CN110514716A - For detecting the preparation method of the current type aptamer sensor of pesticide residue - Google Patents
For detecting the preparation method of the current type aptamer sensor of pesticide residue Download PDFInfo
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- G—PHYSICS
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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Abstract
This application provides a kind of for detecting the preparation method of the current type aptamer sensor of pesticide residue, utilize nano material ordered mesopore carbon, titanium dioxide modified glassy carbon electrode, nano material is dissolved as homogeneous suspension liquid by the good biocompatibility of chitosan and dispersibility, effective fixed adaptation body, stablize effective aptamer sensor to form one, cyclic voltammetry is used respectively, differential pulse voltammetry characterizes the assembling of aptamer sensor and detection in kind, the amperometric biosensor prepared has preparation simple, response quickly, high sensitivity, stability is high, selectivity is high, it is easy to operate, it is low in cost, the feature of quick detection convenient for pesticide residue etc..
Description
Technical field
The present invention relates to electrochemica biological sensor technical fields, more particularly, to a kind of for detecting the electricity of pesticide residue
The preparation method of flow pattern aptamer sensor.
Background technique
Organophosphorus pesticide is common insecticidal/acaricidal agent in agricultural production.Chlopyrifos (Chlorpyrifos) is that China is common
One of organophosphorus pesticide suitable for preventing and treating the pest on the crops such as rice, wheat, cotton be substitution high poison, persistent pesticide
Such as the optimal selection of acephatemet, parathion, parathion-methyl, Azodrin and phosphamidon, it is widely used in agricultural and urban health evil
The prevention and treatment of worm.
But chlopyrifos can brain development or function to the mankind under the action of no overt toxicity effect or metabolin generate
It can have an impact, low dosage causes DNA and protein to synthesize specificity inhibition, and subtoxic dose is given in long-term or repetition can be to thin
Born of the same parents, neural axon have an impact, or even cause behavior change.
Therefore, real there is an urgent need to develop Fast Determination of Pesticide Residue technology to ensure quality of residents'life and food safety
Live, the quick detection of existing pesticide residue, effective monitoring pesticide reasonable employment, reduce food because agriculture it is residual it is exceeded caused by food
Poison and economic loss.
Electrochemica biological sensor has response quickly, high sensitivity, selectivity as a kind of novel rapid detection method
The remarkable advantages such as high, easy to operate, low in cost, are widely applied to environmental pollution analyte detection, food safety detection, drug sieve
Choosing, disease detection etc..
In electrochemica biological sensor, using the biological substances such as enzyme, antibody, aptamer, cell as identification substance, this
Fixed amount, orientation and the activity of a little bio-identification substances directly affect the sensitivity and selection of electrochemica biological sensor
Property.In order to improve the sensitivity and selectivity of electrochemica biological sensor, so that bio-identification substance is preferably played its effect, make
Bio-identification substance is effectively secured on sensing interface, and nano material is caused to be widely used: 1) mesoporous carbon Yin Qigao
Specific surface area, orderly porosity, low-density, high electric conductivity, chemical stability, good biocompatibility and by extensive
Concern, frequently as the candidate material of building novel electrochemical sensor；2) nano-titanium dioxide is more active as research at present
One of inorganic nano material, not only have that particle is small, large specific surface area, light absorption is good, surface-active is big, high catalytic efficiency
The advantages that good with dispersion performance, while also having both good bio-compatibility and excellent chemical stability and being widely used in structure
It builds biosensor and prepares new energy materials.Nano-titanium dioxide has been successfully used to the bioactivity such as immobilized enzyme, DNA at present
Substance can be improved the adsorbance of the bioactive substances such as enzyme and stability and enhance its bioactivity；3) chitosan has
Excellent film forming, adsorptivity, gas permeability and permeability has good adsorptivity, stability and good biology after film forming
Compatibility, amino abundant, cellular structure make it be widely used in the fixation of biomolecule and the modification of electrode.
Therefore, how to provide and a kind of prepare that simple, response quickly, high sensitivity, stability is high, selectivity is high, operation is simple
Single, low-cost amperometric biosensor for being used to detect pesticide residue, is that the art technology urgently to be solved is asked
Topic.
Summary of the invention
Being designed to provide for the embodiment of the present invention is a kind of for detecting the current type aptamer sensor of pesticide residue
Preparation method.
In order to solve the above technical problems, technical solution provided by the invention are as follows:
It is a kind of for detecting the preparation method of the current type aptamer sensor of pesticide residue, including next coming in order carry out
Step:
1) glass-carbon electrode polished, cleaned, activated and tested, obtain pretreatment glass-carbon electrode after the completion；
2) ordered mesopore carbon is added in chitosan solution and is mixed to prepare ordered mesopore carbon-chitosan suspension, then
Ordered mesopore carbon-chitosan hanging drop is coated on the surface of step 1) pretreatment glass-carbon electrode obtained, is then done
Dry, ordered mesopore carbon-chitosan suspension becomes ordered mesopore carbon-chitosan layer after drying；
3) nano-titanium dioxide is added in chitosan solution and is mixed to prepare titanium dioxide-chitosan suspension, then
By titanium dioxide-chitosan hanging drop be coated in step 2) it is dry after ordered mesopore carbon-chitosan layer obtained upper surface on,
Then it is dried, titanium dioxide-chitosan suspension becomes titanium dioxide-chitosan layer after drying；
4) the upper table of titanium dioxide-chitosan layer made from step 3) by the adaptation liquid solution drop coating of pesticide to be detected
On face, then it is dried；
5) the semi-finished product sensor that step 4) obtains after dry is immersed in bovine serum albumen solution, then passes semi-finished product
Sensor takes out from bovine serum albumen solution, and the semi-finished product sensor for being then coated with bovine serum albumen solution is dried,
The current type aptamer sensor is made after the completion.
Preferably, step 1) specifically: glass-carbon electrode is polished, is cleaned, is activated and is detected with cyclic voltammetry,
Until the spike potential difference in the cyclic voltammetry curve of glass-carbon electrode is in 64mV~80mV.
Preferably, in step 2), ordered mesopore carbon-chitosan hanging drop that 7 μ L concentration are 0.25mg/mL is coated in step
On the surface of rapid pretreatment glass-carbon electrode 1) obtained, the then naturally dry in 20 DEG C~30 DEG C of air.
Preferably, in step 3), titanium dioxide-chitosan hanging drop that 7 μ L concentration are 0.25mg/mL is coated in step
2) after dry on the upper surface of ordered mesopore carbon-chitosan layer obtained, the then naturally dry in 20 DEG C~30 DEG C of air.
Preferably, in step 4), the adaptation liquid solution drop coating of the pesticide to be detected of 2 μm of ol/L is obtained in step 3)
On titanium dioxide-chitosan layer upper surface, the then naturally dry in 4 DEG C of air.
Preferably, in step 5), the semi-finished product sensor that step 4) obtains after dry is immersed in bovine serum albumen solution,
Then semi-finished product sensor is taken out from bovine serum albumen solution, the semi-finished product for being then coated with bovine serum albumen solution pass
Sensor is put in naturally dry and preservation in 4 DEG C of air.
Preferably, the pesticide is chlopyrifos, and the adaptation liquid solution of the pesticide is the adaptation liquid solution of chlopyrifos.
This application provides a kind of for detecting the preparation method of the current type aptamer sensor of pesticide residue, wherein institute
Cheap with ordered mesopore carbon and nano-titanium dioxide, preparation method is simple, to simplify current type aptamer sensing
The preparation process of device has good sensitivity and stability at the same time, can be used for the inspection of organophosphorus pesticide in actual sample
It surveys, provides new method for the fast slowdown monitoring in scene of food；It is a kind of pesticide template in this chlopyrifos, if replacing other pesticides
Aptamers can be used to the detection of other pesticides；A kind of simple, response quickly, high sensitivity, stability are prepared to provide
High, high, easy to operate, the low-cost amperometric biosensor for being used to detect pesticide residue of selectivity.
Detailed description of the invention
Fig. 1 is the preparation process of the current type aptamer sensor in the application；
Fig. 2 is that current type aptamer sensor cyclic voltammetric during the preparation process in the application characterizes curve (Fig. 2 is
Cyclic voltammogram, lines and following lines above are a figures, and general relatively good a part for selecting lines separated makes
With the application selects the lines of lower half portion in Fig. 2, so only denoting letter in the lines of lower half portion in Fig. 2)；
Fig. 3 is that the current type aptamer sensor in the application detects the differential pulse voltammetry map of chlopyrifos and works bent
Line.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still
It should be appreciated that these descriptions only further illustrate feature and advantage of the invention, rather than to the limit of the claims in the present invention
System.
This application provides a kind of for detecting the preparation method of the current type aptamer sensor of pesticide residue, including with
Under the step of successively carrying out:
1) glass-carbon electrode polished, cleaned, activated and tested, obtain pretreatment glass-carbon electrode after the completion；
2) ordered mesopore carbon is added in chitosan solution and is mixed to prepare ordered mesopore carbon-chitosan suspension, then
Ordered mesopore carbon-chitosan hanging drop is coated on the surface of step 1) pretreatment glass-carbon electrode obtained, is then done
Dry, ordered mesopore carbon-chitosan suspension becomes ordered mesopore carbon-chitosan layer after drying；
3) nano-titanium dioxide is added in chitosan solution and is mixed to prepare titanium dioxide-chitosan suspension, then
By titanium dioxide-chitosan hanging drop be coated in step 2) it is dry after ordered mesopore carbon-chitosan layer obtained upper surface on,
Then it is dried, titanium dioxide-chitosan suspension becomes titanium dioxide-chitosan layer after drying；
4) the upper table of titanium dioxide-chitosan layer made from step 3) by the adaptation liquid solution drop coating of pesticide to be detected
On face, then it is dried；
5) the semi-finished product sensor that step 4) obtains after dry is immersed in bovine serum albumen solution, then passes semi-finished product
Sensor takes out from bovine serum albumen solution, and the semi-finished product sensor for being then coated with bovine serum albumen solution is dried,
The current type aptamer sensor is made after the completion.
In one embodiment of the application, on the basis of the above embodiments, further, step 1) specifically: will
Glass-carbon electrode is polished, is cleaned, activated and is detected with cyclic voltammetry, until in the cyclic voltammetry curve of glass-carbon electrode
Spike potential difference is in 64mV~80mV.
It is on the basis of the above embodiments, further, in step 2), 7 μ L are dense in one embodiment of the application
Ordered mesopore carbon-chitosan hanging drop that degree is 0.25mg/mL is coated in the surface of step 1) pretreatment glass-carbon electrode obtained
On, the then naturally dry in 20 DEG C~30 DEG C of air.
It is on the basis of the above embodiments, further, in step 3), 7 μ L are dense in one embodiment of the application
Titanium dioxide-chitosan hanging drop that degree is 0.25mg/mL is coated in ordered mesopore carbon-chitosan obtained after step 2) drying
On the upper surface of layer, the then naturally dry in 20 DEG C~30 DEG C of air.
In one embodiment of the application, on the basis of the above embodiments, further, in step 4), by 2 μ
The adaptation liquid solution drop coating of the pesticide to be detected of mol/L is on titanium dioxide-chitosan layer upper surface made from step 3),
Then the naturally dry in 4 DEG C of air.
In one embodiment of the application, on the basis of the above embodiments, further, in step 5), by step
4) the semi-finished product sensor obtained after dry immerses in bovine serum albumen solution, then by semi-finished product sensor from bovine serum albumin
Taken out in solution, be then coated with bovine serum albumen solution semi-finished product sensor be put in 4 DEG C of air naturally dry with
And it saves.
In one embodiment of the application, on the basis of the above embodiments, further, the pesticide is to poison with poison
Tick, the adaptation liquid solution of the pesticide are the adaptation liquid solution of chlopyrifos.
Above-mentioned steps 1) specifically: the working electrode is glass-carbon electrode, and the glass-carbon electrode of diameter 3mm is first used polishing cloth
Polishing, then moves into and cleans 5min in ultrasonic water bath, then the glass-carbon electrode after water-bath is moved into the Piranha solution (body of sulfuric acid
Product: volume=7:3 of hydrogen peroxide) in ultrasound 15min, it is then clean with distilled water flushing；
Then glass-carbon electrode is polished to mirror surface on polishing cloth with the alumina powder that partial size is 0.5~0.7 μm, then
Surface contaminants are washed away, then moves into and cleans 3min in ultrasonic water bath, then place into ultrasound 5min in Piranha solution, then
It is clean wash with distilled water, then glass-carbon electrode is polished on polishing cloth with the alumina powder that partial size is 30~50nm again
Then mirror surface washes away surface contaminants, be then cleaned by ultrasonic 5min with ultrapure water again；
Then the HNO of 6mol/L is used respectively3, dehydrated alcohol and deionized water, be respectively cleaned by ultrasonic 5min；
Then by glass-carbon electrode, first drying is activated with cyclic voltammetry again, by cleaned glass-carbon electrode in 0.5mol/L's
H2SO4Cyclic voltammetric (- 1~1V) is carried out in solution and scans several sections, until scanning curve represents the sulphur of electrode close to being completely coincident
Acid activation terminates；
Finally in the KNO of 0.20mol/L3Middle record l × l0-3The K of mol/L3Fe(CN)6The cyclic voltammetric of solution is bent
Line, to test the performance of obtained working electrode, scanning speed 50mV/S, scanning range is -0.2~0.6V, is followed when described
Spike potential difference in ring volt-ampere curve is in 80mV or less and glass-carbon electrode when as close possible to 64mV just belongs to and meets use and want
It asks, the preparation of the current type aptamer sensor of the application can be used for, otherwise to return to above-mentioned steps again to glass carbon
Electrode is pre-processed, until the spike potential difference in the cyclic voltammetry curve of glass-carbon electrode 80mV or less and as close possible to
64mV。
Above-mentioned chitosan solution are as follows: it is in 1.0% acetic acid solution, at room temperature that 0.25g chitosan, which is added to 50mL concentration,
Magnetic agitation makes chitosan be completely dissolved to obtain 0.5% chitosan solution.
In above-mentioned steps, respectively it disperses 0.25mg ordered mesopore carbon and nano-titanium dioxide in that 1.0mL is above-mentioned to be prepared
Chitosan solution in, for ultrasonic disperse 50min to obtain stable dispersion liquid, high degree of dispersion suspension obtained is respectively to have
Sequence mesoporous carbon-chitosan suspension and titanium dioxide-chitosan suspension.
Embodiment 1: the cyclic voltammetric characterization of current type aptamer sensor preparation process
The glass-carbon electrode modified to bare glassy carbon electrode and each step carries out CV electrochemical Characterization, curve as shown in Fig. 2,
Curve a is the CV curve that the glass-carbon electrode pre-processed measures in figure, does not do any processing on glass-carbon electrode at this time, electric conductivity compared with
Difference, the electric current measured are smaller；
(the curve b) in Fig. 2, due to mesoporous when ordered mesopore carbon-chitosan hanging drop is coated in glassy carbon electrode surface
Carbon has satisfactory electrical conductivity, and electric current significantly increases；
(curve c), electric current obviously increase in Fig. 2 when titanium dioxide-chitosan hanging drop is coated on above-mentioned glass-carbon electrode
Greatly, illustrate that nano-titanium dioxide has played good electron channel in bottom liquid (mesoporous carbon-chitosan suspension) and electrode surface
Effect；
Then it is added dropwise after adaptation liquid solution, resistance increases, and electric current reduces, such as figure (curve d), CV curve superiors in Fig. 2
Value reduces；
Glass-carbon electrode is then immersed in the non-specific binding position in bovine serum albumin (BSA) with enclosed-electrode surface
The addition of point, BSA further hinders the transmission of electronics so that the peak value of current-responsive reduce (curve e) in Fig. 2, so far
BSA/Apt/TiO2The preparation of/OMC/GCE current type aptamer sensor is completed.
The current type aptamer sensor prepared is hung on to phosphate buffer solution (PBS, the pH=of 0.1mol/L
7.5) it above, is saved backup at 4 DEG C.
Embodiment 2: the working curve of current type aptamer sensor detection chlopyrifos
As shown in Figure 3A under the experimental condition of optimization, electrode is incubated for 40min in various concentration chlopyrifos standard solution
It is tested afterwards using DPV.
It can be seen that the difference and chlopyrifos concentration of current peak are in good linear relationship in Fig. 3 B, obtain arriving in 0ng/mL
Within the scope of 90ng/mL be in preferable linear dependence, equation of linear regression Y=4.637X+1.7, linear correlation number be
0.994, detection is limited to 0.04ng/mL (S/N=3).
Embodiment 3: current type aptamer sensor performance test
(1) regenerability of current type aptamer sensor
The regenerability of sensor is the important indicator evaluated biosensor and whether have practical value.Aptamers
Specific binding with target substance mainly dissociates the two by the solution of certain density acid, alkali and high ionic strength.
Current type aptamer sensor after being incubated in chlopyrifos pesticides in this experiment is in the glycine-HCI buffer of pH2.8
Successively dissociation five times, the relative deviation of peak point current only has 8.0%, illustrates that the recycling performance of sensor is preferable.
(2) stability of current type aptamer sensor
The current type aptamer sensor prepared is hung on to phosphate buffer solution (PBS, the pH=of 0.1mol/L
7.5) top saves at 4 DEG C, primary every detection in 1 week, and the response current of current type aptamer sensor is still after the two weeks
Keep original 95.3%；The response current of current type aptamer sensor keeps original 90.1% after 4 weeks, illustrates the electricity
Flow pattern aptamer sensor has good stability.
Embodiment 4: the precision and accuracy of current type aptamer sensor
In order to further study the practical value of the current type aptamer sensor, to the TIANZHU XINGNAO Capsuls of three kinds of vegetables into
Row detection, result is as shown in table 1 below, calculates the rate of recovery of addition pesticide, the rate of recovery can be used between 92%-104%
The detection of actual sample.
The precision of the current type aptamer sensor of 1 the application of table preparation and the test result data of accuracy
The method and apparatus of the not detailed description of the present invention are the prior art, are repeated no more.
Principle and implementation of the present invention are described for specific embodiment used herein, above embodiments
Illustrate to be merely used to help understand method and its core concept of the invention.It should be pointed out that for the common skill of the art
, without departing from the principle of the present invention, can be with several improvements and modifications are made to the present invention for art personnel, these change
It is also fallen within the protection scope of the claims of the present invention into modification.
Claims (7)
1. a kind of for detecting the preparation method of the current type aptamer sensor of pesticide residue, which is characterized in that including following
The step of successively carrying out:
1) glass-carbon electrode polished, cleaned, activated and tested, obtain pretreatment glass-carbon electrode after the completion；
2) ordered mesopore carbon is added in chitosan solution and is mixed to prepare ordered mesopore carbon-chitosan suspension, then will had
Sequence mesoporous carbon-chitosan hanging drop is coated on the surface of step 1) pretreatment glass-carbon electrode obtained, is then dried, has
Sequence mesoporous carbon-chitosan suspension becomes ordered mesopore carbon-chitosan layer after drying；
3) nano-titanium dioxide is added in chitosan solution and is mixed to prepare titanium dioxide-chitosan suspension, then by two
Titanium oxide-chitosan hanging drop be coated in step 2) it is dry after ordered mesopore carbon-chitosan layer obtained upper surface on, then
It is dried, titanium dioxide-chitosan suspension becomes titanium dioxide-chitosan layer after drying；
4) titanium dioxide-chitosan layer upper surface made from step 3) by the adaptation liquid solution drop coating of pesticide to be detected
On, then it is dried；
5) the semi-finished product sensor that step 4) obtains after dry is immersed in bovine serum albumen solution, then by semi-finished product sensor
It is taken out from bovine serum albumen solution, the semi-finished product sensor for being then coated with bovine serum albumen solution is dried, and completes
After the current type aptamer sensor is made.
2. preparation method according to claim 1, which is characterized in that step 1) specifically: polished glass-carbon electrode,
Cleaning, activation and detected with cyclic voltammetry, until glass-carbon electrode cyclic voltammetry curve in spike potential difference 64mV~
80mV。
3. preparation method according to claim 1, which is characterized in that be 0.25mg/mL's by 7 μ L concentration in step 2)
Ordered mesopore carbon-chitosan hanging drop is coated on the surface of step 1) pretreatment glass-carbon electrode obtained, then 20 DEG C~
Naturally dry in 30 DEG C of air.
4. preparation method according to claim 1, which is characterized in that be 0.25mg/mL's by 7 μ L concentration in step 3)
Titanium dioxide-chitosan hanging drop be coated in step 2) it is dry after ordered mesopore carbon-chitosan layer obtained upper surface on, so
The naturally dry in 20 DEG C~30 DEG C of air afterwards.
5. preparation method according to claim 1, which is characterized in that in step 4), by the pesticide to be detected of 2 μm of ol/L
Adaptation liquid solution drop coating on titanium dioxide-chitosan layer upper surface made from step 3), then in 4 DEG C of air from
So dry.
6. preparation method according to claim 1, which is characterized in that in step 5), will step 4) it is dry after obtain half
Finished product sensor immerses in bovine serum albumen solution, then takes out semi-finished product sensor from bovine serum albumen solution, then
The semi-finished product sensor for being coated with bovine serum albumen solution is put in naturally dry and preservation in 4 DEG C of air.
7. preparation method described in any one of -6 according to claim 1, which is characterized in that the pesticide is chlopyrifos, institute
The adaptation liquid solution for stating pesticide is the adaptation liquid solution of chlopyrifos.
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| LEI ZHOU等: "Femtomolar sensitivity of bisphenol A photoelectrochemical aptasensor induced by visible light-driven TiO2 nanoparticle-decorated nitrogen-doped graphene", 《J. MATER. CHEM. B》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175959A (en) * | 2020-10-12 | 2021-01-05 | 广东石油化工学院 | Long-acting phosphorus aptamer, aptamer derivative and application thereof |
| CN112175959B (en) * | 2020-10-12 | 2023-06-20 | 广东石油化工学院 | Long-acting phosphorus nucleic acid aptamer, aptamer derivative and application thereof |
| WO2023024205A1 (en) * | 2021-08-25 | 2023-03-02 | 鱼多康(广州)生物科技有限公司 | Photoelectric co-catalysis water purifier for field of aquatic products |
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| CN110514716B (en) | 2022-06-28 |
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