阅读说明：本技术 双信号-双识别印迹电化学传感器及其制备方法和卵清蛋白的检测方法 (Double-signal-double-recognition imprinting electrochemical sensor, preparation method thereof and ovalbumin detection method ) 是由 阚显文 柴绒 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种双信号-双识别印迹电化学传感器及其制备方法和卵清蛋白的检测方法,该制备方法包括：1)在电极载体的表面进行电化学聚合形成聚对氨基苯磺酸膜pABSA以得到pABSA/电极载体；2)在pABSA/电极载体的表面进行电化学聚合形成聚硫堇膜pTH以得到pTH/pABSA/电极载体；3)将pTH/pABSA/电极载体置于混合溶液中进行电化学聚合,然后将电极进行洗脱去除卵清蛋白OVA形成印迹聚合物MIP,以得到MIP/pTH/pABSA/电极载体；其中,混合溶液中含有卵清蛋白OVA、3-噻吩硼酸3-TBA。该双信号-双识别印迹电化学传感器对卵清蛋白的检测具有优异的灵敏度和选择性。(The invention discloses a double-signal-double-recognition imprinting electrochemical sensor, a preparation method thereof and an ovalbumin detection method, wherein the preparation method comprises the following steps: 1) carrying out electrochemical polymerization on the surface of the electrode carrier to form a poly-p-aminobenzene sulfonic acid membrane pABSA so as to obtain pABSA/electrode carrier; 2) performing electrochemical polymerization on the surface of the pABSA/electrode carrier to form a polythionine membrane pTH so as to obtain pTH/pABSA/electrode carrier; 3) putting the pTH/pABSA/electrode carrier into a mixed solution for electrochemical polymerization, and then eluting the electrode to remove ovalbumin OVA to form an imprinted polymer MIP so as to obtain the MIP/pTH/pABSA/electrode carrier; wherein the mixed solution contains ovalbumin OVA and 3-thiopheneboronic acid 3-TBA. The double-signal and double-recognition imprinting electrochemical sensor has excellent sensitivity and selectivity for detecting ovalbumin.)

1. A preparation method of a double-signal and double-recognition imprinting electrochemical sensor is characterized by comprising the following steps:

1) carrying out electrochemical polymerization on the surface of the electrode carrier to form a poly-p-aminobenzene sulfonic acid membrane pABSA so as to obtain pABSA/electrode carrier;

2) performing electrochemical polymerization on the surface of the pABSA/electrode carrier to form a polythionine membrane pTH so as to obtain pTH/pABSA/electrode carrier;

3) placing the pTH/pABSA/electrode carrier in a mixed solution for electrochemical polymerization, and then eluting the electrode to remove ovalbumin OVA to form an imprinted polymer MIP so as to obtain the MIP/pTH/pABSA/electrode carrier;

wherein the mixed solution contains ovalbumin OVA and 3-thiopheneboronic acid 3-TBA.

2. The production method according to claim 1, wherein, in step 1), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the number of scanning circles is 12-18 circles, the polymerization potential is-1.5- +2.5V, and the scanning rate is 80-120 mV/s.

3. The preparation method according to claim 1, wherein, in step 1), the electrochemical polymerization is performed in a PBS solution with pH of 6.5-7.5, and the concentration of sulfanilic acid ABSA is 1 x 10^-4-3×10^-4mol/L。

4. The production method according to claim 1, wherein, in step 2), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the number of scanning circles is 28-32 circles, the polymerization potential is-0.4- +0.4V, and the scanning rate is 80-120 mV/s.

5. The preparation method according to claim 1, wherein in step 2), the electrochemical polymerization is performed in a PBS solution having a pH of 4.5-5.5, and the concentration of thionine TH is 4-6 mmol/L.

6. The production method according to claim 1, wherein, in step 3), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the scanning cycle is 18-22 cycles, the polymerization potential is-0.2- +1.2V, and the scanning rate is 120-130 mV/s.

7. The preparation method according to claim 1, wherein in step 3), the electrochemical polymerization is performed in a PBS solution with pH of 7.5-8.5, the concentration of 3-thiopheneboronic acid 3-TBA is 8-12mmol/L, and the concentration of ovalbumin OVA is 0.3-0.5 mmol/L.

8. The production method according to claim 1, wherein, in step 3), the elution is: soaking the electrode in HCl solution with pH of 0.2-0.4 for 15-25 min;

preferably, after step 1) and step 2), the preparation method further comprises: washing the electrode with deionized water;

preferably, the electrode support is selected from one of a glassy carbon electrode GCE, a carbon paper electrode and a screen printed electrode.

9. A dual-signal-dual-recognition imprinted electrochemical sensor, which is prepared by the preparation method of any one of claims 1 to 8.

10. A detection method of ovalbumin OVA is characterized by comprising the following steps:

1) performing a first incubation of the dual-signal-dual recognition blot electrochemical sensor of claim 9 in a buffer solution;

2) performing secondary incubation on the double-signal and double-recognition imprinting electrochemical sensor in a buffer solution, and then performing electrochemical detection;

wherein the buffer solution of the first incubation contains ovalbumin OVA, and the buffer solution of the second incubation contains ferroceneboronic acid FcBA;

preferably, the first incubation and the second incubation each independently satisfy the following condition: incubating for 10-20 min;

more preferably, the concentration of the ferroceneboronic acid FcBA in the buffer solution of the second incubation is 0.08-0.12 mmol/L;

further preferably, the pH of the buffer solution of the first incubation and the second incubation is 8-9;

still more preferably, the electrochemical detection is performed by using Differential Pulse Voltammetry (DPV), and the voltage is-0.5 to + 0.5V.

Technical Field

The invention relates to an imprinting electrochemical sensor, in particular to a double-signal and double-recognition imprinting electrochemical sensor, a preparation method thereof and an ovalbumin detection method.

Background

Ovalbumin is a high-quality protein and is an important component of eggs. Researches show that the eggs have the food therapy health-care effects of improving the intelligence of human bodies, reducing the content of cholesterol, treating iron-deficiency anemia, detoxifying and resisting cancers and the like. Therefore, it is very important to develop highly selective and efficient analytical methods for identifying and quantifying ovalbumin in biological samples. However, severe matrix interference and low concentrations of complex biological samples provide a significant obstacle to the identification and detection of ovalbumin.

Many techniques for ovalbumin analysis have been developed so far, such as enzyme-linked immunosorbent assay, liquid chromatography, etc., but these methods generally have disadvantages of being expensive, time-consuming, poor in selectivity, low in sensitivity, etc.

Disclosure of Invention

The invention aims to provide a double-signal-double-recognition imprinting electrochemical sensor, a preparation method thereof and an ovalbumin detection method.

In order to achieve the above object, the present invention provides a preparation method of a double-signal and double-recognition imprinted electrochemical sensor, the preparation method comprising:

1) carrying out electrochemical polymerization on the surface of the electrode carrier to form a poly-p-aminobenzene sulfonic acid membrane pABSA so as to obtain pABSA/electrode carrier;

2) performing electrochemical polymerization on the surface of the pABSA/electrode carrier to form a polythionine membrane pTH so as to obtain pTH/pABSA/electrode carrier;

3) putting the pTH/pABSA/electrode carrier into a mixed solution for electrochemical polymerization, and then eluting the electrode to remove ovalbumin OVA to form an imprinted polymer MIP so as to obtain the MIP/pTH/pABSA/electrode carrier;

wherein the mixed solution contains ovalbumin OVA and 3-thiopheneboronic acid 3-TBA.

The invention also provides a double-signal and double-recognition imprinting electrochemical sensor which is prepared by the preparation method.

The invention further provides a detection method of ovalbumin OVA, which comprises the following steps:

1) performing first incubation on the double-signal-double-recognition blotting electrochemical sensor in a buffer solution;

2) performing secondary incubation on the double-signal and double-recognition imprinting electrochemical sensor in a buffer solution, and then performing electrochemical detection;

wherein, the buffer solution of the first incubation contains ovalbumin OVA, and the buffer solution of the second incubation contains ferroceneboronic acid FcBA.

According to the technical scheme, the invention is prepared by electropolymerizing and modifying a poly-p-aminobenzene sulfonic acid membrane pABSA, a poly-thionine membrane pTH and an imprinted polymer (MIP) on the surface of an electrode carrier in sequence; the MIP is mainly poly-3-thiopheneboronic acid, when OVA is eluted, imprinting holes complementary to OVA are formed on the surface of the poly-3-thiopheneboronic acid, and TH in the inner layer generates a TH electric signal through the holes.

MIP/pTH/pABSA/electrode carrier is placed in a buffer solution containing OVA and a buffer solution containing ferroceneboronic acid (FcBA) for continuous incubation, the electric signal of TH is reduced because OVA enters a blot hole to block an electronic channel, and meanwhile OVA combined in the hole has boric acid affinity with FcBA (under neutral or alkaline conditions, a boric acid group can generate a reversible covalent bond with a compound containing cis-diol) to generate an electric signal of FcBA. The electric signals of TH and FcBA are combined to form a double signal, and the boric acid affinity is combined with the molecular imprinting space matching cavity to form double recognition, so that the constructed double-signal-double-recognition imprinting electrochemical sensor can realize high-selectivity and high-sensitivity detection on OVA.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

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 principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the results of selective detection of MIP/pTH/pABSA/GCE in example 1;

FIG. 2A is a DPV graph of MIP/pTH/pABSA/GCE detecting OVA at different concentrations in example 1;

fig. 2B is a linear fit plot of fig. 2A.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a preparation method of a double-signal and double-recognition imprinting electrochemical sensor, which comprises the following steps:

1) carrying out electrochemical polymerization on the surface of the electrode carrier to form a poly-p-aminobenzene sulfonic acid membrane pABSA so as to obtain pABSA/electrode carrier;

2) performing electrochemical polymerization on the surface of the pABSA/electrode carrier to form a polythionine membrane pTH so as to obtain pTH/pABSA/electrode carrier;

3) putting the pTH/pABSA/electrode carrier into a mixed solution for electrochemical polymerization, and then eluting the electrode to remove ovalbumin OVA to form an imprinted polymer MIP so as to obtain the MIP/pTH/pABSA/electrode carrier;

wherein the mixed solution contains ovalbumin OVA and 3-thiopheneboronic acid 3-TBA.

In step 1) of the above preparation method, the conditions of electrochemical polymerization can be selected within a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 1), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the number of scanning circles is 12-18 circles, the polymerization potential is-1.5- +2.5V, and the scanning rate is 80-120 mV/s.

In step 1) of the above preparation method, the reaction system of electrochemical polymerization can be selected in a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 1), the electrochemical polymerization is carried out in a PBS solution with pH of 6.5-7.5, and the concentration of the sulfanilic acid ABSA is 1 x 10^-4-3×10^-4mol/L。

In step 2) of the above preparation method, the conditions of electrochemical polymerization can be selected within a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 2), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the number of scanning circles is 28-32 circles, the polymerization potential is-0.4- +0.4V, and the scanning rate is 80-120 mV/s.

In step 2) of the above preparation method, the reaction system of electrochemical polymerization can be selected in a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 2), the electrochemical polymerization is carried out in a PBS solution with a pH of 4.5-5.5, and the concentration of thionine TH is 4-6 mmol/L.

In step 3) of the above preparation method, the conditions of electrochemical polymerization can be selected within a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 3), the electrochemical polymerization satisfies the following condition: the method is carried out by adopting cyclic voltammetry CV, the scanning cycle is 18-22 cycles, the polymerization potential is-0.2- +1.2V, and the scanning rate is 120-130 mV/s.

In step 3) of the above preparation method, the reaction system of electrochemical polymerization can be selected in a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector have more excellent selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 3), electrochemical polymerization is carried out in a PBS solution with pH of 7.5-8.5, the concentration of 3-thiopheneboronic acid 3-TBA is 8-12mmol/L, and the concentration of ovalbumin OVA is 0.3-0.5 mmol/L.

In step 3) of the above preparation method, the conditions for elution can be selected within a wide range, but in order to make the prepared MIP/pTH/pABSA/electrode vector more excellent in selectivity and sensitivity for the detection of ovalbumin OVA; preferably, in step 3), the elution is: and soaking the electrode in HCl solution with the pH value of 0.2-0.4 for 15-25 min.

On the basis of the above embodiment, in order to further improve the selectivity and sensitivity of the MIP/pTH/pABSA/electrode vector for detecting ovalbumin OVA, it is preferable that the preparation method further includes, after the steps 1) and 2): the electrodes were rinsed by deionized water.

In addition to the above embodiments, the kind of the electrode carrier can be selected from a wide range, but in order to further improve the selectivity and sensitivity of the MIP/pTH/pABSA/electrode carrier for the detection of ovalbumin OVA, it is preferable that the electrode carrier is selected from one of a glassy carbon electrode GCE, a carbon paper electrode, and a screen printing electrode.

The invention also provides a double-signal and double-recognition imprinting electrochemical sensor which is prepared by the preparation method.

The invention further provides a detection method of ovalbumin OVA, which comprises the following steps:

1) performing first incubation on the double-signal-double-recognition blotting electrochemical sensor in a buffer solution;

2) performing secondary incubation on the double-signal and double-recognition imprinting electrochemical sensor in a buffer solution, and then performing electrochemical detection;

wherein, the buffer solution of the first incubation contains ovalbumin OVA, and the buffer solution of the second incubation contains ferroceneboronic acid FcBA.

In the above detection method, the incubation time may be selected within a wide range, but in order to improve the accuracy of the detection result, it is preferable that the first incubation and the second incubation each independently satisfy the following conditions: the incubation time is 10-20 min.

In the above detection method, the concentration of ferroceneboronic acid FcBA in the buffer solution of the second incubation can be selected within a wide range, but in order to improve the accuracy of the detection result, it is preferable that the concentration of ferroceneboronic acid FcBA in the buffer solution of the second incubation is 0.08 to 0.12 mmol/L.

In the above-mentioned detection method, the pH of the buffer solution can be selected within a wide range, but in order to improve the accuracy of the detection result, it is further preferable that the pH of the buffer solution of the first incubation and the second incubation is 8 to 9.

Among the above-mentioned detection methods, the method of electrochemical detection can be selected within a wide range, but in order to improve the accuracy of the detection result, it is preferable that the electrochemical detection is performed using differential pulse voltammetry DPV at a voltage of-0.5 to + 0.5V.

The present invention will be described in detail below by way of examples.