CN107389923A - A kind of preparation method for exempting from mark type aptamer sensor and the method with the sensor quick detection ochratoxin A - Google Patents
A kind of preparation method for exempting from mark type aptamer sensor and the method with the sensor quick detection ochratoxin A Download PDFInfo
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- CN107389923A CN107389923A CN201710646584.3A CN201710646584A CN107389923A CN 107389923 A CN107389923 A CN 107389923A CN 201710646584 A CN201710646584 A CN 201710646584A CN 107389923 A CN107389923 A CN 107389923A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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Abstract
The invention discloses a kind of preparation method for exempting from mark type aptamer sensor and the method with the sensor quick detection ochratoxin A, belong to electrochemical sensor detection field, to solve the problems, such as existing transducer production method complexity, less stable.Comprise the following steps：Gold electrode is processed by shot blasting；Gold electrode after processing is soaked in the 2 mercaptoethylmaine aqueous solution, AET is self-assembled to gold electrode surfaces with S Au keys and forms decorative layer；Afterwards, gold nano self-assembled film is formed by amino combination gold nano；And then the sulfydryl by being modified in OTA aptamers is attached to gold nano modification layer surface with S Au keys；Then nonactive site is closed using bovine serum albumin, obtains exempting from mark type aptamer sensor.Detection method is simple, quick, and sulfydryl modification aptamers and gold nano stable bond, ensure that the stability of sensor.
Description
Technical field
The invention belongs to electrochemical sensor detection field, and in particular to one kind, which utilizes, exempts from mark type aptamer sensor to reddish brown
The method that aspertoxin A is used for quickly detecting.
Background technology
Ochratoxin is a kind of widely distributed, stronger mycotoxin of toxicity, is produced by several subspecies of mould and aspergillus
Raw secondary metabolite, international cancer research institution (International Agency for Research on
Cancer, IARC) it is set to II B class carcinogenic substances.The World Health Organization, food and agricultural organization food additives joint specialist committee member
The daily tolerance intake of meeting (JECFA, 1995) regulation（TDI）For 14 ng/kg BW.Even part businessman, such as Finland,
Some supermarkets of Britain, all to ochratoxin A（OTA ）Limited the quantity, some are already below 0.5 μ g/kg.It is reddish brown
Pollutions of the aspertoxin A to agricultural product has broad spectrum activity, including corn, rice, soybean, wheat, grape, cocoa, coffee, tune
The crops such as material, vegetables and its product.From a structural point, parts and one and 7- carboxyl base of the OTA by chlorination isocoumarin
The connected L- β of group-phenylalanine composition, therefore, there is inhibitory action to some phenylalanines for the enzyme of substrate.OTA is a kind of line
Plastochondria toxin, injury of mitochondria can be caused, oxygen is quick-fried, lipid peroxidation, and disturbs oxidative phosphorylation.Its hepatotoxicity wind agitation and renal toxicity are made
With lactation liver and nephrotoxicity can be triggered, cancer and teratogenesis, mutagenesis are induced.But because OTA has well
Heat endurance, want to reduce OTA content and infeasible by way of food processing.
Due to traditional OTA detection methods, such as High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD), LC-MS
(LC-MS), the detection of gas chromatography mass spectrometry (GC-MS) generally existing need to rely on the instrument of costliness, there is higher requirement to device operator,
The shortcomings of sample pre-treatments are complicated, and time-consuming, and specificity and sensitivity are poor.Aptamers are a kind of artificial nucleus of chemical synthesis
Acid, there is high specific and compatibility.Compared with antibody, aptamers are more stable, easily obtain, and cost performance is higher.It is similar with antibody,
Aptamers bio-identification mechanism, which is similar to, to be immunized, and the different type biology sensor such as optics, electrochemistry is built with this.These methods
It is simple to operate, it is time-consuming short, there are higher sensitivity and selectivity, so as to be widely used in OTA quick detection.
Electrochemistry has specific good, high sensitivity, the small volume of required sample, miniaturization and an automation, low cost and
The advantages that interference is few.Therefore, electrochemistry has been widely used for the structure of OTA aptamer sensors.Electrochemical method, which is divided into, exempts to mark
Type and marking type detection method, these methods largely use chronoamperometry（voltammetry）And AC impedence method（EIS）.
Although marking type sensor has good test limit, but because detection process needs to introduce the labels such as enzyme, inorganic molecules,
Cause sensor preparation process complicated, less stable, price is also more expensive.Difficulty is fixed and also there are aptamers in existing method,
Some methods need the participation of toxic organic solvents, and the problem of less stable.
The content of the invention
It is an object of the invention to provide a kind of preparation method for exempting from mark type aptamer sensor, to solve existing sensor system
The problem of Preparation Method is complicated, less stable.
It is a further object to provide a kind of method with the sensor quick detection ochratoxin A.
Technical solution of the present invention is as follows：A kind of preparation method for exempting from mark type aptamer sensor, comprises the following steps：
A, gold electrode is processed by shot blasting；
B, gold electrode after processing is soaked in the 2-MEA aqueous solution, AET is self-assembled to gold electrode surfaces shape with S-Au keys
Into decorative layer；Afterwards, gold nano self-assembled film is formed by amino combination gold nano；And then by modifying in OTA aptamers
Sulfydryl is attached to gold nano modification layer surface with S-Au keys；Then nonactive site is closed using bovine serum albumin, is exempted from
Mark type aptamer sensor.
Further, the processing step of the step A gold electrodes is that gold electrode is on polishing flannelette successively with 1.0,0.05
μm Al2O3Polishing, taken out after being then cleaned by ultrasonic 10min with water, be placed in ultrasonic 3min in Piranha solution, going respectively
Ultrasonic 3min in ionized water and absolute ethyl alcohol, then in 0.5 mol/L H in -0.2-1.6V potential ranges2SO4Enter in solution
Row cyclic voltammetry scan, deionized water rinsing and drying.
Further, the detailed process of the step B is：
A, the gold electrode handled well is placed in 5.0 × 10-3In the mol/L mercaptoethylmaine aqueous solution, under the conditions of 4 DEG C of temperature lucifuges
Self assembly is carried out in refrigerator, is taken out after 36 hours, obtains the gold electrode of mercaptoethylmaine modification；After being cleaned by ultrasonic 5min with water
It is standby；
B, step A electrode is soaked 8 hours in 13nm gold nano colloidal sols, flushing obtains AuNPs/AET/Au electrodes；
C, 25 μ L 0.35 μM of sulfydryl modification OTA adaptation liquid solutions are added dropwise in step B the electrode obtaineds surface, are incubated in constant incubator
Educate 90 minutes, with deionized water rinsing electrode surface to remove the aptamers not combined with gold nano after taking-up；
D, μ L of step C the electrode obtaineds surface drop coating 25, the bovine serum albumen solution of content 1%, 60 points are incubated in constant incubator
Clock, deionized water rinsing electrode surface is taken out to remove unnecessary bovine serum albumen solution.
Further, the adaptation liquid solution in the step C is prepared in the buffer solution of pH values 7.4, and it contains 2.7 mM
KCl, 1 mM MgCl2, 140 mM NaCl, 0.1 mM Na2HPO4With 1.8 mM KH2PO4。
With the method for the obtained sensor quick detection ochratoxin A, comprise the following steps：25 μ L OTA are molten
Drop be applied to prepare exempt from mark type aptamer sensor surface, and be incubated 60 minutes in constant incubator, with secondary after taking-up
Water rinses OTA of the surface not with aptamers specific binding.
The principle of the invention is to simplify preparation method, afterwards sulfydryl in gold electrode surfaces first by mercaptoethylmaine self assembly
Ethamine combination gold nano carrys out amplified signal, improves sensitivity, sulfydryl modification aptamers, cow's serum egg are then combined in gold nano
After white closing nonactive site, and then aptamers are specifically bound with OTA, with sensor surface electronics transfer resistance value to OTA
Quantified, finally with AC impedence method quick detection OTA, detection method is simple, quick, and sulfydryl modification aptamers and Jenner
Rice stable bond, ensure that the stability of sensor.
The invention is characterized in that amplify strategy using gold nano as signal, using the sulfydryl modified in OTA aptamers with S-
Au key stable bond gold nanos exempt from mark type aptamer sensor to build, and are realized using overdelicate AC impedence method to detection
OTA quick detection, sensor structure is simple, Stability Analysis of Structures, and detection is quick.Detection process uses hypersensitive electrochemical impedance
Method, without label, the detection method is quick, efficiently, high specific, cost it is low, reach the quick detection of ochratoxin A.
Effect of the present invention is as follows：
1st, the present invention builds sensor using the method for LBL self-assembly, and method is simple, is easily mastered；
2nd, the present invention is that signal amplification is tactful using gold nano, modifies aptamers with sulfydryl, utilizes the good stability of S-Au keys
It ensure that the stability of sensor.
3rd, present invention selection AC impedence method detects the change of electrode surface electric current after aptamers combination OTA, due to trace
Amount OTA combinations aptamers can produce larger signal intensity, so as to which this method obtains higher high sensitivity.
Brief description of the drawings
Fig. 1 is to exempt from mark type aptamer sensor preparation process phenogram；
Fig. 2 is ochratoxin A detection figure.
Embodiment
The following examples can further illustrate the present invention, but be not easy any mode and limit the present invention.
Embodiment 1：Exempt from the preparation of mark type aptamer sensor
1st, the processing method of gold electrode is that gold electrode is on polishing flannelette successively with 1.0,0.05 μm of Al2O3Polishing, then
Taken out after being cleaned by ultrasonic 10min with water, be placed in Piranha solution（The volume of mixture of the concentrated sulfuric acid and 30% hydrogen peroxide ratio is 7:
3）Middle ultrasonic 3min, the respectively ultrasonic 3min in deionized water and absolute ethyl alcohol, then in -0.2-1.6V potential ranges in
0.5 mol/L H2SO4Cyclic voltammetry scan (100mV/s, 80 circles), deionized water rinsing and drying are carried out in solution.
2nd, exempt from being prepared as mark type aptamer sensor, the gold electrode handled well is placed in 5.0 × 10-3Mol/L sulfydryl second
In amine (AET) aqueous solution, self assembly is carried out under the conditions of 4 DEG C of lucifuges in refrigerator, is taken out after 36 hours, obtains mercaptoethylmaine
The gold electrode of modification, with water be cleaned by ultrasonic 5min after it is standby；The sequence of aptamers is as follows：
5’-GATCGGGTGT GGGTGGCGTA AAGGGAGCAT CGGACA-3’, 5’-SH
The electrode of mercaptoethylmaine modification is in 13nm gold nano colloidal sols（AuNPs）Middle immersion 8 hours, flushing obtains AuNPs/AET/
Au electrodes；
25 μ L, 0.35 μM of sulfydryl modification OTA adaptation liquid solution is added dropwise in the electrode obtained surface, is incubated 90 minutes in constant incubator,
With deionized water rinsing electrode surface to remove the aptamers not combined with gold nano after taking-up.
The BSA solution of modified electrode surface 25 μ L of drop coating 1%, it is incubated 60 minutes in constant incubator, takes out deionized water punching
Electrode surface is washed to remove unnecessary BSA.
Embodiment 2：Adaptation liquid solution is prepared in the buffer solution of pH values 7.4, and it contains 2.7 mM KCl, 1 mM
MgCl2, 140 mM NaCl, 0.1 mM Na2HPO4With 1.8 mM KH2PO4。
Embodiment 3：Ochratoxin A standard curve determination
25 μ L OTA solution drop coatings are exempted from into mark type aptamer sensor surface in what is prepared, and 60 points are incubated in constant incubator
Clock, OTA of the surface not with aptamers specific binding is rinsed with secondary water after taking-up.
Using three-electrode system, the mark type aptamer sensor of exempting from for being combined with OTA is working electrode, determines ochratoxin
A standard liquids, Ag/AgCl are reference electrode；Platinum electrode is to electrode, and electronics transfer resistance value is obtained using AC impedence method
（Ret）, OTA and Ret response relation is obtained, and establish standard curve.
Condition of work is specific as follows in detection：Probe solution is the 0.5mol/L K of the KCl containing 0.1mol/L3[Fe(CN)6]/
K4[Fe(CN)6] solution, its pH=7.42, AC impedance uses OCP, sinusoidal voltage 5mV（RMS）Ac potential disturbs,
Frequency range 0.1-104 Hz。
《Effect test》
Fig. 1 is to exempt from mark type aptamer sensor preparation process phenogram；Wherein a:Exempt from the friendship of mark type aptamer sensor preparation process
Flow impedance phenogram：Electronics transfer resistance value increases after modifying AET as seen from the figure, and modifies electronics transfer resistance value after gold nano
Reduce on the contrary, illustrate that gold nano layer forms the more preferably golden face of a new electric conductivity；After being modified in OTA aptamers, due to it
Electronegativity, hinder diffusion of the probe molecule to electrode surface, arc radius increase；It is nonactive that BAS blocks SPCE surfaces completely
Behind site, arc radius further becomes big；OTA is combined with the aptamer of electrode surface causes the increase of electronics transfer resistance value, with
Upper result fully demonstrates each step building process of sensor, it was demonstrated that the sensor successfully constructs.
b:Exempt from mark type aptamer sensor preparation process cyclic voltammetric phenogram：Shown in figure as AET group of molecules is mounted in
Gold electrode surfaces, current reduction.After self assembly gold nano layer is formed, redox peak current increase, it was demonstrated that new gold electrode table
Face is formed.Aptamers self-assembled film forms negative valency decorative layer, and electrostatic barrier, peak are formed between electrode surface and molecular probe
Current reduction.BAS is used to block SPCE surfaces nonactive site completely, and peak current further reduces.OTA and electrode surface it is suitable
Gamete combines, and due to the presence of electronegativity group in OTA structural formulas, and aptamer forms the stranded structures of G- tetra- with OTA and answered
Compound, space bit resistive is big, and peak current reduces.Above results proved that the successful structure of sensor.
《Contrast test》
It is respectively 3.8 ng/ml, 11.4 ng/ml, 20 ng/ml, 40 ng/ml, 80 ng/ml, 120 ng/ by 25 μ L concentration
Ml, 160 ng/ml, 200 ng/ml OTA solution drop coatings exempt from mark type aptamer sensor surface in what is prepared, and are trained in constant temperature
Support case to be incubated 60 minutes, OTA of the surface not with aptamers specific binding is rinsed with secondary water after taking-up.Using three electrode bodies
System, the mark type aptamer sensor of exempting from for being combined with OTA is working electrode, and Ag/AgCl is reference electrode；Platinum electrode is to electricity
Pole, it is measured using AC impedence method, is as a result shown in Fig. 2, Fig. 2 is ochratoxin A detection figure；Wherein a:It is different dense
Spend the impedance diagram of ochratoxin：As illustrated, as the increase of OTA standard sample concentration, arc radius increase, solution electronics turn
Move resistance increase.b:Ochratoxin A standard curve.Data fitting and normalization are carried out to electronics transfer resistance in Fig. 2 a,
Normalization data ratio maps to obtain Fig. 2 b to OTA concentration, has obtained linear when OTA concentration ranges are 3.8-200 ng/ml
Quantitative relationship, detection are limited to 3.8ng/ml.
Claims (5)
1. a kind of preparation method for exempting from mark type aptamer sensor, it is characterised in that comprise the following steps：
(1), gold electrode is processed by shot blasting；
(2), gold electrode after processing is soaked in the 2-MEA aqueous solution, AET is self-assembled to gold electrode surfaces with S-Au keys
Form decorative layer；Afterwards, gold nano self-assembled film is formed by amino combination gold nano；And then by being modified in OTA aptamers
Sulfydryl with S-Au keys be attached to gold nano modification layer surface；Then nonactive site is closed using bovine serum albumin, obtained
Exempt from mark type aptamer sensor.
A kind of 2. preparation method for exempting from mark type aptamer sensor as claimed in claim 1, it is characterised in that：The step
(1) processing step of gold electrode is that gold electrode is on polishing flannelette successively with 1.0,0.05 μm of Al2O3Polishing, then uses water
Taken out after being cleaned by ultrasonic 10min, be placed in ultrasonic 3min in Piranha solution, it is ultrasonic in deionized water and absolute ethyl alcohol respectively
3min, then in 0.5 mol/L H in -0.2-1.6V potential ranges2SO4Cyclic voltammetry scan, deionization are carried out in solution
Water is rinsed and dried.
A kind of 3. preparation method for exempting from mark type aptamer sensor as claimed in claim 1 or 2, it is characterised in that：The step
Suddenly the detailed process of (2) is：
A, the gold electrode handled well is placed in 5.0 × 10-3In the mol/L mercaptoethylmaine aqueous solution, under the conditions of 4 DEG C of temperature lucifuges
Self assembly is carried out in refrigerator, is taken out after 36 hours, obtains the gold electrode of mercaptoethylmaine modification；After being cleaned by ultrasonic 5min with water
It is standby；
B, step A electrode is soaked 8 hours in 13nm gold nano colloidal sols, flushing obtains AuNPs/AET/Au electrodes；
C, 25 μ L 0.35 μM of sulfydryl modification OTA adaptation liquid solutions are added dropwise in step B the electrode obtaineds surface, are incubated in constant incubator
Educate 90 minutes, with deionized water rinsing electrode surface to remove the aptamers not combined with gold nano after taking-up；
D, μ L of step C the electrode obtaineds surface drop coating 25, the bovine serum albumen solution of content 1%, 60 points are incubated in constant incubator
Clock, deionized water rinsing electrode surface is taken out to remove unnecessary bovine serum albumen solution.
A kind of 4. preparation method for exempting from mark type aptamer sensor as claimed in claim 3, it is characterised in that：The step C
In adaptation liquid solution prepared in the buffer solution of pH values 7.4, it contains 2.7 mM KCl, 1 mM MgCl2, 140 mM
NaCl, 0.1 mM Na2HPO4With 1.8 mM KH2PO4。
5. the method for sensor quick detection ochratoxin A made from claim 1, it is characterised in that including following step
Suddenly：25 μ L OTA solution drop coatings are exempted from into mark type aptamer sensor surface in what is prepared, and 60 points are incubated in constant incubator
Clock, OTA of the surface not with aptamers specific binding is rinsed with secondary water after taking-up.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116381018A (en) * | 2023-04-06 | 2023-07-04 | 华北理工大学 | Modified screen printing electrode, ochratoxin aptamer competitive biosensor and detection method |
| CN116953047A (en) * | 2023-06-29 | 2023-10-27 | 甘肃农业大学 | Current type T-2 aptamer sensor and detection method for T-2 toxin |
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| CN1523346A (en) * | 2003-09-03 | 2004-08-25 | 中国科学院长春应用化学研究所 | Nano immunological biosensor |
| CN1525163A (en) * | 2003-09-17 | 2004-09-01 | 中国科学院长春应用化学研究所 | Preparation method of deoxyribonucleic acid electrochemical nanometer sensor |
| 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 |
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2017
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| CN1523346A (en) * | 2003-09-03 | 2004-08-25 | 中国科学院长春应用化学研究所 | Nano immunological biosensor |
| CN1525163A (en) * | 2003-09-17 | 2004-09-01 | 中国科学院长春应用化学研究所 | Preparation method of deoxyribonucleic acid electrochemical nanometer sensor |
| 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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116381018A (en) * | 2023-04-06 | 2023-07-04 | 华北理工大学 | Modified screen printing electrode, ochratoxin aptamer competitive biosensor and detection method |
| CN116953047A (en) * | 2023-06-29 | 2023-10-27 | 甘肃农业大学 | Current type T-2 aptamer sensor and detection method for T-2 toxin |
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