Free testosterone detection method based on molecularly imprinted electrochemical sensor

Abstract

According to the method, a molecularly imprinted membrane modified electrode is used as a working electrode, a gold electrode is used as an auxiliary electrode, an Ag / AgCl electrode is used as a reference electrode, the molecularly imprinted electrochemical sensor of a three-electrode system is formed, and the free testosterone is detected by combining an electrochemical workstation. Analyzing and detecting the free testosterone. The molecularly imprinted membrane is prepared by taking testosterone as a template molecule and o-phenylenediamine as a functional monomer through an electropolymerization method, and is a specific detection and recognition component of the sensor. Aiming at the problems that the content of free testosterone in a serum sample is extremely low, and the free testosterone detection operation in the existing detection means is complicated, low in sensitivity and the like, the trace free testosterone can be detected at high sensitivity, and the kit is wide in detection range, simple to operate, portable and reusable.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical analysis and detection, and specifically relates to a free testosterone detection method based on a molecular imprinting electrochemical sensor. Background Art

[0002] Testosterone (TESTO) is a steroid hormone and the most important androgenic hormone in the human body. Male testosterone is mainly secreted by interstitial cells of the testicles, while female testosterone is mainly derived from the peripheral conversion of hormone precursors from the ovaries, adrenal cortex and non-endocrine tissues. Testosterone can effectively promote the growth and development of male external reproductive organs and secondary sexual characteristics, affect sperm production, and has the effects of enhancing sexual desire, strength, immune function, and fighting osteoporosis. In addition, testosterone also has important physiological effects such as promoting protein synthesis, promoting bone growth, promoting the production of erythropoietin in the kidneys, fighting estrogen, and maintaining normal sexual function and mental sexual orientation. Therefore, the specific detection of testosterone is of great significance.

[0003] After secreted testosterone enters the blood, only 2% exists in free form, and the rest exists in bound form. Free testosterone is in an active state, and its concentration is relatively stable under specific physiological and pathological conditions. Compared with total testosterone, it can more reliably reflect the hormone state. At present, commercial free testosterone detection methods include liquid chromatography-tandem mass spectrometry (LC-MS / MS) and kit detection. The instruments required for LC-MS / MS are expensive, bulky, and the sample pretreatment process is complicated, causing serious environmental pollution; the kit detection has the problem of low detection sensitivity. These methods have certain limitations in actual clinical operations. Therefore, the development of a free testosterone detection method that is simple, fast, portable, and highly sensitive has important market application value. Summary of the invention

[0004] In view of the technical problems existing in the detection of free testosterone by existing analytical methods, the purpose of the present invention is to provide a method for detecting free testosterone based on a molecular imprinting electrochemical sensor. Compared with the existing detection methods, this method has the advantages of good selectivity, high sensitivity, less time consumption, simple and fast operation, portability, and rapid response.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] In the first aspect, the present invention seeks protection for a molecular imprinting membrane modified electrode for detecting free testosterone, wherein the molecular imprinting membrane modified electrode comprises an electrode material and a molecular imprinting membrane for specifically recognizing testosterone located on the surface of the electrode material; the electrode material is a screen-printed gold electrode; the template molecule in the molecular imprinting membrane for specifically recognizing testosterone is testosterone, and the functional monomer is o-phenylenediamine; and the solvent is acetate buffer.

[0007] In a second aspect, the present invention claims a method for preparing the molecular imprinted membrane modified electrode for detecting free testosterone, the method comprising the following steps:

[0008] (1) Pretreatment of the screen-printed gold electrode: the screen-printed gold electrode is immersed in dilute sulfuric acid and activated by cyclic voltammetry to remove impurities on its surface, and then ultrasonically cleaned in anhydrous ethanol to obtain a pretreated screen-printed gold electrode;

[0009] (2) preparing a modified electrode: using acetate buffer as a solvent, testosterone as a template molecule, and o-phenylenediamine as a functional monomer to prepare a molecular imprinting polymer solution; placing the pretreated screen-printed gold electrode in the molecular imprinting polymer solution, electropolymerizing the molecular imprinting polymer on the electrode using cyclic voltammetry to obtain a modified electrode, and then taking out and rinsing it for use;

[0010] (3) The modified electrode prepared in (2) is eluted in a mixture of ethanol and water until the testosterone molecules are completely removed, thereby obtaining a molecular imprinting membrane modified electrode.

[0011] Further: The operating parameters of the cyclic voltammetry method in step (1) of the above method are: scanning voltage range: -2V-2V, scanning rate 100mV / s, scanning number 10-30 circles.

[0012] Further: The pH value of the acetate buffer in step (2) of the above method is 4-5, the molar ratio of testosterone to o-phenylenediamine is 1:10-30; the operating parameters of the cyclic voltammetry are: scanning voltage range: 0V-1V, scanning rate 50mV / s, and scanning number of circles is 10-30 circles.

[0013] Further: the elution conditions in step (3) of the above method are: the eluent is 30%-50% ethanol aqueous solution, and the elution time is 20-30 minutes.

[0014] In a specific embodiment of the present invention, the dilute sulfuric acid is 0.5M dilute sulfuric acid, but is not limited thereto.

[0015] In the third aspect, the present invention claims a molecular imprinting electrochemical sensor for detecting free testosterone, which uses the above-mentioned molecular imprinting membrane modified electrode or the molecular imprinting membrane modified electrode prepared by the above-mentioned method as a working electrode, a gold electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a molecular imprinting electrochemical sensor of a three-electrode system. The sensor uses the above-mentioned molecular imprinting membrane modified electrode as a specific detection and recognition component.

[0016] In a fourth aspect, the present invention claims protection for a method for detecting free testosterone based on a molecular imprinting electrochemical sensor, which uses the above-mentioned molecular imprinting membrane modified electrode or the molecular imprinting membrane modified electrode prepared by the above-mentioned method as a working electrode, a gold electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a molecular imprinting electrochemical sensor with a three-electrode system, and combines it with an electrochemical workstation to complete the analysis and detection of free testosterone.

[0017] Furthermore, the detection method specifically comprises the following steps:

[0018] (1) Prepare testosterone solutions of different concentrations using phosphate buffered saline (PBS);

[0019] (2) placing the molecular imprinting membrane modified electrode in testosterone solutions of different concentrations for adsorption for 20-30 minutes;

[0020] (3) using the molecular imprinting membrane modified electrode after adsorption as the working electrode, the gold electrode as the auxiliary electrode, and the Ag / AgCl electrode as the reference electrode to form a molecular imprinting electrochemical sensor with a three-electrode system, and placing the sensor in a potassium ferrocyanide solution;

[0021] (4) Using [Fe(CN)6] 3- / 4- As a redox probe, differential pulse voltammetry was used to obtain the current values ​​corresponding to the adsorption of different concentrations of testosterone, and a standard curve was drawn with the ratio of the peak current difference to the current value after adsorption as the ordinate and the logarithm of the concentration as the abscissa;

[0022] (5) The molecular imprinting membrane modified electrode is placed in a testosterone solution to be tested for adsorption, and then the constructed molecular imprinting electrochemical sensor is placed in a potassium ferrocyanide solution to detect its corresponding current value, and the testosterone concentration is obtained according to the standard curve.

[0023] Further: The electrochemical parameters of the differential pulse voltammetry described in the above detection method are: scanning potential range: -0.1V-0.4V, scanning rate is 100mV.

[0024] In a specific embodiment of the present invention, the potassium ferrocyanide solution is a 2.5 mmol / L potassium ferrocyanide solution, but is not limited thereto.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In view of the extremely low free testosterone content in serum samples, the existing detection methods have complex free testosterone detection operations and low sensitivity. The present invention forms a molecular imprinting membrane by electropolymerization to prepare a molecular imprinting electrochemical sensor on the surface of a screen-printed gold electrode. Due to the signal amplification effect of gold nanoparticles and the high selectivity of the molecular imprinting membrane, a method for highly selective detection of free testosterone by a molecular imprinting electrochemical sensor is provided. The sensor prepared by the present invention is used to detect free testosterone. Compared with other detection methods, it has the advantages of high sensitivity detection, wide detection range, simple operation, portability and reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart for the preparation of molecular imprinted membrane modified electrodes.

[0028] Figure 2 Cyclic voltammograms of different modified electrodes; A is a screen-printed gold electrode, B is a molecularly imprinted polymer modified electrode (before elution), C is a molecularly imprinted membrane modified electrode (after elution), and D is a molecularly imprinted membrane modified electrode adsorbing testosterone (10 pg / mL testosterone).

[0029] Figure 3 Differential pulse voltammograms of different modified electrodes; A is a screen-printed gold electrode, B is a molecularly imprinted polymer modified electrode (before elution), C is a molecularly imprinted membrane modified electrode (after elution), and D is a molecularly imprinted membrane modified electrode adsorbing testosterone (10 pg / mL testosterone).

[0030] Figure 4 The standard curve of the sensor response signal versus testosterone concentration. DETAILED DESCRIPTION

[0031] The present invention is further described below by means of specific examples. The following examples are helpful for those skilled in the art to understand the present invention in detail, but in no way limit the scope of protection of the present invention.

[0032] Example 1 Preparation of molecular imprinted membrane modified electrode

[0033] like Figure 1 As shown, the preparation method of the molecular imprinted membrane modified electrode specifically includes the following steps:

[0034] (1) Pretreatment of screen-printed gold electrodes: The screen-printed gold electrodes were soaked in 0.5 M dilute sulfuric acid, and the electrodes were activated by cyclic voltammetry to remove impurities on the surface (scanning voltage range: -2 V to 2 V, scanning rate 100 mV / s, scanning number 10 to 30 circles), and then ultrasonically cleaned in anhydrous ethanol to obtain pretreated screen-printed gold electrodes;

[0035] (2) using acetate buffer as solvent, testosterone as template molecule, and o-phenylenediamine as functional monomer to prepare molecular imprinting polymer solution, wherein the pH value of acetate buffer is 4.5, and the molar ratio of testosterone to o-phenylenediamine is 1:10; placing a screen-printed gold electrode in the molecular imprinting polymer solution, and electropolymerizing the molecular imprinting polymer on the electrode by cyclic voltammetry to obtain a modified electrode (scanning voltage range: 0 V-1 V, scanning rate 50 mV / s, scanning number of 10 to 30 circles), and then taking out and rinsing for standby use;

[0036] (3) The modified electrode prepared in (2) was eluted in a 40% ethanol aqueous solution for 20 min to obtain a molecular imprinting membrane modified electrode.

[0037] Example 2 Preparation of Molecularly Imprinted Membrane Modified Electrode

[0038] The preparation method of the molecular imprinted membrane modified electrode specifically comprises the following steps:

[0039] (1) Pretreatment of screen-printed gold electrodes: The screen-printed gold electrodes were soaked in 0.5 M dilute sulfuric acid, and the electrodes were activated by cyclic voltammetry to remove impurities on the surface (scanning voltage range: -2 V to 2 V, scanning rate 100 mV / s, scanning number 10 to 30 circles), and then ultrasonically cleaned in anhydrous ethanol to obtain pretreated screen-printed gold electrodes;

[0040] (2) using acetate buffer as solvent, testosterone as template molecule, and o-phenylenediamine as functional monomer to prepare molecular imprinting polymer solution, wherein the pH value of acetate buffer is 4.5, and the molar ratio of testosterone to o-phenylenediamine is 1:20; placing a screen-printed gold electrode in the molecular imprinting polymer solution, and electropolymerizing the molecular imprinting polymer on the electrode by cyclic voltammetry to obtain a modified electrode (scanning voltage range: 0 V-1 V, scanning rate 50 mV / s, scanning number of 10 to 30 circles), and then taking out and rinsing for standby use;

[0041] (3) The modified electrode prepared in (2) was eluted in a 30% ethanol aqueous solution for 30 minutes to obtain a molecular imprinting membrane modified electrode.

[0042] Example 3 Preparation of Molecularly Imprinted Membrane Modified Electrode

[0043] The preparation method of the molecular imprinted membrane modified electrode specifically comprises the following steps:

[0044] (1) Pretreatment of screen-printed gold electrodes: The screen-printed gold electrodes were soaked in 0.5 M dilute sulfuric acid, and the electrodes were activated by cyclic voltammetry to remove impurities on the surface (scanning voltage range: -2 V to 2 V, scanning rate 100 mV / s, scanning number 10 to 30 circles), and then ultrasonically cleaned in anhydrous ethanol to obtain pretreated screen-printed gold electrodes;

[0045] (2) using acetate buffer as solvent, testosterone as template molecule, and o-phenylenediamine as functional monomer to prepare molecular imprinting polymer solution, wherein the pH value of acetate buffer is 4.5, and the molar ratio of testosterone to o-phenylenediamine is 1:30; placing a screen-printed gold electrode in the molecular imprinting polymer solution, and electropolymerizing the molecular imprinting polymer on the electrode by cyclic voltammetry to obtain a modified electrode (scanning voltage range: 0 V-1 V, scanning rate 50 mV / s, scanning number of 10 to 30 circles), and then taking out and rinsing for standby use;

[0046] (3) The modified electrode prepared in (2) was eluted in a 40% ethanol aqueous solution for 20 min to obtain a molecular imprinting membrane modified electrode.

[0047] Example 4

[0048] The molecular imprinted membrane modified electrode obtained in Example 3 was used as the working electrode, the gold electrode was used as the auxiliary electrode, and the Ag / AgCl electrode was used as the reference electrode to form a molecular imprinted electrochemical sensor with a three-electrode system, and combined with an electrochemical workstation, the analysis and detection of free testosterone was completed. Specifically, the following steps were included:

[0049] (1) Prepare testosterone solutions of different concentrations using phosphate buffer (pH 7.4);

[0050] (2) placing the molecular imprinting membrane modified electrode in testosterone solutions of different concentrations for adsorption for 30 minutes;

[0051] (3) A molecular imprinting electrochemical sensor with a three-electrode system was constructed by using the molecular imprinting film modified electrode after adsorption as the working electrode, the gold electrode as the auxiliary electrode, and the Ag / AgCl electrode as the reference electrode. The sensor was placed in a 2.5 mmol / L potassium ferrocyanide solution.

[0052] (4) Using [Fe(CN)6] 3- / 4- As a redox probe, differential pulse voltammetry (scanning potential range: -0.1V-0.4V, scan rate: 100mV) was used to obtain the current values ​​corresponding to the adsorption of different concentrations of testosterone, and a standard curve was drawn with the ratio of the peak current difference to the current value after adsorption as the ordinate and the logarithm of the concentration as the abscissa;

[0053] (5) The molecular imprinting membrane modified electrode is placed in a testosterone solution to be tested for adsorption, and then the constructed molecular imprinting electrochemical sensor is placed in a potassium ferrocyanide solution to detect its corresponding current value, and the testosterone concentration is obtained according to the standard curve.

[0054] The sensor obtained in Example 4 was used for electrochemical testing:

[0055] (1) Cyclic voltammetry test of the sensor:

[0056] The molecular imprinted membrane modified electrode before elution, after elution, and after adsorption in testosterone solution were placed in a 2.5mmol / L potassium ferrocyanide solution for cyclic voltammetry scanning. The scanning parameters were: voltage range -0.4V-0.6V, scanning rate 50mV / s. The cyclic voltammetry diagram is shown in Figure 2 ,from Figure 2 It can be seen that before the molecular imprinting membrane was eluted, the molecular imprinting membrane occupied the electrode surface, which hindered the electron transfer between the redox probe and the electrode; after the molecular imprinting membrane was eluted, the peak current signal of potassium ferrocyanide increased significantly, because the template molecule testosterone in the imprinting membrane was eluted, leaving specific recognition holes, making electron exchange easier; after the eluted molecular imprinting membrane modified electrode was immersed in the testosterone solution, some specific holes were occupied by testosterone, which hindered the electron transfer, so the peak current became smaller again.

[0057] (2) Differential pulse voltammetry test of the sensor:

[0058] The system was placed in a potassium ferrocyanide solution for differential pulse voltammetry scanning, and the scanning parameters were: scanning potential range: -0.1V-0.4V, scanning rate 100mV / s. The eluted molecular imprinted membrane modified electrode was placed in a 2.5mmol / L potassium ferrocyanide solution, and the blank current I0 was obtained by scanning; then the electrode was placed in a testosterone solution of a certain concentration for adsorption, and the current I was obtained by scanning again. The measurement results are shown in Figure 3 , Figure 4 The linear range of testosterone determination was 0.001-10pg / mL, and the detection limit was 0.05fg / mL. The ratio of the peak current difference to the current value after adsorption was used as the ordinate, and the logarithm of the concentration was used as the abscissa to draw the standard curve, y=0.07x+0.0655, R 2 =0.9919. The molecular imprinting membrane modified electrode was placed in the serum sample to be tested for adsorption, and then the constructed molecular imprinting electrochemical sensor was placed in a potassium ferrocyanide solution to detect its corresponding current value, and the concentration of testosterone was obtained according to the standard curve.

Claims

1. A molecular imprinted membrane modified electrode for detecting free testosterone, characterized in that: The molecular imprinting membrane modified electrode comprises an electrode material and a molecular imprinting membrane for specifically recognizing testosterone located on the surface of the electrode material; the electrode material is a screen-printed gold electrode; the template molecule in the molecular imprinting membrane for specifically recognizing testosterone is testosterone, the functional monomer is o-phenylenediamine; and the solvent is an acetate buffer solution.

2. The method for preparing the molecular imprinted membrane modified electrode for detecting free testosterone according to claim 1, characterized in that: The method comprises the following steps: (1) Pretreatment of the screen-printed gold electrode: the screen-printed gold electrode was immersed in 0.5 M dilute sulfuric acid and activated by cyclic voltammetry to remove impurities on its surface, and then ultrasonically cleaned in anhydrous ethanol to obtain a pretreated screen-printed gold electrode; (2) preparing a modified electrode: using acetate buffer as a solvent, testosterone as a template molecule, and o-phenylenediamine as a functional monomer to prepare a molecular imprinting polymer solution; placing the pretreated screen-printed gold electrode in the molecular imprinting polymer solution, electropolymerizing the molecular imprinting polymer on the electrode using cyclic voltammetry to obtain a modified electrode, and then taking out and rinsing it for use; (3) The modified electrode prepared in (2) is eluted in a mixture of ethanol and water until the testosterone molecules are completely removed, thereby obtaining a molecular imprinting membrane modified electrode.

3. The preparation method according to claim 1, characterized in that: The operating parameters of the cyclic voltammetry method in step (1) are: scanning voltage range: -2V-2V, scanning rate 100mV / s, and scanning number 10-30 circles.

4. The preparation method according to claim 1, characterized in that: The pH value of the acetate buffer in step (2) is 4-5, the molar ratio of testosterone to o-phenylenediamine is 1:10-30; the operating parameters of the cyclic voltammetry are: scanning voltage range: 0V-1V, scanning rate 50mV / s, and scanning number of 10-30 circles.

5. The preparation method according to claim 1, characterized in that: The elution conditions in step (3) are: the eluent is 30%-50% ethanol aqueous solution, and the elution time is 20-30 minutes.

6. A molecular imprinting electrochemical sensor for detecting free testosterone, characterized in that: A molecular imprinting electrochemical sensor with a three-electrode system is formed by using the molecular imprinting membrane modified electrode described in claim 1 or the molecular imprinting membrane modified electrode prepared by any method in claims 2-5 as the working electrode, a gold electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode.

7. A method for detecting free testosterone based on molecular imprinting electrochemical sensor, characterized in that: The method uses the molecular imprinted membrane modified electrode described in claim 1 or the molecular imprinted membrane modified electrode prepared by any method in claims 2-5 as a working electrode, a gold electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a molecular imprinted electrochemical sensor with a three-electrode system, and combines it with an electrochemical workstation to complete the analysis and detection of free testosterone.

8. The detection method according to claim 7, characterized in that: The method specifically comprises the following steps: (1) preparing testosterone solutions of different concentrations using phosphate buffer; (2) placing the molecular imprinting membrane modified electrode in testosterone solutions of different concentrations for adsorption for 20-30 minutes; (3) using the molecular imprinting membrane modified electrode after adsorption as the working electrode, the gold electrode as the auxiliary electrode, and the Ag / AgCl electrode as the reference electrode to form a molecular imprinting electrochemical sensor with a three-electrode system, and placing the sensor in a potassium ferrocyanide solution; (4) Using [Fe(CN)6] 3- / 4- As a redox probe, differential pulse voltammetry was used to obtain the current values ​​corresponding to the adsorption of different concentrations of testosterone, and a standard curve was drawn with the ratio of the peak current difference to the current value after adsorption as the ordinate and the logarithm of the concentration as the abscissa; (5) The molecular imprinting membrane modified electrode is placed in a testosterone solution to be tested for adsorption, and then the constructed molecular imprinting electrochemical sensor is placed in a potassium ferrocyanide solution to detect its corresponding current value, and the testosterone concentration is obtained according to the standard curve.

9. The method according to claim 8, characterized in that: The electrochemical parameters of the differential pulse voltammetry are: scanning potential range: -0.1V-0.4V, scanning rate: 100mV.

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