhodamine B labeling amplification was introduced to replace the enzyme with high cost, which greatly improved the sensitivity and reduced the detection cost. The change of the electrochemiluminescent signal is achieved by the competition of the gibberellin A3 in the test solution to replace the incubated Rhodamine B-labeled gibberellin A3 on the molecular imprinted membrane. The greater the concentration of gibberellin A3 in the solution to be tested, the stronger the competitive ability, which reduces the amount of gibberellin A3 labeled with rhodamine B on the electrode surface, that is, the amount of reaction between rhodamine B and luminol in the bottom solution decreases, The generated luminescent intermediates are reduced, thereby detecting that the electrochemiluminescent signal is weakened, so that the purpose of indirect detection of gibberellin A3 can be achieved.
本发明涉及罗丹明B标记放大效应的分子印迹技术电化学发光增敏技术。当待测分子赤霉素A3与电极表面的分子印迹膜上罗丹明B标记的赤霉素A3进行竞争取代时，罗丹明B与底液中的鲁米诺在金电极上的电化学发光信号发生变化，电化学发光强度变化与待测赤霉素A3的浓度在1×10-11 ~ 3×10-9 mol/L范围内呈良好的线性关系。 The invention relates to the molecular imprinting technology electrochemiluminescence sensitization technology of rhodamine B label amplification effect. Electrochemiluminescent signal of rhodamine B and luminol in the bottom solution on the gold electrode when the molecule gibberellin A3 to be tested competes with rhodamine B-labeled gibberellin A3 on the molecular imprinted membrane on the electrode surface for substitution There is a good linear relationship between the change of electrochemiluminescence intensity and the concentration of gibberellin A3 to be tested in the range of 1×10 -11 ~ 3×10 -9 mol/L.
具体步骤如下： Specific steps are as follows:
（1）金电极的处理： (1) Treatment of gold electrodes:
依次用1.0、0.3和0.05μm氧化铝粉将金电极抛光后，分别在无水乙醇和水中各超声洗涤5分钟，然后在0.5 mol/L H2SO4中于-0.2 ~ 1.0 V进行循环伏安(CV)电化学处理，直到获得稳定的CV响应。 After the gold electrode was polished with 1.0, 0.3 and 0.05 μm alumina powder in sequence, it was ultrasonically washed in absolute ethanol and water for 5 minutes, and then cyclic voltammetry was performed at -0.2 to 1.0 V in 0.5 mol/L H 2 SO 4 (CV) electrochemical treatment until a stable CV response is obtained.
（2）赤霉素A3分子印迹电化学发光传感器的制备： (2) Preparation of gibberellin A3 molecularly imprinted electrochemiluminescence sensor:
先将0.009 ~ 0.036g邻苯二胺用10 mLpH=5.2的醋酸缓冲液溶解，然后加入2×10-4 ~ 8×10-4 mol/L的赤霉素A3(GA3)溶液，充分混合均匀，用循环伏安法扫描20 ~ 40圈，扫描范围-0.2 ~ 1.0V，扫速50 mV/s；聚合完成后将金电极取出，用二次蒸馏水冲洗干净后，放入体积比为7 ~ 9:1的无水甲醇-冰醋酸体系中，在搅拌下洗涤金电极表面的聚合膜5 ~15分钟，除去聚合物膜中的赤霉素A3以及吸附在分子印迹膜上的其它吸附物，制成保留有模板分子构型孔穴的赤霉素A3分子印迹电化学发光传感器； Dissolve 0.009 ~ 0.036g o-phenylenediamine in 10 mL of acetic acid buffer solution with pH=5.2, then add 2×10 -4 ~ 8×10 -4 mol/L gibberellin A3 (GA3) solution, and mix well , using cyclic voltammetry to scan 20 to 40 cycles, the scanning range is -0.2 to 1.0V, and the scanning speed is 50 mV/s; after the polymerization is completed, the gold electrode is taken out, rinsed with double distilled water, and placed in a volume ratio of 7 ~ In a 9:1 anhydrous methanol-glacial acetic acid system, wash the polymer film on the surface of the gold electrode for 5 to 15 minutes under stirring to remove gibberellin A3 in the polymer film and other adsorbates adsorbed on the molecularly imprinted film, Making gibberellin A3 molecularly imprinted electrochemiluminescent sensors with template molecular configuration holes;
（3）检测方法： (3) Detection method:
首先将步骤(2)制得的赤霉素A3分子印迹电化学发光传感器置于罗丹明B标记的赤霉素A3的溶液中孵化18分钟，然后取出赤霉素A3分子印迹电化学发光传感器冲洗表面；将孵化完全的赤霉素A3分子印迹电化学发光传感器分别放入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 mol/L的赤霉素A3标准溶液中进行竞争吸附10 ~ 20分钟；最后接入检测体系，检测体系为：10 mL 含1.2×10-3 mol/L鲁米诺的0.05 mol/L Tris-HCl缓冲溶液，Tris-HCl缓冲溶液pH=7.8；用MPI-E型电致化学发光分析系统进行电致化学发光扫描，扫描电压-0.3 ~ 0.8 V，扫速为100 mV/s，光电倍增管高压900 V，采样速率10 T/S，放大倍数4，测量时间90 s；
First, place the gibberellin A3 molecularly imprinted electrochemiluminescent sensor prepared in step (2) in a solution of rhodamine B-labeled gibberellin A3 and incubate for 18 minutes, then take out the gibberellin A3 molecularly imprinted electrochemiluminescent sensor and rinse Surface; put the fully incubated gibberellin A3 molecularly imprinted electrochemiluminescent sensor into 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 mol/L gibberellin A3 Perform competitive adsorption in the standard solution for 10 to 20 minutes; finally connect to the detection system, the detection system is: 10 mL of 0.05 mol/L Tris-HCl buffer solution containing 1.2×10 -3 mol/L luminol, Tris-HCl buffer The pH of the solution was 7.8; the electrochemiluminescence scanning was carried out with an MPI-E electrochemiluminescence analysis system, the scanning voltage was -0.3 ~ 0.8 V, the scanning speed was 100 mV/s, the high voltage of the photomultiplier tube was 900 V, and the sampling rate was 10 T /S,
（4）标准工作曲线的绘制： (4) Drawing of standard working curve:
在15 mL小烧杯中加入10 mL含1.2×10-3 mol/L鲁米诺的0.05 mol/L Tris-HCl缓冲溶液，Tris-HCl缓冲溶液pH=7.8；将已在罗丹明B标记赤霉素A3溶液中孵化完全的赤霉素A3分子印迹电化学发光传感器浸入10 mL赤霉素A3标准溶液中竞争吸附15分钟，进行电致化学发光检测；赤霉素A3在1.0×10-11 ~ 3.0×10-9 mol/L浓度范围内与电化学发光强度减少值 ?I F 呈良好的线性关系，线性方程分别为：?I F = 11.51 C (10-11 mol/L) + 134.44，线性相关系数r =0.999； Add 10 mL of 0.05 mol/L Tris-HCl buffer solution containing 1.2×10 -3 mol/L luminol to a 15 mL small beaker, the pH of Tris-HCl buffer solution is 7.8; The fully incubated gibberellin A3 molecularly imprinted electrochemiluminescence sensor was immersed in 10 mL of gibberellin A3 standard solution for 15 minutes to perform electrochemiluminescence detection; Within the concentration range of 3.0×10 -9 mol/L, there is a good linear relationship with the reduction value of electrochemiluminescence intensity ? IF , and the linear equations are: ? IF = 11.51 C (10 -11 mol/L) + 134.44, linear Correlation coefficient r = 0.999;
（5）样品中赤霉素A3含量的测定： (5) Determination of gibberellin A3 content in samples:
在15 mL小烧杯中加入10 mL含1.2×10-3 mol/L鲁米诺的0.05 mol/L Tris-HCl缓冲溶液，Tris-HCl缓冲溶液pH=7.8；将在罗丹明B标记赤霉素A3溶液中孵化完全的赤霉素A3分子印迹电化学发光传感器浸入10 mL赤霉素A3溶液中竞争吸附15分钟；利用电致化学发光分析系统对待测液进行电致化学发光扫描，扫描电压扫描电压-0.3 ~ 0.8 V，扫速为100 mV/s，光电倍增管高压900 V，采样速率10 T/S，放大倍数4，测量时间90 s，得到电化学发光强度I F ；根据校正曲线计算出赤霉素A3的浓度C；
Add 10 mL of 0.05 mol/L Tris-HCl buffer solution containing 1.2×10 -3 mol/L luminol to a 15 mL small beaker, the pH of the Tris-HCl buffer solution is 7.8; The fully incubated gibberellin A3 molecularly imprinted electrochemiluminescence sensor was immersed in 10 mL of gibberellin A3 solution for 15 minutes to compete for adsorption; the electrochemiluminescence analysis system was used to scan the solution to be tested by electrochemiluminescence, and the scanning voltage was scanned. Voltage -0.3 ~ 0.8 V,
本发明克服了已有技术在检测时存在过于方法繁琐，步骤复杂等诸多缺点，提高了灵敏度和选择性，对于低浓度的赤霉素A3的检测易于自动化。 The invention overcomes many shortcomings of the prior art such as cumbersome methods and complicated steps in detection, improves sensitivity and selectivity, and is easy to automate the detection of low-concentration gibberellin A3.
附图说明 Description of drawings
图1为本发明实施例金电极上赤霉素A3分子印迹膜在不同的条件下的电化学发光图。 FIG. 1 is an electrochemiluminescence diagram of a gibberellin A3 molecularly imprinted membrane on a gold electrode in an embodiment of the present invention under different conditions.
其中a.裸金电极; b.去除模板分子的赤霉素A3分子印迹电化学发光传感器在鲁米诺底液中; c.重新吸附赤霉素A3后的赤霉素A3分子印迹电化学发光传感器在鲁米诺底液中; d.去除模板分子的赤霉素A3分子印迹电化学发光传感器在含罗丹明B底液中; e.已在赤霉素A3标准溶液中竞争后的赤霉素A3分子印迹电化学发光传感器在不含鲁米诺的底液中; f.在赤霉素A3标准溶液中竞争后的赤霉素A3分子印迹电化学发光传感器在鲁米诺底液中。 Among them a. bare gold electrode; b. gibberellin A3 molecularly imprinted electrochemiluminescence sensor after removal of template molecules in luminol bottom solution; c. gibberellin A3 molecularly imprinted electrochemiluminescence after re-adsorption of gibberellin A3 The sensor is in the luminol base solution; d. The gibberellin A3 molecularly imprinted electrochemiluminescence sensor that removes the template molecule is in the rhodamine B-containing base solution; e. The gibberellin has been competed in the gibberellin A3 standard solution Molecularly imprinted ECL sensor for GA3 in the base solution without luminol; f. Molecularly imprinted ECL sensor for GA3 in luminol base solution after competition in GA standard solution.
图2为本发明实施例赤霉素A3含量与电化学发光强度的关系图。 Fig. 2 is a graph showing the relationship between the content of gibberellin A3 and the intensity of electrochemiluminescence in an example of the present invention.
具体实施方式 Detailed ways
实施例:Example:
（1）金电极的处理： (1) Treatment of gold electrodes:
依次用1.0、0.3和0.05μm 氧化铝粉将金电极抛光后，分别在无水乙醇和水中各超声洗涤5分钟，然后在0.5 mol/L H2SO4中于-0.2 ~1.0 V进行循环伏安(CV)电化学处理，直到获得稳定的CV响应。 After the gold electrode was polished with 1.0, 0.3 and 0.05 μm alumina powder in sequence, it was ultrasonically washed in absolute ethanol and water for 5 minutes, and then cyclic voltammetry was performed at -0.2 ~ 1.0 V in 0.5 mol/L H 2 SO 4 (CV) electrochemical treatment until a stable CV response is obtained.
（2）赤霉素A3分子印迹电化学发光传感器的制备： (2) Preparation of gibberellin A3 molecularly imprinted electrochemiluminescence sensor:
先将0.018 g邻苯二胺用10 mLpH=5.2醋酸缓冲液溶解，然后加入5×10-4 mol/L的GA3溶液，充分混合均匀，用循环伏安法扫描30圈，扫描范围-0.2V～1.0V，扫速50 mV/s；聚合完成后将金电极取出，用二次蒸馏水冲洗干净后，放入体积比为8:1的无水甲醇-冰醋酸体系中，在搅拌下洗涤金电极表面的聚合膜10分钟，除去聚合物膜中的赤霉素A3以及吸附在分子印迹膜上的其它吸附物，制成保留有模板分子构型孔穴的赤霉素A3分子印迹电化学发光传感器；
Dissolve 0.018 g o-phenylenediamine in 10 mL pH=5.2 acetic acid buffer solution, then add 5×10 -4 mol/L GA3 solution, mix well, and scan 30 cycles with cyclic voltammetry, the scanning range is -0.2V ～1.0V,
（3）检测方法： (3) Detection method:
首先将步骤（2）制得的赤霉素A3分子印迹电化学传感器置于罗丹明B标记的赤霉素A3的溶液中孵化18分钟，然后取出赤霉素A3分子印迹电化学发光传感器冲洗表面；然后将其分别放入含有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 mol/L的赤霉素A3标准溶液中进行竞争吸附15分钟，最后接入检测体系；检测体系为：10 mL含1.2×10-3 mol/L鲁米诺的0.05 mol/L Tris-HCl缓冲溶液，Tris-HCl缓冲溶液pH=7.8；利用MPI-E型电致化学发光分析系统对待测液进行电致化学发光扫描，扫描电压扫描电压-0.3 V到0.8 V，扫速为100 mV/s，光电倍增管高压900V，采样速率10 T/S，放大倍数4，测量时间90 s； First, place the molecularly imprinted gibberellin A3 electrochemical sensor prepared in step (2) in the rhodamine B-labeled gibberellin A3 solution and incubate for 18 minutes, then take out the gibberellin A3 molecularly imprinted electrochemical luminescence sensor and wash the surface ; and then put them into the 11 , 120×10 -11 , 160×10 -11 , 200×10 -11 , 250×10 -11 , 300×10 -11 mol/L standard solution of gibberellin A3 for 15 minutes for competitive adsorption, and finally into the detection system; the detection system is: 10 mL of 0.05 mol/L Tris-HCl buffer solution containing 1.2×10 -3 mol/L luminol, the pH of Tris-HCl buffer solution is 7.8; The luminescence analysis system performs electrochemiluminescence scanning of the liquid to be tested, the scanning voltage is from -0.3 V to 0.8 V, the scanning speed is 100 mV/s, the high voltage of the photomultiplier tube is 900 V, the sampling rate is 10 T/S, the magnification is 4, and the measurement time 90 s;
（4）标准工作曲线的绘制： (4) Drawing of standard working curve:
在15 mL小烧杯中加入10 mL含1.2×10-3 mol/L鲁米诺的0.05 mol/LTris-HCl缓冲溶液，Tris-HCl缓冲溶液pH=7.8；将已在罗丹明B标记赤霉素A3溶液中孵化完全的赤霉素A3分子印迹电化学发光传感器浸入10 mL赤霉素A3标准溶液中竞争吸附15分钟，进行电化学发光扫描；赤霉素A3在1×10-11 mol/L～3×10-9 mol/L浓度范围内与电化学发光强度减少值?I F 呈良好的线性关系，线性方程为：?I F = 11.51 C (10-11 mol/L) + 134.44，线性相关系数r =0.999； Add 10 mL of 0.05 mol/LT Tris-HCl buffer solution containing 1.2×10 -3 mol/L luminol into a 15 mL small beaker, the pH of Tris-HCl buffer solution is 7.8; The fully incubated gibberellin A3 molecularly imprinted electrochemiluminescence sensor in the A3 solution was immersed in 10 mL gibberellin A3 standard solution for 15 minutes for competitive adsorption, and the electrochemiluminescence scanning was performed; Within the concentration range of ~3×10 -9 mol/L, it has a good linear relationship with the reduction value of electrochemiluminescence intensity ? IF , and the linear equation is: ? IF = 11.51 C (10 -11 mol/L) + 134.44, linear Correlation coefficient r = 0.999;
（5）啤酒样品中赤霉素A3含量的测定：用于样品检测的啤酒购于超市，同时进行用加标回收实验。在15 mL小烧杯中加入含含1.2×10-3 mol/L鲁米诺的0.05 mol/L Tris-HCl缓冲溶（pH=7.8）；将在罗丹明B标记赤霉素A3溶液中孵化完全的赤霉素A3分子印迹电化学发光传感器浸入10 mL赤霉素A3溶液中竞争吸附15分钟；利用电致化学发光分析系统对待测液进行电致化学发光扫描，扫描电压扫描电压-0.3 ~ 0.8 V，扫速为100 mV/s，光电倍增管高压900 V，采样速率10 T/S，放大倍数4，测量时间90 s，得到电化学发光强度I F ；根据校正曲线计算出赤霉素A3的浓度C和回收率，结果如表1： (5) Determination of gibberellin A3 content in beer samples: The beer used for sample detection was purchased from a supermarket, and the recovery experiment with standard addition was carried out at the same time. Add 0.05 mol/L Tris-HCl buffer solution (pH=7.8) containing 1.2×10 -3 mol/L luminol to a 15 mL small beaker; incubate completely in rhodamine B-labeled gibberellin A3 solution The gibberellin A3 molecularly imprinted electrochemiluminescence sensor was immersed in 10 mL gibberellin A3 solution for 15 minutes to compete for adsorption; the electrochemiluminescence analysis system was used to scan the liquid to be tested, and the scanning voltage was -0.3 ~ 0.8 V, the scan rate is 100 mV/s, the high voltage of the photomultiplier tube is 900 V, the sampling rate is 10 T/S, the magnification factor is 4, and the measurement time is 90 s, the electrochemiluminescence intensity I F is obtained; the gibberellin A3 is calculated according to the calibration curve Concentration C and recovery rate, the results are shown in Table 1:
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100858770A CN103163124A (en) | 2013-03-18 | 2013-03-18 | Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100858770A CN103163124A (en) | 2013-03-18 | 2013-03-18 | Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103163124A true CN103163124A (en) | 2013-06-19 |
Family
ID=48586389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100858770A Pending CN103163124A (en) | 2013-03-18 | 2013-03-18 | Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103163124A (en) |
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 |
| 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 |
| CN115236064A (en) * | 2016-01-29 | 2022-10-25 | 梁庆耀 | Detection of organic chemicals |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999037815A1 (en) * | 1998-01-22 | 1999-07-29 | Akzo Nobel N.V. | ISOTHERMAL TRANSCRIPTION BASED ASSAY FOR THE DETECTION AND QUANTIFICATION OF CHEMOKINE RANTES, MIP-1α AND MIP-1$g(b) |
| CN102121903A (en) * | 2010-12-03 | 2011-07-13 | 江南大学 | Sensor for detecting drugs by molecular imprinting (MI)-electrochemiluminescence (ECL) method |
| CN102706927A (en) * | 2012-04-11 | 2012-10-03 | 桂林理工大学 | Method for measuring gibberellin in fruits by aid of molecularly imprinted electrochemically modified electrode |
-
2013
- 2013-03-18 CN CN2013100858770A patent/CN103163124A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999037815A1 (en) * | 1998-01-22 | 1999-07-29 | Akzo Nobel N.V. | ISOTHERMAL TRANSCRIPTION BASED ASSAY FOR THE DETECTION AND QUANTIFICATION OF CHEMOKINE RANTES, MIP-1α AND MIP-1$g(b) |
| CN102121903A (en) * | 2010-12-03 | 2011-07-13 | 江南大学 | Sensor for detecting drugs by molecular imprinting (MI)-electrochemiluminescence (ECL) method |
| CN102706927A (en) * | 2012-04-11 | 2012-10-03 | 桂林理工大学 | Method for measuring gibberellin in fruits by aid of molecularly imprinted electrochemically modified electrode |
Non-Patent Citations (3)
| Title |
|---|
| JIANPING LI, ET AL: "Molecularly Imprinted Electrochemical Luminescence Sensor Based On Signal Amplification for Selective Determination of Trace Gibberellin A3", 《ANALYTICAL CHEMISTRY》 * |
| 云雯等: "基于电化学聚合固定抗体的电化学发光免疫分析法检测人免疫球蛋白", 《分析化学》 * |
| 蒋复阳等: "利用分子印迹传感器选择性测定绿麦隆", 《分析测试学报》 * |
Cited By (7)
| 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 |
| CN115236064A (en) * | 2016-01-29 | 2022-10-25 | 梁庆耀 | Detection of organic chemicals |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ahmadi et al. | Monitoring of Aflatoxin M1 in milk using a novel electrochemical aptasensor based on reduced graphene oxide and gold nanoparticles | |
| Akbar et al. | Electrochemical determination of folic acid: A short review | |
| CN113075269B (en) | An electrochemiluminescence aptasensor for specific detection of chloramphenicol and its preparation method and application | |
| CN103149268B (en) | Method for detection of micro gibberellin A3 using molecularly imprinted electrochemical sensor | |
| CN103163124A (en) | Method for detecting trace amount of gibberellin A3 by molecular imprinting electrochemical luminescence sensor | |
| CN103940872B (en) | Detect preparation method and the application of two kinds of acute leukemia mark electrochemical sensors simultaneously | |
| CN110618185A (en) | Ratiometric electrochemical detection method of ochratoxin A | |
| CN106706748B (en) | Detect the aptamers electrochemica biological sensor and preparation method thereof of malachite green | |
| Li et al. | A molecularly imprinted sensor based on an electrochemiluminescent membrane for ultratrace doxycycline determination | |
| CN102375021A (en) | Electrochemical method employing DNA as probe to detect environmental pollutant | |
| CN108344792B (en) | Method for rapidly detecting total arsenic in water body | |
| CN105738441B (en) | A kind of modified glassy carbon electrode and its preparation method and application | |
| CN106290537A (en) | The method of L-type Tryptophan concentration in detection solution | |
| CN104677962B (en) | Peroxide polydopamine modified electrode and chloramphenicol electrochemical detection method based on electrode | |
| CN104165909B (en) | Biological electrochemical detection method of fumaric acid | |
| CN103033548B (en) | Fabrication method of ACHE biosensor based on composite nano-material | |
| CN102608188A (en) | Preparation method of current mode immunosensor used for detecting pesticide residue | |
| CN113358716A (en) | Histamine electrochemical sensor based on aptamer, preparation method thereof and application thereof in river crab detection | |
| CN105891309B (en) | The method for detecting L-type Tryptophan concentration in solution | |
| CN108776160A (en) | Method that is a kind of while detecting bisphenol-A and bisphenol S | |
| CN116429865A (en) | Electrochemical sensor for sensitively detecting trefoil glycoside and preparation method and application thereof | |
| CN110618176A (en) | Method for electrochemically detecting selenium (IV) by using dendritic nano-gold modified glassy carbon electrode | |
| CN103196898B (en) | Method for determining trace levamisole by bipyridine ruthenium electrochemiluminescence method | |
| CN101975774B (en) | A method for detecting hydrazine hydrate by electrochemiluminescence with ruthenium terpyridyl | |
| CN115575470A (en) | A method for rapid in situ detection of nicotine in tobacco leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130619 |