CN101936940A - A kind of method of electrochemiluminescence aptamers sensor ochratoxin A - Google Patents
A kind of method of electrochemiluminescence aptamers sensor ochratoxin A Download PDFInfo
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- CN101936940A CN101936940A CN2010102712479A CN201010271247A CN101936940A CN 101936940 A CN101936940 A CN 101936940A CN 2010102712479 A CN2010102712479 A CN 2010102712479A CN 201010271247 A CN201010271247 A CN 201010271247A CN 101936940 A CN101936940 A CN 101936940A
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Abstract
A kind of method of electrochemiluminescence aptamers sensor ochratoxin A belongs to the electrochemical luminous sensor technical field, is used for wheat, cereal, the detection of ochratoxin A content in feed and the goods thereof.Basis of the present invention is specific recognition and the hydrogen peroxide (H of aptamers to ochratoxin A
2O
2) react with the electrochemiluminescence of different luminol (ABEI) under alkali condition.Nano Au particle is modified naked gold electrode surfaces, the electrochemiluminescence signal is played amplification, single stranded DNA on working electrode surface is modified again, and modify electrode surface by the aptamers that base pairing will be marked with different luminol (ABEI), add hydrogen peroxide (H
2O
2), detecting the electroluminescence signal, electrochemiluminescence signal intensity and ochratoxin A concentration are proportional relatively, and then set up the typical curve of ochratoxin A detection by quantitative.The present invention is intended to set up highly sensitive, workable ochratoxin A quantitative detecting method.
Description
Technical field
The present invention relates to utilize electrochemical luminous sensor that ochratoxin A is detected, characteristics are the application of ochratoxin A aptamers and different luminol electrochemiluminescence label, the effect of nano Au particle amplifying signal in the electrochemiluminescence process, and the high sensitivity of this detection method and ease for operation, belong to the electrochemical luminous sensor technical field.
Background technology
(ochratoxin A, OTA) extensive in distributed in nature, strong toxicity damages greatly human and animals and plants ochratoxin A.OTA mainly is present in cereal, beans and bean product, thousand fruits, coffee, grape and grape wine, fragrant section, oil crops, beer, the tealeaves etc.Toxicologic study shows that ochratoxin A has Toxicity of Kidney, hepatotoxicity, immunotoxicity, and multiple toxicity such as teratogenesis, carcinogenic and mutagenesis, and animal and human's body health is had very big potential hazard.(The International Agency for Research onCancer IARC) has classified OTA as human possibility carcinogenic substance to international cancer research institution in 1993.In view of the OTA strong toxicity, big to human health damage, countries in the world are made OTA limit standard in grain and the feed one after another.As European Union's regulation cereal materials OTA maximum 5 μ g/kg that limit the quantity of, the cereal fabricated product maximum 3 μ g/kg that limit the quantity of, infant and have special medical purpose food to be no more than 0.5 μ g/kg; China's feed OTA maximum 100 μ g/kg that limit the quantity of.
The OTA analyzing detecting method mainly comprises thin layer chromatography (TLC), high pressure liquid chromatography-fluorescence detection (HPLC-FD), capillary electrophoresis technique (CE), high pressure liquid chromatography-mass spectrometry (HPLC-MS), enzyme linked immunosorbent assay (Enzyme-linked immunosorbentassay is called for short ELISA), time resolved fluoro-immunoassay method (TRFIA), colloidal gold immunochromatographimethod analytic approach (GICA) and radioimmunology (RIA) etc. at present.Then use at most with ELISA and GICA method in the on-the-spot screening method fast, these two kinds of methods all are based on the Ag-Ab compatible reaction, with antibody as the identification molecule.But antibody is subject to especially Temperature Influence of external condition, and storage conditions is required harshness, has limited the flexible Application of method to a great extent.In addition, Antibody Preparation need be passed through zoopery or cell experiment, and is loaded down with trivial details time-consuming, the antibody cost height of preparation, and it is also corresponding higher that the method for development detects cost.So be necessary to develop a kind of quick and convenient, good stability, highly sensitive, high specificity, the novel detection method that use cost is low.
The electrochemiluminescence biology sensor is meant then that by biomaterial as sensitive element, electrode is as conversion element, with the sensor of light intensity as the feature detection signal.Biology sensor is meant various biological components (enzyme, antigen, antibody, hormone etc.) or biosome itself (cell, organelle, the tissue etc.) sensor as sensitive element.Aptamer (aptamer) is phyletic evolution (systematicevolution of ligands by exponential enrichment, SELEX) technology screening and come by the index concentration part.Compare with antibody, that aptamer has is easily synthetic, easily modify, easily fixing, can use repeatedly and the advantage of long preservation, thereby aptamer is used widely at aspects such as protein research, drug test, toxin detection, medical diagnosiss for the biology sensor of identification molecule construction.
The present invention is applied to electrochemical luminous sensor to the ochratoxin A aptamers, and on electrode face finish, can strengthen the nano Au particle of light signal, improved the sensitivity of sensor greatly, this sensor is working electrode with the gold electrode, silver electrode is a contrast electrode, platinum electrode is to electrode, with hydrogen peroxide (H
2O
2) light signal that reacts under alkali condition with different luminol (ABEI) is detection signal, by the detection to variable concentrations ochratoxin A standard items, sets up typical curve, reach containing the purpose that the ochratoxin A sample carries out detection by quantitative.This invention can be used for wheat, cereal, the detection of ochratoxin A content in feed and the goods thereof.
The present invention compares with CN 1011699277A (method that a kind of electrochemical sensor detects micro ochratoxin A), different fully on the making of aptamers sensor and detecting pattern, CN 1011699277 A utilize electrochemical method with current value as quantitative signal, and the present invention adopts the electrochemiluminescence detection to obtain higher sensitivity, and detectability can be low to moderate 0.007ng/mL.
Summary of the invention
A kind of method of electrochemiluminescence aptamers sensor ochratoxin A: nano Au particle is modified naked gold electrode surfaces, electrochemical signals is played amplification, the single stranded DNA of sulfhydrylation on working electrode surface is modified in addition, and the aptamers dna modification that will be marked with different luminol (ABEI) by base pairing is assembled into the aptamers electrochemical luminous sensor to electrode surface.Add hydrogen peroxide (H
2O
2), detect luminous signal, when adding the target analytes ochratoxin A, aptamers DNA combines with ochratoxin A generation specificity, make the space conformation of aptamers DNA change, can not mate with complementary single-stranded dna, thereby the aptamers DNA that is marked with different luminol (ABEI) comes off from electrode surface, causes the electrochemiluminescence signal weakening.With this phenomenon the ochratoxin A standard items are detected, set up typical curve, to reach containing the purpose that the ochratoxin A sample carries out detection by quantitative; Step is:
(1) polishing of naked gold electrode: naked gold electrode is carried out grinding process with the alumina powder of 1 μ m, 0.3 μ m, 0.05 μ m successively; Placed aqueous acetone solution then ultrasonic several minutes.The electrode that polishing is good is 0.5mol/L H in concentration
2SO
4In, 0～1.5V (standard calomel electrode) 100mV/s carries out cyclic voltammetry scan until the cyclic voltammogram that obtains reappearing.With containing the PBS solution of the potassium ferricyanide, under-0.2～0.6V (standard calomel electrode) 100mV/s, carry out cyclic voltammetry scan and characterize then, until obtain shape good, the peak-peak difference is less than the cyclic voltammogram of 74mV.
(2) modification of naked gold electrode: naked gold electrode is placed 2mM 1, incubated at room 20h in 3-dimercaptopropane-ethanolic solution, the sulfydryl unimolecular layer is modified electrode surface, after ethanol and ultrapure water cleaning, be placed on 4 ℃ of reaction 10h in the 200 μ L nano-Au solutions, promptly get the electrode of decorated by nano-gold, it is standby that room temperature dries up the back.
(3) aptamers is modified: 2mg ABEI (purchases in Sigma, USA) be dissolved in hydrochloric acid, add the PBS damping fluid that contains 5% glutaraldehyde then, behind the stirring at room reaction 2h, add 4 ℃ of jolting reactions of aptamers DNA (purchasing) 12h, obtain the aptamers DNA of ABEI mark in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(4) preparation of aptamers electrochemical luminous sensor: it is 2.0 * 10 that the electrode that decorated by nano-gold is good places 1.0mL concentration
-6React 4h in the mol/L single stranded DNA,, be placed on 37 ℃ of hybridization reaction 30min of aptamers DNA of 1.0mL ABEI mark again, cleaning electrode with 0.1mol/L PBS buffer solution for cleaning electrode.Promptly be prepared into this electrochemical luminous sensor.
(5) electrochemical luminous sensor to the preparation gained applies cyclic voltammetric and pulse voltage respectively, because ABEI degree of oxidation under pulse voltage is higher, so the galvanochemistry working signal that pulse voltage obtains when detecting is better than cyclic voltammetry, and the electrochemiluminescence signal that obtains under the pulse voltage also than cyclic voltammetry obtain stable, dimension the present invention selects pulse voltage for use.And under pulse voltage, the electrochemiluminescence signal that utilizes the electrode of decorated by nano-gold to detect to obtain uses naked gold electrode to increase 10 times than simple.
(6) the ochratoxin A standard items are detected, set up typical curve: place the ochratoxin A standard solution to react the electrochemical luminous sensor for preparing, standard solution concentration is followed successively by 0ng/mL, 0.02ng/mL, 0.04ng/mL, 0.06ng/mL, 0.08ng/mL, 0.1ng/mL, 0.2ng/mL, 0.8ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 3ng/mL, selection work voltage is 0.8V, and the working buffer system is Na
2CO
3-NaHCO
3(pH11.0), each concentration is reacted 30min respectively, subsequently the H of Jia Ruing
2O
2Concentration is 1.5mM, sets up the border directrix curve according to relative electrochemiluminescence value with corresponding ochratoxin A standard items concentration.
(7) the ochratoxin A sample is detected: place the ochratoxin A testing sample solution to react the electrochemical luminous sensor for preparing, selection work voltage is 0.8V, and the working buffer system is Na
2CO
3-NaHCO
3(pH 11.0), each example reaction 30min, the H of Jia Ruing subsequently
2O
2Concentration is 1.5mM, according to relative electrochemiluminescence value and the concentration of trying to achieve corresponding ochratoxin A from typical curve.
Described nano Au particle particle diameter is about 10nm.
Described aptamers DNA is the single stranded DNA that ochratoxin A is had specific binding capacity.
The amino aptamers DNA of described mark ABEI is by the glutaraldehyde reaction, and the amino of modifying on ABEI and the aptamers is obtained by the coupled action reaction.
Described single stranded DNA is that the nano Au particle of modifying on the electrode combines by golden mercapto key with the DNA of sulfydryl modification with the end complementary pairing of aptamers DNA and with the DNA of sulfydryl modification.
After the described typical curve of setting up the ochratoxin A detection is meant that the ochratoxin A of aptamers electrochemical luminous sensor and variable concentrations is hatched, carry out electrochemiluminescence reaction with hydrogen peroxide under the condition of pulse voltage applying again, the typical curve of doing according to the relative electrochemiluminescence intensity that obtains under the variable concentrations.
Beneficial effect:
(1) the present invention adopts the aptamers of different luminol (ABEI) mark to make sensor, has improved the sensitivity and the stability that detect.
(2) the present invention modifies electrode surface to nano Au particle, has amplified the electrochemiluminescence signal, has further improved its sensitivity.
(3) the present invention adopts electrochemiluminescence as detection means, and is highly sensitive, convenient and swift.
Description of drawings
Fig. 1: based on the electrochemical luminous sensor of aptamers experimental principle figure to the ochratoxin A detection by quantitative.
Fig. 2: to the electrochemiluminescence working signal figure of ochratoxin A detection by quantitative.A. cyclic voltammetric collection of illustrative plates; B. pulse voltage collection of illustrative plates, wherein (a) figure is that gold electrode surfaces has been modified the signal graph that nano Au particle obtains; (b) figure is the signal graph that naked gold electrode obtains.
Fig. 3: ochratoxin A examination criteria curve map.
Fig. 4: the present invention and national standard method detect the correlativity curve that same actual sample obtains.
Embodiment
Embodiment 1: the foundation of ochratoxin A examination criteria curve and wheat actual sample detect
Place the ochratoxin A standard solution to react the electrochemical luminous sensor for preparing, standard solution concentration is followed successively by 0ng/mL, 0.02ng/mL, 0.04ng/mL, 0.06ng/mL, 0.08ng/mL, 0.1ng/mL, 0.2ng/mL, 0.8ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 3ng/mL, selection work voltage is 0.8V, and the working buffer system is Na
2CO
3-NaHCO
3(pH 11.0), each concentration is reacted 30min respectively, subsequently the H of Jia Ruing
2O
2Concentration is 1.5mM, sets up typical curve according to relative electrochemiluminescence value with corresponding ochratoxin A standard items concentration.
Sample pretreatment: the wheat high speed pulverization sieves, take by weighing 20g in the 100mL flask, add 5g NaCl, 80% ethanol water, fully mixing is placed on the homogenizer high speed and stirs extraction 2min, static a moment, filter, get 10mL filtrate and place the 50mL flask, stir in the homogenizer high speed once more and extract 2min, filter with the ultra-fine fibre glass filter paper static back, until the filtrate clarification, collects filtrate for later use.
Buy 20 kinds of different classes of wheats from the local six tame market of farm produces, utilize the inventive method and national standard method to measure the wherein content of ochratoxin A respectively, the results are shown in Table one, the data that obtain are carried out correlativity relatively, P＜0.0001 illustrates this inventive method fast and reliable as a result, and is highly sensitive, good stability is suitable for the detection of ochratoxin A in the wheat actual sample.
Table one: the wheat actual sample detects, the inventive method and national standard method contrast
Annotate: ND is not for detecting.
Embodiment 2: the detection of ochratoxin A and recovery of standard addition experiment in the corn actual sample
The foundation of ochratoxin A examination criteria curve and sample pretreatment are with embodiment 1.
Choose the different corn of 10 kinds from the market of farm produce, utilize the inventive method that its ochratoxin A content is detected earlier and obtain background values, then choose 0.5 μ g/kg, 1.0 μ g/kg, 2.0 μ g/kg, 5.0 μ g/kg, 10 μ g/kg, the OTA standard items of 5 kinds of variable concentrations add in the determinand, utilize the inventive method to detect the wherein content of OTA once more equally, obtain detected value.Recovery %=(detected value-background values)/addition * 100%.From the result of table two, illustrate that the present invention is stable, sensitivity accurately, is suitable for the detection of ochratoxin A in the corn actual sample.
Table two: the detection of ochratoxin A and recovery of standard addition in the corn actual sample
Claims (5)
1. the method for an electrochemiluminescence aptamers sensor ochratoxin A, it is characterized in that: to naked gold electrode polishing, nano Au particle is modified on the naked gold electrode then, then single stranded DNA is modified on the electrode, the aptamers that will be marked with different luminol (ABEI) by base pairing is modified electrode surface at last, be assembled into electrochemical luminous sensor based on aptamers, the ochratoxin A standard items are detected, set up typical curve, to reach to containing the purpose that the ochratoxin A sample carries out detection by quantitative.
2. the method for a kind of electrochemiluminescence aptamers sensor ochratoxin A as claimed in claim 1 is characterized in that the polishing of naked gold electrode: naked gold electrode is carried out grinding process with the alumina powder of 1 μ m, 0.3 μ m, 0.05 μ m successively; Placed aqueous acetone solution then ultrasonic several minutes.
3. the method for a kind of electrochemiluminescence aptamers sensor ochratoxin A as claimed in claim 1, it is characterized in that modifying nano Au particle on the naked gold electrode: naked gold electrode is placed 2mM 1, incubated at room 20h in 3-dimercaptopropane-ethanolic solution, the sulfydryl unimolecular layer is modified electrode surface, after ethanol and ultrapure water cleaning, be placed on 4 ℃ of reaction 10h in the 200 μ L nano-Au solutions, promptly get the electrode of decorated by nano-gold, it is standby that room temperature dries up the back.
4. the method for a kind of electrochemiluminescence aptamers sensor ochratoxin A as claimed in claim 1, it is characterized in that the aptamers dna modification: 2mg ABEI is dissolved in hydrochloric acid, add the PBS damping fluid that contains 5% glutaraldehyde then, behind the stirring at room reaction 2h, add DNA4 ℃ of jolting reaction of aptamers 12h, obtain the aptamers DNA of ABEI mark.
5. the method for an electrochemiluminescence aptamers sensor ochratoxin A utilizes pulse voltage to be electromotive force, optimizes every condition of work: operating voltage is 0.8V, and the working buffer system is Na
2CO
3-NaHCO
3(pH 11.0), the H of adding
2O
2Concentration is 1.5mM, guarantees sensitivity, accuracy and the stability of this method.
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102735740A (en) * | 2012-06-14 | 2012-10-17 | 合肥工业大学 | One-step rapid detection method of ochratoxin A by using electrochemical aptamer sensor |
| CN103512931A (en) * | 2013-07-26 | 2014-01-15 | 江苏大学 | Method for detection of ochratoxin A with ultralow concentration by label-free aptamer sensor |
| CN103592291A (en) * | 2013-10-09 | 2014-02-19 | 青岛科技大学 | Method for measuring abscisic acid based on nano-gold marking and tyramine signal amplifying technology |
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN104017679A (en) * | 2014-05-18 | 2014-09-03 | 中国食品发酵工业研究院 | Method for reducing OTA (ochratixin A) in beer malting process |
| CN104155289A (en) * | 2014-06-25 | 2014-11-19 | 宁波大学 | Solid electrochemical luminescence sensor for detecting mercury ions and preparation method and application of solid electrochemical luminescence sensor for detecting mercury ions |
| CN105039519A (en) * | 2015-06-12 | 2015-11-11 | 青岛科技大学 | Novel DNA detecting method based on station point region hybridization |
| CN105241869A (en) * | 2015-09-29 | 2016-01-13 | 江南大学 | Bisphenol A electrochemiluminescent aptamer sensor based on upper conversion nano material |
| CN105510420A (en) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | Method for determining ATP content on basis of magnetic bead separation and DNA marker gold nanoparticle probe |
| CN106053439A (en) * | 2016-06-01 | 2016-10-26 | 江南大学 | Method for simultaneously detecting oxytetracycline, tetracycline and kanamycin based on ABEI modified flower-shaped nanogold |
| CN106383110A (en) * | 2016-09-09 | 2017-02-08 | 南昌大学 | OTA chemiluminiscence detecting method based on nano-gold label aptasensor |
| CN106442480A (en) * | 2016-09-09 | 2017-02-22 | 南昌大学 | OTA chemiluminescence detection method based on horseradish peroxidase marker aptasensor |
| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN106706737A (en) * | 2016-12-10 | 2017-05-24 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | Rapid detection method for ochratoxin A |
| CN112877406A (en) * | 2021-01-20 | 2021-06-01 | 南京师范大学 | Preparation method and application of organic framework material taking Ce as metal center |
| CN113340881A (en) * | 2021-06-04 | 2021-09-03 | 安徽师范大学 | Target substance and redox dual-response aptamer sensor, preparation method and application thereof, and quantitative detection method of anabaena toxin |
| CN117825685A (en) * | 2024-01-08 | 2024-04-05 | 云南省农业科学院质量标准与检测技术研究所 | Ochratoxin A colloidal gold marker and preparation method and application thereof |
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| CN103512931A (en) * | 2013-07-26 | 2014-01-15 | 江苏大学 | Method for detection of ochratoxin A with ultralow concentration by label-free aptamer sensor |
| CN103512931B (en) * | 2013-07-26 | 2015-10-28 | 江苏大学 | A kind of method exempting from mark aptamers sensing detection super low concentration ochratoxin A |
| CN103592291A (en) * | 2013-10-09 | 2014-02-19 | 青岛科技大学 | Method for measuring abscisic acid based on nano-gold marking and tyramine signal amplifying technology |
| CN104017679A (en) * | 2014-05-18 | 2014-09-03 | 中国食品发酵工业研究院 | Method for reducing OTA (ochratixin A) in beer malting process |
| CN104007152B (en) * | 2014-06-18 | 2016-05-25 | 青岛科技大学 | Measure the method for electrochemical sensor and the mensuration DNA of DNA based on nano platinum particle catalytic electrochemical cycle signal amplifying technique |
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN104155289A (en) * | 2014-06-25 | 2014-11-19 | 宁波大学 | Solid electrochemical luminescence sensor for detecting mercury ions and preparation method and application of solid electrochemical luminescence sensor for detecting mercury ions |
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| CN105039519A (en) * | 2015-06-12 | 2015-11-11 | 青岛科技大学 | Novel DNA detecting method based on station point region hybridization |
| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN105241869A (en) * | 2015-09-29 | 2016-01-13 | 江南大学 | Bisphenol A electrochemiluminescent aptamer sensor based on upper conversion nano material |
| CN105510420A (en) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | Method for determining ATP content on basis of magnetic bead separation and DNA marker gold nanoparticle probe |
| CN105510420B (en) * | 2015-12-20 | 2017-12-12 | 青岛科技大学 | A kind of method based on Beads enrichment and DNA marker gold nanoparticle probe detection ATP contents |
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| CN106442480A (en) * | 2016-09-09 | 2017-02-22 | 南昌大学 | OTA chemiluminescence detection method based on horseradish peroxidase marker aptasensor |
| CN106383110A (en) * | 2016-09-09 | 2017-02-08 | 南昌大学 | OTA chemiluminiscence detecting method based on nano-gold label aptasensor |
| CN106442480B (en) * | 2016-09-09 | 2019-05-24 | 南昌大学 | OTA chemical luminescence detection method based on HRP label aptamer sensor |
| CN106383110B (en) * | 2016-09-09 | 2019-05-24 | 南昌大学 | OTA chemical luminescence detection method based on nano gold mark aptamer sensor |
| CN106706737A (en) * | 2016-12-10 | 2017-05-24 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | Rapid detection method for ochratoxin A |
| CN106706737B (en) * | 2016-12-10 | 2019-03-26 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | A kind of ochratoxin A rapid detection method |
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| CN117825685A (en) * | 2024-01-08 | 2024-04-05 | 云南省农业科学院质量标准与检测技术研究所 | Ochratoxin A colloidal gold marker and preparation method and application thereof |
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