阅读说明：本技术 甲砜霉素分子印迹电化学传感器的应用 (Application of thiamphenicol molecularly imprinted electrochemical sensor ) 是由 杨光明 刘卫 徐世娟 陈显兰 石玲 于 2016-11-02 设计创作，主要内容包括：本发明公开了一种甲砜霉素分子印迹电化学传感器的应用,将分子印迹膜修饰的L型玻碳电极浸入到含有甲砜霉素的样品中,进行识别,之后用水冲洗去非特异性吸附的分子；再以识别后的分子印迹膜修饰的L型玻碳电极为工作电极、饱和甘汞电极为参比电极,铂丝电极为对电极,组成三电极系统,以2cm比色皿为光电检测池,浸入到含有0.1mol L<Sup>-1</Sup>抗坏血酸的pH＝7.0的磷酸盐缓冲液中；将405nm激光照射工作电极表面,AA产生光电流,根据光电流的变化量与甲砜霉素的浓度成正比,得到工作曲线。传感器可快速精准地对肉类样品及饲料样品甲砜霉素检测。(The invention discloses an application of a thiamphenicol molecular imprinting electrochemical sensor, wherein an L-shaped glassy carbon electrode modified by a molecular imprinting film is immersed into a sample containing thiamphenicol for identification, and then water is used for washing to remove non-specifically adsorbed molecules; then, the identified molecular imprinting membrane modified L-shaped glassy carbon electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode to form a three-electrode system, a 2cm cuvette is used as a photoelectric detection cell, and the three-electrode system is immersed into a solution containing 0.1mol L of the identified molecular imprinting membrane modified L-shaped glassy carbon electrode ‑1 Ascorbic acid in phosphate buffer at pH 7.0; and irradiating the surface of the working electrode by 405nm laser, wherein AA generates photocurrent, and a working curve is obtained according to the direct proportion of the variation of the photocurrent to the concentration of thiamphenicol. The sensor can quickly and accurately detect the thiamphenicol in meat samples and feed samples.)

1. The application of the thiamphenicol molecularly imprinted electrochemical sensor is characterized by being implemented according to the following detection steps: immersing the L-shaped glassy carbon electrode modified by the molecularly imprinted membrane into a sample containing thiamphenicol for identification, and then washing with water to remove non-specifically adsorbed molecules; then, the identified molecular imprinting membrane modified L-shaped glassy carbon electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode to form a three-electrode system, a 2cm cuvette is used as a photoelectric detection cell, and the three-electrode system is immersed into a solution containing 0.1mol L of the identified molecular imprinting membrane modified L-shaped glassy carbon electrode^-1Ascorbic acid in phosphate buffer at pH 7.0; irradiating the surface of the working electrode with 405nm laser, wherein AA generates photocurrent, and a working curve is obtained according to the direct proportion of the variation of the photocurrent to the concentration of thiamphenicol; wherein

The preparation method of the molecularly imprinted membrane modified L-shaped glassy carbon electrode comprises the following steps:

Step 1, preparing porous Pt-Pd nano particles;

Step 2, preparing a porous graphene-molybdenum disulfide nano flower-like compound;

Step 3, modifying the L-shaped glassy carbon electrode by using the porous graphene-molybdenum disulfide nano flower-shaped compound, the aminated multi-walled carbon nanotube and the porous Pt-Pd nano particles;

Step 4, preparing the modified L-shaped glassy carbon electrode into a molecularly imprinted modified electrode by using o-phenylenediamine as a functional monomer and thiamphenicol as a template molecule through cyclic voltammetry; and then washing to remove the template molecules in the polymeric membrane to obtain the thiamphenicol molecularly imprinted membrane modified electrode.

2. the application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 1, wherein the preparation of the porous Pt-Pd nanoparticles in step 1 is specifically as follows: mixing cetyl pyridine and Na₂PdCl₄and H₂PtCl₆Adding the mixture into a round-bottom flask according to the volume ratio of 3:1:1-8:1:1 to form uniform dispersion liquid; then, quickly adding a freshly prepared ascorbic acid solution into the solution, wherein the volume ratio of the ascorbic acid solution to the cetylpyridinium solution is 1:25-1:5, dispersing the ascorbic acid solution uniformly by slight earthquake, and placing the round-bottom flask into an oil bath at the temperature of 80-90 ℃ for reacting for 2.5-3.5 h; then, centrifuging the obtained sol, and washing with water for multiple times to obtain dendritic porous Pt-Pd nanoparticles; cetyl pyridine, Na₂PdCl₄、H₂PtCl₆The concentrations of (A) are as follows: 10mol L of^-1。

3. The application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 1, characterized in that the modification in step 3 adopts the following steps: dripping N, N-dimethylformamide dispersed liquid of the porous graphene-molybdenum disulfide nanoflower composite on the surface of an L-shaped glassy carbon electrode, and drying at 80 ℃; then, the N, N-dimethylformamide dispersion liquid containing the aminated multi-walled carbon nanotube and the porous Pt-Pd nanoparticle is dripped on the surface of the electrode, and the drying is continued at 80 ℃.

4. The application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 3,

The concentration of the N, N-dimethylformamide dispersion liquid of the porous graphene-molybdenum disulfide nano flower-like compound is 5mg mL^-1(ii) a The concentration of the aminated multi-walled carbon nanotube in the DMF dispersion liquid containing the aminated multi-walled carbon nanotube and the porous Pt-Pd nano-particles is 0.2mg mu L^-1the concentration of the porous Pt-Pd nanoparticles is 5mg mu L^-1The volume ratio of the N, N-dimethylformamide dispersion liquid of the porous graphene-molybdenum disulfide nanoflower composite to the N, N-dimethylformamide dispersion liquid containing the aminated multiwalled carbon nanotube and the porous Pt-Pd nanoparticle is 1: 1.

5. The application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 1, wherein in the step 4, the modified L-shaped glassy carbon electrode takes o-phenylenediamine as a functional monomer and thiamphenicol as a template molecule, and the molecularly imprinted modified electrode is prepared by cyclic voltammetry; and then washing to remove the template molecules in the polymeric membrane to obtain the thiamphenicol molecularly imprinted membrane modified electrode, which specifically comprises the following steps: immersing the modified L-shaped glassy carbon electrode into acetate buffer solution containing template molecules and functional monomers, wherein the template molecules are thiamphenicol, the functional monomers are o-phenylenediamine, and obtaining the molecularly imprinted modified electrode embedded with the thiamphenicol by cyclic voltammetry, wherein the scanning potential of the cyclic voltammetry is 0mV-1.2mV, the number of scanning cycles is 10, and the scanning speed is 100mV s^-1。

6. The application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 5, wherein the molar concentration ratio of o-phenylenediamine to thiamphenicol in the acetic acid buffer solution is 2:1-8:1, the pH of the acetic acid buffer solution is 5.2, the eluent is a methanol/acetic acid solution with a volume ratio of 9:1, and the elution time is 30 minutes.

7. the application of the thiamphenicol molecularly imprinted electrochemical sensor according to claim 6, characterized in that the sample containing thiamphenicol is prepared by the following method: accurately weighing 1.0000g of a sample containing thiamphenicol, adding 20mL of methanol, and swirling for 15min on a high-speed swirl mixer; subsequently, the resulting suspension was centrifuged at 10000r/min for 10min, and the supernatant was taken out and concentrated in a constant temperature water bath at 40 ℃. When the methanol was completely volatilized, the solution was dissolved in a small amount of phosphate buffer solution having a pH of 7.0, and then filtered through a 0.45 μm organic membrane, and the filtrate was transferred to a 10.0mL volumetric flask and subjected to constant volume.

8. The application of the thiamphenicol molecularly imprinted electrochemical sensor in detecting thiamphenicol in meat samples and feed samples as claimed in claim 7, wherein the linear range of detection of the molecularly imprinted electrochemical sensor on the thiamphenicol is 1.0 x 10^-9-3.5×10^-6mol L^-1regression equation is i_Δ(μA)＝0.4981C(μmol L^-1)+0.5012,(r²0.9912), detection limit of 5.0 × 10^-9mol L^-1。

Technical Field

the invention belongs to the technical field of molecular imprinting, and particularly relates to an application of a thiamphenicol molecular imprinting electrochemical sensor.

Background

Thiamphenicol is a commonly used antibiotic, belongs to a broad-spectrum antibiotic with chloramphenicol and florfenicol, has good antibacterial effect, and is widely applied to human disease treatment and animal food production, and residues are formed. Meanwhile, in the background of the banned use of chloramphenicol in the production of animal-derived foods in countries such as china, canada, the united states and the european union, chloramphenicol is gradually becoming a substitute for chloramphenicol in the production of animal-derived foods. However, excessive intake can have a significant impact on human health. Therefore, countries such as the united states, canada, and china have prohibited their use in the production of animal-derived foods and set maximum residual amounts in food safety management systems. In view of this, the construction of the sensor for electrochemically detecting thiamphenicol has certain research work with practical significance.

In the construction of the molecular imprinting electrochemical sensor, the electropolymerization method is one of the common methods for preparing a Molecular Imprinting Polymer (MIP) modified electrode, realizes the simultaneous completion of the preparation and modification of the MIP film, and has the characteristics of controllable thickness, convenience, simplicity, practicability and the like. It is well known that the imprinted substrate is one of the important factors determining the performance of such electrochemical sensors. Meanwhile, the conductive nano material modified interface can improve the imprinting site number, the conductivity and the catalytic performance of the imprinting film, and the three-dimensional imprinting substrate can obviously improve the performances, is very beneficial to improving the sensitivity of the sensor, and is good for selecting the substrate for preparing the MIP film in situ by electropolymerization. Meanwhile, the detection of the non-electroactive target by combining photoelectric current is also one of the research hotspots of the current molecularly imprinted electrochemical sensor.

Disclosure of Invention

In view of the above, the invention provides a thiamphenicol molecularly imprinted electrochemical sensor, and a preparation method and application thereof, and the invention adopts porous graphene (P-r-GO-MoS)₂) Nanoflower composite and aminated porous carbon Nanotube (NH)₂MWCNTs) and porous Pt-Pd nanoparticles (Pt-Pd NPs) construct a three-dimensional porous imprinting substrate; then, o-phenylenediamine is used as a functional monomer, thiamphenicol is imprinted by cyclic voltammetry, and photocurrent generated by an Ascorbic Acid (AA) electrochemical probe is used as an electric signal to construct an electrochemical sensor for detecting electrochemical detection thiamphenicol. As far as we know, a method for electrochemically detecting thiamphenicol is not reported, and the method for applying the three-dimensional modified electrode to the technical field of molecular imprinting is not reported.

In order to solve the technical problem, the invention discloses a preparation method of a thiamphenicol molecularly imprinted electrochemical sensor, which comprises the following steps:

Step 1, preparing porous Pt-Pd nano particles;

Step 2, preparing a porous graphene-molybdenum disulfide nano flower-like compound;

Step 3, modifying the L-shaped glassy carbon electrode by using the porous graphene-molybdenum disulfide nano flower-shaped compound, the aminated multi-walled carbon nanotube and the porous Pt-Pd nano particles;

Step 4, preparing the modified L-shaped glassy carbon electrode into a molecularly imprinted modified electrode by using o-phenylenediamine as a functional monomer and thiamphenicol as a template molecule through cyclic voltammetry; and then washing to remove the template molecules in the polymeric membrane to obtain the thiamphenicol molecularly imprinted membrane modified electrode.

Further, the preparation of the porous Pt-Pd nanoparticles in step 1 is specifically as follows: mixing cetylpyridinium (HDPC) and Na₂PdCl₄And H₂PtCl₆Adding the mixture into a round-bottom flask according to the volume ratio of 3:1:1-8:1:1 to form uniform dispersion liquid; then, quickly adding a freshly prepared Ascorbic Acid (AA) solution into the solution, wherein the volume ratio of the AA solution to HDPC is 1:25-1:5, dispersing the AA solution uniformly by slight earthquake, and placing the round-bottom flask into an oil bath at the temperature of 80-90 ℃ for reacting for 2.5-3.5 h; then thecentrifuging the obtained sol, and washing with water for multiple times to obtain dendritic porous Pt-Pd nanoparticles; cetyl pyridine, Na₂PdCl₄、H₂PtCl₆The concentrations of (A) are as follows: 10mol L of^-1。

Further, the preparation of the porous graphene-molybdenum disulfide nanoflower composite in the step 2 specifically comprises the following steps:

step 2.1, preparing porous graphene oxide:

KMnO was added under magnetic stirring₄Adding the Graphene Oxide (GO) into the Graphene Oxide (GO) dispersion liquid to react for 12h, wherein KMnO₄the mass ratio of the GO to the GO dispersion liquid is 8:1-15: 1; then, HCl and H are added₂O₂adding into the reaction solution, and continuing to react for 3H, wherein GO dispersion liquid, HCl and H₂O₂The volume ratio of (A) to (B) is 2:1-4: 1; after the reaction is finished, centrifugally separating and washing the obtained product, and drying the product in a vacuum drier at the temperature of 55-60 ℃; preparing a porous graphene oxide dispersion liquid;

Step 2.2, preparing the porous graphene-molybdenum disulfide nanoflower compound:

Will be (NH)₄)₆Mo₇O₂·4H₂Dissolving O and thiourea in the porous graphene solution, transferring the mixed solution into a reaction kettle, and reacting for 12 hours at 210-230 ℃; wherein (NH)₄)₆Mo₇O₂·4H₂The mass ratio of the O to the porous graphene oxide is 4:1-1: 1; the mass ratio of the thiourea to the porous graphene oxide is 50:1-30: 1; after the reaction is finished, centrifugally separating and washing the obtained product, and drying the product in a vacuum drier at the temperature of 55-65 ℃ to prepare the porous graphene-molybdenum disulfide nano flower-like compound (P-r-GO-MoS)₂) (ii) a HCl and H₂O₂The mass percentage concentration of (A) is 36% and 30% respectively.

Further, the modification in step 3 employs the following steps: dropping DMF dispersed liquid of the porous graphene-molybdenum disulfide nanoflower composite onto the surface of an L-shaped GCE electrode, and drying at 80 ℃; then, multi-wall carbon nano-tube (NH) containing amino₂-MWCNTs) with DMF dispersed droplets of porous Pt-Pd nanoparticlesdrying the surface of the electrode at 80 ℃;

The concentration of the N, N-dimethylformamide dispersion liquid of the porous graphene-molybdenum disulfide nano flower-like compound is 5mg mL^-1(ii) a The concentration of the aminated multi-walled carbon nanotube in the DMF dispersion liquid containing the aminated multi-walled carbon nanotube and the porous Pt-Pd nano-particles is 0.2mg mu L^-1The concentration of the porous Pt-Pd nanoparticles is 5mg mu L^-1The volume ratio of the N, N-dimethylformamide dispersion liquid of the porous graphene-molybdenum disulfide nanoflower composite to the DMF dispersion liquid containing the aminated multiwalled carbon nanotube and the porous Pt-Pd nanoparticles is 1: 1.

Further, in the step 4, the modified L-shaped glassy carbon electrode takes o-phenylenediamine as a functional monomer and thiamphenicol as a template molecule, and a molecular imprinting modified electrode is prepared by cyclic voltammetry; and then washing to remove the template molecules in the polymeric membrane to obtain the thiamphenicol molecularly imprinted membrane modified electrode, which specifically comprises the following steps: immersing the modified L-shaped glassy carbon electrode into acetate buffer solution containing template molecules and imprinting monomers, wherein the template molecules are thiamphenicol, the functional monomers are o-phenylenediamine, and obtaining the molecularly imprinted modified electrode embedded with the thiamphenicol by cyclic voltammetry, wherein the scanning potential of the cyclic voltammetry is 0mV-1.2mV, the number of scanning cycles is 10, and the scanning speed is 100mV s^-1。

Further, the molar concentration ratio of o-phenylenediamine to thiamphenicol in the acetic acid buffer solution is 2:1-8:1, the pH value of the acetic acid buffer solution is 5.2, the eluent is a methanol/acetic acid solution with the volume ratio of 9:1, and the elution time is 30 minutes.

the invention also provides the thiamphenicol molecularly imprinted electrochemical sensor obtained by the preparation method.

The invention also provides an application of the thiamphenicol molecularly imprinted electrochemical sensor in detecting thiamphenicol in meat samples and feed samples, which is implemented according to the following detection steps: immersing the L-shaped glassy carbon electrode modified by the molecularly imprinted membrane into a sample containing thiamphenicol for identification, and then washing with water to remove non-specifically adsorbed molecules; then working with the identified molecular engram film modified L-shaped glassy carbon electrodeAs an electrode, a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode to form a three-electrode system, and a 2cm cuvette as a photoelectric detection cell immersed in a solution containing 0.1mol L of mercury^-1Ascorbic Acid (AA) in Phosphate Buffered Saline (PBS) at pH 7.0; and irradiating the surface of the working electrode by 405nm laser, wherein AA generates photocurrent, and a working curve is obtained according to the direct proportion of the variation of the photocurrent to the concentration of thiamphenicol.

Further, a sample containing thiamphenicol was prepared by: accurately weighing 1.0000g of a sample containing thiamphenicol, adding 20mL of methanol, and swirling for 15min on a high-speed swirl mixer; subsequently, the resulting suspension was centrifuged at 10000r/min for 10min, and the supernatant was taken out and concentrated in a constant temperature water bath at 40 ℃. When the methanol was completely volatilized, the solution was dissolved in a small amount of PBS (pH 7.0), and then filtered through a 0.45 μm organic membrane, and the filtrate was transferred to a 10.0mL volumetric flask and subjected to constant volume.

Further, the linear range of detection of the molecularly imprinted electrochemical sensor on thiamphenicol is 1.0 multiplied by 10^-9-3.5×10^-6mol L^-1regression equation is i_Δ(μA)＝0.4981C(μmol L^-1)+0.5012,(r²0.9912), detection limit of 5.0 × 10^-9mol L^-1。

Compared with the prior art, the invention can obtain the following technical effects:

1) The sensor has good response to thiamphenicol, and the linear range of the sensor is 1.0 multiplied by 10^-9-3.5×10^-6mol L^-1The lower detection limit is 5.0 × 10^-9mol L^-1。

2) the invention adopts MoS₂、NH₂The MWCNTs and the porous Pt-Pd NPs construct a three-dimensional porous imprinting substrate, and the electrochemical sensor for detecting thiamphenicol is prepared by combining the MIP technology and the photoelectric sensing technology. The three-dimensional porous imprinting substrate has a larger specific surface and a faster mass transfer speed, which is very beneficial to improving the sensitivity of the sensor, and the MIP improves the selectivity of the sensor.

3) The sensor can be used for detecting thiamphenicol in practical samples, particularly for detecting thiamphenicol in meat samples and feed samples, widens the detection channel, and has certain practical significance.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a technical flow chart of the sensor preparation of the present invention. Wherein, A is the preparation process of the imprinted sensor, and B is the electrochemical detection device of the sensor;

FIG. 2 is a representation of a microscopic material according to the present invention; wherein A represents a porous graphene-molybdenum disulfide projection electron microscope image; b represents a high-power projection electron microscope image of the porous graphene; c represents a high-power transmission electron microscope image of the molybdenum disulfide; d represents a projection electron micrograph of the porous Pt-Pd nanoparticles;

FIG. 3 is a microscopic representation of a scanning electron microscope during electrode modification according to the present invention; wherein A represents a scanning electron microscope image of a porous graphene-molybdenum disulfide modified L-glassy carbon electrode (L-GCE), and B represents a porous Pt-Pd-aminated multi-walled carbon nanotube modified interface; c represents a scanning electron microscope image after the molecularly imprinted polymer is polymerized on the modified interface;

FIG. 4 shows that the imprinted sensor and the non-imprinted sensor of the present invention contain 0.1mol L^-1Comparison of photocurrent generated in phosphate buffered saline (PBS, pH 7.0) of Ascorbic Acid (AA); a and b represent photocurrent generated by AA when thiamphenicol (concentration) is not recognized by the imprinted electrode and the non-imprinted electrode respectively; a 'and b' represent the recognition of thiamphenicol by the imprinted electrode and the non-imprinted electrode respectively (1.75 multiplied by 10)^-6mol L^-1) The photocurrent generated by AA;

FIG. 5 shows a different blot sensor of the invention containing 0.1mol L^-1comparison of photocurrent generated in phosphate buffered saline (PBS, pH 7.0) of Ascorbic Acid (AA). a. b, c and d respectively replace the imprinting sensor of the naked L-shaped glassy carbon electrode, porous graphene,The imprinting sensor prepared by using the electrode modified by the porous graphene-molybdenum disulfide and the aminated multi-walled carbon nanotube-porous graphene-molybdenum disulfide contains 0.1mol L of L^-1Photocurrent generated in phosphate buffered saline (PBS, pH 7.0) of Ascorbic Acid (AA); a ', b', c 'and d' respectively represent a print sensor of a bare L-shaped glassy carbon electrode, porous graphene-molybdenum disulfide and an aminated multi-walled carbon nanotube-porous graphene-molybdenum disulfide modified electrode prepared print sensor enriched with 1.75 multiplied by 10^-6mol L^-1After thiamphenicol, in a solution containing 0.1mol L^-1Photocurrent generated in phosphate buffered saline (PBS, pH 7.0) with Ascorbic Acid (AA);

FIG. 6 is a graph of the ratio of functional monomers to template molecules of the present invention as a function of the response current of the sensor;

FIG. 7 is a graph of the aggregate time versus response current of the sensor of the present invention;

FIG. 8 is a graph of enrichment time versus response current of a sensor according to the present invention;

FIG. 9 is a graph of the sensorlinear response of the present invention; the concentration of thiamphenicol is: 1.0X 10^-9-3.5×10^-6mol L^-1The inset is a calibration curve, and the error bars represent standard deviations (n ═ 3);

FIG. 10 is an interference plot of a sensor of the present invention; wherein, a represents a sensing pair of 1.75 × 10^-6mol L^-1Photocurrent response value of thiamphenicol; b. c represents a sensor pair of 1.75X 10, respectively^-6mol L^-1+3.5×10^-5mol L^-1Chloramphenicol and 1.75X 10^-6mol L^-1+3.5×10^-5mol L^-1Photocurrent response values of florfenicol.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.