Molecularly imprinted electrochemical sensor as well as preparation method and application thereof

Abstract

The invention discloses a molecularly imprinted electrochemical sensor and a preparation method and application thereof.The sensor comprises an electrode and a pyrrolyl molecularly imprinted polymer film synthesized on the surface of the electrode in situ, and the preparation method comprises the following steps that 1, pyrrolyl compound polymeric monomers, metal nanoparticles and cortisol are added into a PBS buffer solution, mixing to obtain an electro-polymerization solution; (2) preparing a pyrrole-based molecularly imprinted polymer containing a cortisol template on the surface of an electrode by taking the electropolymerization solution as a raw material and adopting a CV electrochemical polymerization method to obtain a molecularly imprinted modified electrode; and (3) removing cortisol template molecules from the molecularly imprinted modified electrode in an eluent, thereby detecting the content of cortisol in a to-be-detected solution. The molecularly imprinted electrochemical sensor disclosed by the invention can be used for detecting the concentration of cortisol, is simple in preparation method and low in cost, has relatively high sensitivity and selectivity to cortisol, and has relatively good practicability and safety.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection of specific substances, and in particular to a molecular imprinting electrochemical sensor with high sensitivity, good selectivity, practicality and safety, and a preparation method and application thereof. Background Art

[0002] Cortisol is a stress hormone secreted by the hypothalamus-pituitary-adrenal (HPA) system. Under normal circumstances, its secretion behavior has strict regularity. When the body is subjected to physical trauma or under great mental stress, it will show obvious changes, which is called "stress hormone". When the body's cortisol is in an abnormal range for a long time, it will cause various diseases, such as depression, cardiovascular disease and metabolic disorders, which directly threaten human physical and mental health. It is widely present in various body fluids of the human body, such as blood, saliva, sweat and urine (the content is at the nM level), and has the characteristics of easy sample acquisition and high detectability.

[0003] The detection method based on electrochemical method is easy to operate and can be used in daily life. Since the redox activity of cortisol molecules themselves is weak, the main detection methods are to capture them on the electrode surface by specific binding methods (such as antibodies, aptamers, molecular imprinting, etc.), thereby realizing indirect detection of cortisol concentration. Compared with antibodies and aptamers, molecular imprinting materials are easy to synthesize, highly stable, and low-cost, and have great application prospects.

[0004] At present, there are few studies on molecular imprinting materials for cortisol. In the existing studies, molecular imprinting materials are mainly poly(o-phenylenediamine). However, o-phenylenediamine is highly toxic, and sensors made of such molecular imprinting materials may pose a great safety hazard. Summary of the invention

[0005] The present invention provides a molecular imprinting electrochemical sensor and a preparation method and application thereof, which are used to solve the technical problems of insufficient selectivity and sensitivity and poor biocompatibility of the existing electrochemical sensors for detecting cortisol concentration.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0007] A molecular imprinting electrochemical sensor comprises an electrode and a molecular imprinting polymer synthesized in situ on the surface of the electrode, wherein the molecular imprinting polymer is a pyrrole-based molecular polymer.

[0008] The design idea of ​​the above technical solution is that when detecting the concentration of cortisol, the inventors found that the selectivity and sensitivity of the existing electrochemical sensors for cortisol are not easy to achieve high-precision detection of trace amounts of cortisol, and there is a problem of poor biocompatibility, so there is a motivation to optimize it. The inventors unexpectedly found that there is a strong intermolecular interaction between cortisol and pyrrole. When pyrrole-based molecular polymers are used as molecular imprinting polymers of electrochemical sensors, the selectivity and sensitivity of the electrochemical sensor to cortisol molecules are significantly improved. At the same time, the molecular imprinting polymers supported by pyrrole-based molecular polymers have better biocompatibility than the existing poly(o-phenylenediamine), so they have better practicality and safety in the application of biosensors.

[0009] Based on the same technical concept, the present invention also provides a method for preparing the above-mentioned molecular imprinting electrochemical sensor, comprising the following steps:

[0010] (1) adding pyrrole compound polymerization monomers, metal nanoparticles and cortisol into PBS buffer and mixing to obtain an electropolymerization solution;

[0011] (2) using the electropolymerization solution as a raw material, preparing a pyrrole-based molecularly imprinted polymer containing a cortisol template on the electrode surface by a CV electrochemical polymerization method to obtain a molecularly imprinted modified electrode;

[0012] (3) The molecular imprinted modified electrode is eluted in an elution solution to obtain the molecular imprinted electrochemical sensor, which can then be used to detect the cortisol content in a test solution.

[0013] As a further preferred embodiment of the above technical solution, the pyrrole compound polymer monomer in step (1) includes one or both of pyrrole and carboxyl pyrrole; and the metal nanoparticles include one or both of gold nanoparticles and silver nanoparticles.

[0014] As a further preferred embodiment of the above technical solution, the concentration of the pyrrole compound polymerization monomer in step (1) is 0.03-0.8 M. When the concentration of the polymer monomer is low, a polymer film cannot be formed on the electrode. When the monomer concentration is too high, the density of the resulting polymer film is too high, so the cortisol therein cannot be eluted, making the sensing electrode ineffective.

[0015] As a further preferred embodiment of the above technical solution, the concentration of cortisol in step (1) is 0.5-5 mM. The concentration of cortisol is determined based on the content of cortisol in the body (the maximum content is several hundred nanomoles per liter). If the concentration of cortisol used in the preparation of the cortisol molecular imprinting polymer is significantly lower than this interval, elution is difficult and requires a lot of time for elution. In addition, the detection signal may not be obtained due to the small number of holes. When the interval is exceeded, excessive holes will be generated, and it is difficult to obtain the phenomenon of current change when detecting cortisol in body fluids.

[0016] As a further preferred embodiment of the above technical solution, the setting parameters of the CV electrochemical polymerization method in step (2) are: 20 to 100 mV / s, potential -1 to 1 V, and 5 to 20 cycles. The scanning speed will determine the compactness of the obtained molecular imprinted film. The faster the scanning speed, the looser the molecular imprinted film, and the slower the scanning speed, the tighter the film. Films that are too loose or too tight cannot achieve a good molecular imprinting effect. The same applies to the number of cycles. The potential is mainly determined by the voltage required for the electropolymerization of pyrrole, and the electropolymerization reaction of pyrrole cannot occur beyond this range.

[0017] As a further preferred embodiment of the above technical solution, the eluent includes at least one of ethanol, sodium hydroxide solution and acetic acid solution.

[0018] As a further preferred embodiment of the above technical solution, the elution time in step (3) is 2 to 15 minutes.

[0019] Based on the same technical concept, the present invention also provides an application of the above molecular imprinting electrochemical sensor or the molecular imprinting electrochemical sensor prepared by the above preparation method, wherein the molecular imprinting electrochemical sensor is used to detect the concentration of cortisol.

[0020] As a further preferred embodiment of the above technical solution, when the molecular imprinting electrochemical sensor is used to detect the concentration of cortisol, the sensor comprises the following steps:

[0021] (1) dropping cortisol standard solutions of different concentrations onto the surface of the molecularly imprinted electrochemical sensor for incubation, so that the cortisol molecules are fully in contact with the molecularly imprinted polymer of the molecularly imprinted electrochemical sensor;

[0022] (2) washing the molecular imprinting electrochemical sensor to remove excess cortisol molecules adsorbed on the surface;

[0023] (3) adding a detection solution containing a certain concentration of electrochemical probe molecules to the molecular imprinting electrochemical sensor, and performing cyclic voltammetry scanning to obtain cyclic voltammetry curves of cortisol standard solutions with different concentrations;

[0024] (4) correlating the peak value in the cyclic voltammetry curve with the concentration of cortisol in the standard solution to obtain a cortisol standard curve;

[0025] (5) Before each test of the sample to be tested, the molecular imprinted electrochemical sensor is eluted, and the sample to be tested is measured according to steps (1) to (3) to obtain a cyclic voltammetry curve, and the peak value in the cyclic voltammetry curve is substituted into the cortisol standard curve to obtain the concentration of cortisol in the sample to be tested.

[0026] As a further preferred embodiment of the above technical solution, the incubation time of the cortisol standard solution or the sample to be tested on the surface of the molecular imprinting electrochemical sensor in step (1) is 2 to 15 minutes, and the drop volume of the cortisol standard solution or the sample to be tested is 50 to 100 μL. The sample volume is considered based on the electrodes used. When the volume is too small, it cannot completely cover the electrodes; when the volume is too large, it will cause a waste of solution. The incubation time in the above range can ensure that cortisol can completely enter the molecular imprinting polymer, thereby producing a detection effect. If the time is too short, detection cannot be achieved, and if the time is too long, a higher time cost will be incurred.

[0027] As a further preferred embodiment of the above technical solution, the electrochemical molecular probe includes one of potassium ferrocyanide, ferrocene and methylene blue.

[0028] As a further preferred embodiment of the above technical solution, the drop volume of the detection solution in step (3) is 50-100 μL; the detection conditions of the cyclic voltammetry scan are: 50-100 mV / s, and the potential is -0.6-0.6 V. The present invention has the following beneficial effects:

[0029] Compared with the prior art, the molecular imprinting electrochemical sensor of the present invention has a simple preparation method, low cost, high sensitivity and selectivity to cortisol, can achieve high-precision detection of cortisol, and has good biocompatibility, and has good practicality and safety in the application of biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the electrochemical sensing principle of the molecular imprinting electrochemical sensor of the present invention.

[0031] Figure 2 Cyclic voltammetry curves of cortisol standard solutions with different concentrations according to the present invention.

[0032] Figure 3 This is the cortisol standard curve of the present invention. DETAILED DESCRIPTION

[0033] The following is a detailed description of the embodiments of the present invention in conjunction with the accompanying drawings, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0034] Embodiment 1:

[0035] The molecular imprinting electrochemical sensor of this embodiment includes an electrode and a molecular imprinting polymer synthesized in situ on the surface of the electrode. The electrode used is a carbon material electrode (in other embodiments, a gold electrode or a glass fiber electrode may also be used), and the molecular imprinting polymer is a pyrrole-based molecular polymer.

[0036] The molecular imprinting electrochemical sensor of this embodiment is prepared by the following method:

[0037] (1) A 0.1 M PBS buffer solution with a pH of 7.4 was prepared, to which 0.3 M carboxypyrrole, 0.5 mM cortisol, 1.5 wt % chitosan, and 0.05 g / L gold nanoparticle dispersion were added to obtain an electropolymerization solution.

[0038] (2) inserting the electrode into the electropolymerization solution in (1) by using a CV electrochemical polymerization method, wherein the setting parameters of the CV electrochemical polymerization method are: 50 mV / s, scanning potential -0.2 to 0.8 V, and 5 cycles, thereby preparing a pyrrole-based molecularly imprinted polymer containing a cortisol template on the electrode surface to obtain a molecularly imprinted modified electrode;

[0039] (3) The prepared molecularly imprinted modified electrode was placed in anhydrous ethanol eluent for elution for 5 min, and then dried with nitrogen to obtain the molecularly imprinted electrochemical sensor of this embodiment.

[0040] The molecular imprinting electrochemical sensor of this embodiment can be used to detect the concentration of cortisol in the sweat sample to be tested. The specific detection method includes the following steps:

[0041] First, determine the cortisol standard curve:

[0042] (1) 50 μL of cortisol standard solution (100 pM-10 μM) was dropped onto the surface of the molecular imprinting electrochemical sensor and incubated for 15 min to allow the cortisol molecules in the solution to fully bind to the molecular imprinting polymer. The electrochemical sensing principle diagram of the molecular imprinting electrochemical sensor is shown in the figure. Figure 1 shown.

[0043] (2) The molecularly imprinted electrochemical sensor was rinsed with a large amount of deionized water and blown dry with nitrogen gas to remove the cortisol physically adsorbed on the surface.

[0044] (3) Add 50 μL of 5 mM potassium ferrocyanide solution onto the surface of the molecular imprinting electrochemical sensor and perform cyclic voltammetry scanning to obtain a cyclic voltammetry curve, such as Figure 2 The experimental conditions of cyclic voltammetry are: 50mV / s, potential -0.6~0.6V.

[0045] (4) Correlate the peak value in the cyclic voltammetry curve with the cortisol concentration in the added standard solution to obtain a cortisol standard curve. The result is as follows: Figure 3 As shown, it can be seen that the prepared material has the following linear relationship between logarithmic concentration and cyclic voltammetry peak current in the range of 1nM to 10μM:

[0046] I = 3.65 × log 10 (c cortisol (nM))-54.58,R 2 =0.99

[0047] Then test the concentration of cortisol in the sample to be tested:

[0048] (5) The molecular imprinting electrochemical sensor of this embodiment was placed in anhydrous ethanol eluent for 5 min and dried with nitrogen to obtain a molecular imprinting electrochemical sensor that can be used for detection;

[0049] (6) 50 μL of the test solution was dropped onto the surface of the molecularly imprinted electrochemical sensor and incubated for 15 min to allow the cortisol molecules in the solution to fully bind to the molecularly imprinted polymer;

[0050] (7) Rinse the molecularly imprinted electrochemical sensor with a large amount of deionized water and blow dry with nitrogen gas to remove the cortisol physically adsorbed on the surface;

[0051] (8) Add 50 μL of 5 mM potassium ferrocyanide solution to the surface of the molecular imprinting electrochemical sensor and perform cyclic voltammetry scanning to obtain cyclic voltammetry results. The cyclic voltammetry experimental conditions are: 50 mV / s, potential -0.6 to 0.6 V;

[0052] (9) The obtained cyclic voltammetry peak value result is substituted into the cortisol standard curve to obtain the cortisol concentration in the sample to be tested. For example, for the prepared 100nM cortisol standard solution, the average CV current of the repeated experiments is -47.19μA. The cortisol concentration calculated by the standard curve is 105.8nM, the recovery rate is 105.8%, and the relative standard deviation RSD is 0.27 (n=3). Based on the obtained linear standard curve, the detection sensitivity of the electrode is 3.65μA / lg(c).

[0053] Embodiment 2:

[0054] The molecular imprinting electrochemical sensor of this embodiment includes an electrode and a molecular imprinting polymer synthesized in situ on the surface of the electrode, wherein the molecular imprinting polymer is a pyrrole-based molecular polymer.

[0055] The molecular imprinting electrochemical sensor of this embodiment is prepared by the following method:

[0056] (1) A 0.1 M PBS buffer solution with a pH of 7.4 was prepared, and 0.03 M carboxypyrrole, 0.5 mM cortisol, chitosan, and metal nanoparticles were added thereto to obtain an electropolymerization solution.

[0057] (2) using a CV electrochemical polymerization method to prepare a pyrrole-based molecularly imprinted polymer containing a cortisol template on the electrode surface to obtain a molecularly imprinted modified electrode;

[0058] (3) The prepared molecularly imprinted modified electrode was placed in anhydrous ethanol eluent for elution for 2 min, and then dried with nitrogen to obtain the molecularly imprinted electrochemical sensor of this embodiment.

[0059] The preparation method and application method of the molecular imprinting electrochemical sensor of this embodiment are the same as those of Example 1. Compared with Example 1, due to the lower concentration of polymer monomer (carboxypyrrole), less molecular imprinting material is synthesized on the electrode surface, and the elution time is less, but the detection ability is relatively weaker. The cortisol detection range of the obtained material is 100nM-1μM, and the detection sensitivity is 3.23μA / lg(c). There is a linear relationship between the logarithmic concentration and the peak current as follows:

[0060] I=-3.23×log 10 (c cortisol (nM))+34.96,R 2 =0.99

[0061] For the prepared 100 nM cortisol standard solution, the average current of repeated experiments was 28.47 μA, and the cortisol concentration calculated by the standard curve was 102.2 nM, the recovery rate was 102.2%, and the relative standard deviation RSD was 0.09 (n=3).

[0062] Comparative Example 1:

[0063] The molecular imprinting electrochemical sensor of this comparative example was prepared by the following method:

[0064] (1) A 0.1 M PBS buffer solution with a pH of 7.4 was prepared, and 0.8 M carboxypyrrole, 5 mM cortisol, chitosan, and metal nanoparticles were added thereto to obtain an electropolymerization solution.

[0065] (2) using a CV electrochemical polymerization method to prepare a pyrrole-based molecularly imprinted polymer containing a cortisol template on the electrode surface to obtain a molecularly imprinted modified electrode;

[0066] (3) The prepared molecularly imprinted modified electrode was placed in anhydrous ethanol eluent for elution for 15 min, and then dried with nitrogen to obtain the molecularly imprinted electrochemical sensor of this embodiment.

[0067] The preparation method and application method of the molecular imprinting electrochemical sensor of this comparative example are the same as those of Example 1. Compared with Example 1, the concentration of the polymer monomer (carboxypyrrole) in this comparative example is too high, and the density of the molecular imprinting material synthesized on the electrode surface is too large, resulting in the inability to wash out the cortisol molecules after a long time, so it is difficult for the electrode to achieve an effective detection effect.

[0068] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A molecular imprinting electrochemical sensor, characterized in that: The invention comprises an electrode and a molecular imprinting polymer synthesized in situ on the surface of the electrode. The molecular imprinting polymer is a pyrrole-based molecular polymer prepared by taking cortisol as a template molecule.

2. A method for preparing a molecular imprinting electrochemical sensor according to claim 1, characterized in that: The following steps are involved: (1) adding a pyrrole compound polymerization monomer (such as pyrrole, carboxypyrrole, etc.), metal nanoparticles and cortisol to a PBS buffer solution, and mixing to obtain an electropolymerization solution; (2) using the electropolymerization solution as a raw material, preparing a pyrrole-based molecularly imprinted polymer containing a cortisol template on the electrode surface by a CV electrochemical polymerization method to obtain a molecularly imprinted modified electrode; (3) Eluting the molecularly imprinted modified electrode in an elution solution to obtain the molecularly imprinted electrochemical sensor.

3. The method for preparing a molecularly imprinted electrochemical sensor according to claim 2, characterized in that: The concentration of the pyrrolyl compound polymerization monomer in step (1) is 0.03-0.8M.

4. The method for preparing a molecularly imprinted electrochemical sensor according to claim 2, characterized in that: The concentration of cortisol in step (1) is 0.5-5 mM.

5. The method for preparing a molecularly imprinted electrochemical sensor according to claim 2, characterized in that: The setting parameters of the CV electrochemical polymerization method in step (2) are: 20 to 100 mV / s, potential -1 to 1 V, and 5 to 20 cycles.

6. The method for preparing a molecularly imprinted electrochemical sensor according to claim 1, characterized in that: The elution time in step (3) is 2 to 15 minutes.

7. Use of the molecular imprinted electrochemical sensor according to claim 1 or the molecular imprinted electrochemical sensor prepared by the preparation method according to any one of claims 2 to 6, characterized in that: The molecular imprinting electrochemical sensor is used to detect the concentration of cortisol.

8. The use of the molecular imprinting electrochemical sensor according to claim 7, characterized in that: When the molecular imprinting electrochemical sensor is used to detect the concentration of cortisol, the method comprises the following steps: (1) dropping cortisol standard solutions of different concentrations onto the surface of the molecularly imprinted electrochemical sensor for incubation, so that the cortisol molecules are fully in contact with the molecularly imprinted polymer of the molecularly imprinted electrochemical sensor; (2) washing the molecular imprinting electrochemical sensor to remove excess cortisol molecules adsorbed on the surface; (3) adding a detection solution containing a certain concentration of electrochemical probe molecules to the molecular imprinting electrochemical sensor, and performing cyclic voltammetry scanning to obtain cyclic voltammetry curves of cortisol standard solutions with different concentrations; (4) correlating the peak value in the cyclic voltammetry curve with the concentration of cortisol in the standard solution to obtain a cortisol standard curve; (5) Before each test of the sample to be tested, the molecular imprinted electrochemical sensor is eluted, and the sample to be tested is measured according to steps (1) to (3) to obtain a cyclic voltammetry curve, and the peak value in the cyclic voltammetry curve is substituted into the cortisol standard curve to obtain the concentration of cortisol in the sample to be tested.

9. The use of the molecular imprinting electrochemical sensor according to claim 8, characterized in that: In step (1), the incubation time of the cortisol standard solution or the sample to be tested on the surface of the molecular imprinting electrochemical sensor is 2 to 15 minutes, and the drop volume of the cortisol standard solution or the sample to be tested is 50 to 100 μL.

10. The use of the molecular imprinting electrochemical sensor according to claim 8, characterized in that: The drop volume of the detection solution in step (3) is 50-100 μL; the detection conditions of the cyclic voltammetry scan are: 50-100 mV / s, and the potential is -0.6-0.6V.

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