CN106706737B - A kind of ochratoxin A rapid detection method - Google Patents
A kind of ochratoxin A rapid detection method Download PDFInfo
- Publication number
- CN106706737B CN106706737B CN201611132457.3A CN201611132457A CN106706737B CN 106706737 B CN106706737 B CN 106706737B CN 201611132457 A CN201611132457 A CN 201611132457A CN 106706737 B CN106706737 B CN 106706737B
- Authority
- CN
- China
- Prior art keywords
- ochratoxin
- methylene blue
- silicon nano
- aptamer
- nano material
- 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.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of ochratoxin A rapid detection method, comprises the following steps that and load a large amount of methylene blue molecule in micropore silicon nano material；The micropore silicon nano material of methylene blue molecule has been loaded with the aptamer closing of ochratoxin A；After ochratoxin A sample is added, since it specifically binds between aptamer, aptamer falls off from microporous silicon nano-material surface, and methylene blue molecule is released.By the electric signal of screen printing electrode and portable electrochemical detector test supernatant Methylene Blue, its relation curve between ochratoxin A concentration is obtained, to realize the indirect quantification detection of ochratoxin A.On this basis, the signal amplification of enzyme is introduced.The G- tetrad structure that nuclease is formed between selective destruction ochratoxin A and aptamer after being added, ochratoxin A are returned in solution with free state again, are repeated the above process again.The sensitivity of detection can and so on be greatly improved.Sensor building process and detection process in this method do not need expensive instrument and reagent, and easy to operate, sensitivity is very high.
Description
Technical field
The present invention relates to a kind of ochratoxin A rapid detection methods, and in particular to one kind based on micropore silicon nano material,
The ochratoxin A rapid detection method of nuclease cycle signal amplification and screen printing electrode.
Background technique
Ochratoxin (ochratoxins) is a kind of compound generated by a variety of aspergillus and Penicillium notatum, according to its discovery
Sequence is referred to as ochratoxin A (OTA), ochratoxin B (OTB) and ochratoxin C (OTC).It is after aspergillus flavus
The mycotoxin that another causes world's extensive concern after toxin.Its Poisoning is maximum, distribution is most wide, produces poison amount highest, to agriculture
Product pollution it is most heavy, with human health it is most close be OTA.It is a kind of colourless crystallization compound, is dissolved inPolarity
Organic solvent and dilute sodium bicarbonate solution, are slightly soluble in water, have very highChemical stabilityAnd thermal stability.This toxin is by more
Kind is grown on the crops such as grain (wheat, corn, barley, oat, rye, rice and broomcorn millet class etc.), peanut, vegetables (beans)
Aspergillus and mould generate.After being taken in by animal, this toxin mainly encroaches on animal's liver and kidney, causes kidney injury；
A large amount of toxin may also cause the intestinal mucosa inflammation and necrosis of animal, in some instances it may even be possible to generate teratogenesis, carcinogenic, mutagenesis and be immunized
Inhibiting effect.It is very slow in the intracorporal metabolism of people after the OTA of residual in animal body enters human body by food chain, partly decline
Phase is up to more than 30 days, and very big damaging effect can be generated to human health.Therefore, multiple countries have formulated relevant health mark
It is quasi-.For example, Switzerland provides that the allowance of OTA in pig and fowl mixed feed must not exceed 200 μ g/kg and 1000 μ g/kg respectively.Beauty
State is also working out related regulations.Domestic GB 2761-2011 provides that cereal, the allowance of OTA must not surpass in beans and its product
Cross 5 μ g/kg.Currently, the classics of detection OTA and authoritative method mainly have thin-layered chromatography and high performance liquid chromatography.Although this
A little method high sensitivities, accuracy are high, but require complicated, expensive instrument, professional operator, and take a long time.Cause
This, it is necessary to develop new method that is simple, quick, highly sensitive, being suitble to on-site test.
Summary of the invention
Sensor building process and inspection the purpose of the present invention is to provide a kind of OTA rapid detection method, in this method
Survey process does not need expensive instrument and reagent, and easy to operate, sensitivity is very high.The present invention is by technical solution below come real
It is existing:
The present invention also provides a kind of OTA rapid detection methods, comprise the following steps that
(1) a large amount of methylene blue molecule is loaded in micropore silicon nano material；
(2) the micropore silicon nano material for having loaded a large amount of methylene blue molecules is closed by aptamer；
(3) mixed solution of OTA sample and nuclease is added；
(4) by screen printing electrode and portable electrochemical detector, the electric signal of supernatant Methylene Blue is detected,
The quantitative detection of OTA is realized indirectly；The concentration of OTA is proportional to the concentration of methylene blue molecule.
Wherein, step (3) is received from microporous silicon because of specific binding very strong between OTA and aptamer, aptamer
Rice material surface falls off, and methylene blue molecule is released.Nuclease DNase I is added simultaneously, because nuclease can selectively be broken
The G- tetrad structure formed between bad OTA and aptamers, OTA are returned in solution with free state again, are repeated the above process again.
And so on, the sensitivity of detection can be improved.
Meanwhile heretofore described method and step (4) can also use the concentration of blood glucose meter detection glucose, to detect
The concentration of OTA.
Preferably, a kind of OTA rapid detection method, specifically includes following step:
(1) 10mg micropore silicon nano material addition 2mL is contained into 30mg mL-1The Tris-HCl buffer of methylene blue
In (10mM, pH 7.0), ambient temperature overnight slight oscillatory makes methylene blue molecule sufficiently be diffused into the hole of micropore silicon nano material
In；
(2) 20 μ L aptamer solution (200 μM) are added in suspension obtained by step (1), 4 DEG C of gentle agitation 8h
Afterwards, it is centrifuged with Tris-HCl buffer (10mM, pH 7.0) and washing 3 times or more (to remove microporous silicon nano-material surface object
Manage the methylene blue of absorption), finally material is distributed in 2mL Tris-HCl buffer；
(3) in the suspension obtained by 400 μ L steps (2), the mixed liquor of 400 μ L OTA samples and nuclease is added
(10 or more concentration are chosen into 2000nM from 0), after slight oscillatory is incubated for 2h at room temperature, by product at 5000rpm from
Heart 5min；
(4) it takes 50 μ L supernatant drops on screen printing electrode, carries out subsequent electrochemistry inspection with differential pulse voltametry
It surveys, design parameter are as follows: pulse voltage range is-500-0.0mV, impulse amplitude 50mV, pulse width 200ms.
Preferably, the quality of micropore silicon nano material described in step (1) and (2) is 10mg；Methylene blue and nucleic acid are suitable
The concentration of ligand is respectively 30mg mL-1With 200 μM；The buffer is Tris-HCl buffer (10mM, pH 7.0).
Preferably, the concentration that nuclease described in step (3) is DNase I is 50U.
Preferably, electrochemical detection method described in step (4) is differential pulse voltametry, design parameter are as follows: pulse electricity
Pressure range is-500-0.0mV, impulse amplitude 50mV, pulse width 200ms；The body of the supernatant for Electrochemical Detection
Product is 50 μ L.
The present invention is directed to the microporous silicon nanometer materials that the aptamer closing load by OTA has a large amount of methylene blue molecules
Material, using specific binding of the OTA between aptamer and nuclease to the G- tetrad formed after in conjunction with the two
Destruction is released a large amount of methylene blue molecules.After the OTA sample of various concentration is added by detection, system supernatant
The electrical signal intensity of liquid methylene cyan molecule, construct it is a kind of based on screen printing electrode can scene, it is simple, quickly, it is high
The model electrochemical detection platform of Sensitive Detection OTA, detection limit can be down to 0.1nM.The entire building process of sensor is not necessarily to
Expensive reagents, the costs such as the aptamers marked using antibody, signaling molecule are substantially reduced；It is good using micropore silicon nano material
The characteristics of biocompatibility, structure repeatability, high load efficiency, size adjustable and easy functionalization, plays one stage signal amplification
Effect；Cycle signal based on nuclease amplifies strategy, and sample is used repeatedly, and methylene blue is constantly discharged, detection spirit
Sensitivity is further enhanced；In detection pattern use the Electrochemical Detection based on screen printing electrode, electrode it is instant throw, at
This is low, and test performance is very stable, and instrument is also easily miniaturized.Detection method provided in the present invention have it is easy to operate, at low cost,
The features such as high sensitivity；The operator of large-scale instrument and profession is needed not rely in the building and use process of sensor simultaneously
Member can be widely used for the rural area of scarcity of resources and the in situ quantitation detection of developing country.
Specific embodiment
Technical solution of the present invention is described in detail below by specific embodiment, but the scope of the present invention
It is not restricted by the embodiments.
Embodiment 1
A kind of OTA rapid detection method is present embodiments provided, is comprised the following steps that
(1) 2mL is added in 10mg aminated micropore silicon nano material and contains 30mg mL-1The Tris-HCl of methylene blue
In buffer (10mM, pH 7.0), ambient temperature overnight slight oscillatory makes methylene blue molecule sufficiently be diffused into micropore silicon nano material
Hole in；
(2) 20 μ L aptamer solution (200 μM) are added in suspension obtained by step (1), 4 DEG C of gentle agitation 8h
Afterwards, it is centrifuged with Tris-HCl buffer (10mM, pH 7.0) and washing 3 times or more (to remove microporous silicon nano-material surface object
Manage the methylene blue of absorption), finally material is distributed in 2mL Tris-HCl buffer；
(3) in the suspension obtained by 400 μ L steps (2), the mixed liquor of 400 μ L OTA samples and nuclease is added
(10 or more concentration are chosen into 2000nM from 0), after slight oscillatory is incubated for 2h at room temperature, by product at 5000rpm from
Heart 5min；
(4) it takes 50 μ L supernatant drops on screen printing electrode, carries out subsequent electrochemistry inspection with differential pulse voltametry
It surveys, design parameter are as follows: pulse voltage range is-500-0.0mV, impulse amplitude 50mV, pulse width 200ms.
Embodiment 2
A kind of OTA rapid detection method is present embodiments provided, is comprised the following steps that
(1) Tris-HCl that 500mL contains 2.0M glucose is added in the positively charged micropore silicon nano material in the surface 5mg to delay
It rushes in solution (pH 7.5), ambient temperature overnight slight oscillatory is diffused into glucose molecule sufficiently in the hole of micropore silicon nano material；
(2) 20 μ L aptamer solution (200 μM) are added in above-mentioned suspension, after 4 DEG C are gently mixed 8h, are used
Tris-HCl buffer (10mM, pH 7.5) is centrifuged and washing 3 times or more (to remove microporous silicon nano-material surface physical absorption
Methylene blue), material is finally distributed to (C in 2mL Tris-HCl buffer[microporous silicon]≈25mg mL-1)；
(3) in the suspension made from 400 μ L steps (2), the OTA sample and nuclease of 400 μ L various concentrations is added
Mixed liquor (chooses 10 or more concentration from 0) into 3000nM, and slight oscillatory is incubated for 2h at room temperature；After having reacted, by product
5min is centrifuged at 5000rpm；
(4) it takes 5 μ L supernatant drops on blood glucose meter test paper, is detected with the Roche blood glucose meter of commercialization.It may be noted that
, it is not the restriction to invention that the two above embodiments, which are explanation of the invention, without prejudice to essence of the invention
In the case where mind, the present invention can make any type of modification, such as replacement, the micropore silicon nano material of simple signaling molecule
The variation of function base, the variation of nucleic acid aptamer sequence and the variation of final detection pattern on surface all should be in the technology of the present invention sides
Within case protection scope.
Claims (5)
1. a kind of ochratoxin A rapid detection method, which is characterized in that include the following steps:
(1) a large amount of methylene blue molecule is loaded in micropore silicon nano material, and the micro- of a large amount of methylene blue molecules of load is made
Hole silicon nano material；
(2) the micropore silicon nano material that a large amount of methylene blue molecules have been loaded obtained by step (1) is closed as aptamer；
(3) mixed solution of ochratoxin A sample and nuclease is added；
(4) by screen printing electrode and portable electrochemical detector, the electric signal of supernatant Methylene Blue is detected, indirectly
Realize the quantitative detection of ochratoxin A；The concentration of ochratoxin A is proportional to the concentration of methylene blue molecule.
2. a kind of ochratoxin A rapid detection method based on claim 1, which is characterized in that include the following steps:
(1) 10mg micropore silicon nano material addition 2mL is contained into 30mg mL-1The Tris-HCl of the 10mM of methylene blue, pH 7.0
In buffer, ambient temperature overnight slight oscillatory is diffused into methylene blue molecule sufficiently in the hole of micropore silicon nano material；
(2) by 20 μ L, 200 μM of aptamer solution are added in suspension obtained by step (1), after 4 DEG C of gentle agitation 8h, are used
The Tris-HCl buffer by centrifugation of 10mM, pH 7.0 and washing 3 times or more, to remove microporous silicon nano-material surface physical absorption
Methylene blue, finally material is distributed in 2mL Tris-HCl buffer；
(3) in the suspension obtained by 400 μ L steps (2), the mixing of 400 μ L ochratoxin A samples and nuclease is added
Liquid chooses 10 or more concentration from 0 into 2000nM, after slight oscillatory is incubated for 2h at room temperature, by product at 5000rpm from
Heart 5min；
(4) it takes 50 μ L supernatant drops on screen printing electrode, carries out subsequent Electrochemical Detection with differential pulse voltametry, have
Body parameter are as follows: pulse voltage range is-500-0.0mV, impulse amplitude 50mV, pulse width 200ms.
3. ochratoxin A rapid detection method as claimed in claim 2, it is characterised in that: in step (1) and step (2)
The quality of the micropore silicon nano material is 10mg；The concentration of the methylene blue and aptamer is respectively 30mg mL-1With
200μM；The buffer is 10mM, the Tris-HCl buffer of pH 7.0.
4. ochratoxin A rapid detection method as claimed in claim 2, it is characterised in that: nuclease described in step (3)
For DNase I.
5. ochratoxin A rapid detection method as claimed in claim 2, it is characterised in that: electrochemistry described in step (4)
Detection method is differential pulse voltametry, design parameter are as follows: pulse voltage range is-500-0.0mV, impulse amplitude 50mV, arteries and veins
Rush width 200ms；The volume of the supernatant for Electrochemical Detection is 50 μ L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611132457.3A CN106706737B (en) | 2016-12-10 | 2016-12-10 | A kind of ochratoxin A rapid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611132457.3A CN106706737B (en) | 2016-12-10 | 2016-12-10 | A kind of ochratoxin A rapid detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106706737A CN106706737A (en) | 2017-05-24 |
| CN106706737B true CN106706737B (en) | 2019-03-26 |
Family
ID=58936809
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611132457.3A Active CN106706737B (en) | 2016-12-10 | 2016-12-10 | A kind of ochratoxin A rapid detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106706737B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107490609B (en) * | 2017-07-18 | 2019-11-01 | 济南大学 | Acetamiprid aptamers electrochemical sensor based on mesoporous silicon dioxde film |
| CN111024791B (en) * | 2019-12-26 | 2021-01-01 | 中国科学院生态环境研究中心 | Electrochemical sensor and method for detecting ochratoxin A |
| CN112557651A (en) * | 2020-11-09 | 2021-03-26 | 武汉市农业科学院 | Rapid detection method for pathogenic microorganisms with double signal outputs |
| CN114518359A (en) * | 2022-03-08 | 2022-05-20 | 山东理工大学 | Preparation method of G-quadruplet-based dual-mode kanamycin aptamer sensor |
| CN115728363B (en) * | 2022-09-20 | 2023-10-03 | 首都医科大学 | Method for detecting nucleic acid by CRISPR-electrochemistry based on mesoporous material release |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936940A (en) * | 2010-09-03 | 2011-01-05 | 江南大学 | A kind of method of electrochemiluminescence aptamers sensor ochratoxin A |
| CN103424448A (en) * | 2013-07-26 | 2013-12-04 | 江苏大学 | Method for detecting trace ochratoxin A (OTA) by adopting electrochemical aptamer sensor |
| CN103808701A (en) * | 2013-09-18 | 2014-05-21 | 河南省农业科学院 | Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching |
| CN105400789A (en) * | 2015-10-26 | 2016-03-16 | 中国农业科学院北京畜牧兽医研究所 | Method for quantitatively detecting ochratoxin A |
-
2016
- 2016-12-10 CN CN201611132457.3A patent/CN106706737B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936940A (en) * | 2010-09-03 | 2011-01-05 | 江南大学 | A kind of method of electrochemiluminescence aptamers sensor ochratoxin A |
| CN103424448A (en) * | 2013-07-26 | 2013-12-04 | 江苏大学 | Method for detecting trace ochratoxin A (OTA) by adopting electrochemical aptamer sensor |
| CN103808701A (en) * | 2013-09-18 | 2014-05-21 | 河南省农业科学院 | Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching |
| CN105400789A (en) * | 2015-10-26 | 2016-03-16 | 中国农业科学院北京畜牧兽医研究所 | Method for quantitatively detecting ochratoxin A |
Non-Patent Citations (2)
| Title |
|---|
| Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification;Shunbi Xie 等;《Biosensors and Bioelectronics》;20131216;第55卷;第324-329页 |
| 适配体传感器结合便携式血糖仪定量检测赭曲霉毒素A的方法研究;张勇等;《中国农业科技导报》;20160131;第18卷(第1期);第182-188页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106706737A (en) | 2017-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106706737B (en) | A kind of ochratoxin A rapid detection method | |
| Tittlemier et al. | Developments in mycotoxin analysis: an update for 2017-2018 | |
| Cao et al. | Recent advances of molecularly imprinted polymer-based sensors in the detection of food safety hazard factors | |
| Riberi et al. | Development of an electrochemical immunosensor to determine zearalenone in maize using carbon screen printed electrodes modified with multi-walled carbon nanotubes/polyethyleneimine dispersions | |
| Prieto-Simón et al. | Emerging biotools for assessment of mycotoxins in the past decade | |
| Zhang et al. | Surface molecularly imprinted polymer capped Mn-doped ZnS quantum dots as a phosphorescent nanosensor for detecting patulin in apple juice | |
| Liu et al. | Recent progress on electrochemical biosensing of aflatoxins: A review | |
| Li et al. | Determination of trace patulin in apple-based food matrices | |
| Mishra et al. | Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor | |
| Arévalo et al. | Citrinin (CIT) determination in rice samples using a micro fluidic electrochemical immunosensor | |
| Hu et al. | Rapid methods for detecting acrylamide in thermally processed foods: A review | |
| Sun et al. | Photoelectrochemical sensor for pentachlorophenol on microfluidic paper-based analytical device based on the molecular imprinting technique | |
| Oracz et al. | New trends in quantification of acrylamide in food products | |
| Hu et al. | Sensitive competitive immunoassay of multiple mycotoxins with non-fouling antigen microarray | |
| CN112280831B (en) | Preparation method and application of electrochemical biosensor based on DNA walker | |
| Perrotta et al. | Development of a very sensitive electrochemical magneto immunosensor for the direct determination of ochratoxin A in red wine | |
| Vinogradova et al. | Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements | |
| CN103424448A (en) | Method for detecting trace ochratoxin A (OTA) by adopting electrochemical aptamer sensor | |
| Chang et al. | Molecularly imprinted polymer-based chemiluminescence array sensor for the detection of proline | |
| Herrera-Chacón et al. | Integrating molecularly imprinted polymer beads in graphite-epoxy electrodes for the voltammetric biosensing of histamine in wines | |
| CN105699650A (en) | Carbofuran bio-barcode immunoassay kit and application thereof | |
| Longobardi et al. | Determination of ochratoxin A in wine by means of immunoaffinity and aminopropyl solid-phase column cleanup and fluorometric detection | |
| CN201184874Y (en) | Molecular engram membrane electrode for detecting mycotoxins | |
| CN112505119A (en) | Electrochemical sensor for detecting aflatoxin B1, detection method and preparation | |
| CN105403612B (en) | A kind of method residual based on plant esterase quick detection agriculture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |