An Electrochemical Biosensor Based on Recombinant Human Renin Molecularly Imprinted Polymer, Preparation Method and Application

Abstract

The present invention provides an electrochemistry biosensor based on a recombinant human renin molecularly imprinted polymer, a preparation method and an application thereof. The electrochemistry biosensor has a recombinant human renin molecularly imprinted polymer loaded on the surface of an electrode. Among them, the molecularly imprinted polymer is prepared by an electrochemical polymerization method using recombinant human renin as a template and methyldopa as a functional monomer. The electrochemistry biosensor has strong specificity for the target molecule recombinant human renin, can specifically detect the target molecule recombinant human renin, and can also treat hypertension, achieving the purpose of integrating detection and treatment. The preparation method of the electrochemistry biosensor is simple, convenient to operate and has a low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and relates to an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, a preparation method and an application thereof. Background Art

[0002] A molecularly imprinted polymer is a polymer synthesized by imprinting technology that has specific recognition and selective adsorption for a specific target molecule and its structural analogs. Molecularly imprinted polymers have gradually attracted wide attention due to their outstanding sensitivity and specificity, especially in the application of electrochemical biosensors.

[0003] Regarding the preparation of an imprinted polymer using methyldopa as a functional monomer, the main research content retrieved so far is to use methyldopa as a functional monomer and the drug darifenacin as a template molecule to prepare a molecularly imprinted polymer by electrochemical polymerization, and then prepare an electrochemical biosensor. This electrochemical biosensor can detect the target molecule darifenacin with high sensitivity and specificity. Currently, there is no report on the preparation of an electrochemical biosensor using recombinant human renin as a template molecule and methyldopa as a functional monomer. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, a preparation method and an application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, wherein the electrochemical biosensor has a recombinant human renin molecularly imprinted polymer loaded on the electrode surface, and the molecularly imprinted polymer is prepared by electrochemical polymerization using recombinant human renin as a template and methyldopa as a functional monomer.

[0007] Methyldopa is an organic compound mainly used as an antihypertensive drug, especially for renal hypertension and hypertension with reduced renal function. Recombinant human renin is a biomarker for hypertension. In this application, a molecularly imprinted polymer is prepared by electrochemical polymerization using recombinant human renin as a template and methyldopa as a functional monomer, and then an electrochemical biosensor is prepared. This electrochemical biosensor can specifically recognize and detect the disease biomarker recombinant human renin of hypertension, and at the same time can functionally treat hypertension, achieving the integration of diagnosis and treatment.

[0008] The present application also provides a preparation method for an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, and the method includes:

[0009] S01: The glassy carbon electrode was polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm, sonicated, and rinsed with ethanol and water respectively.

[0010] The glassy carbon electrode has good electrical conductivity, high chemical stability, and a small thermal expansion coefficient. Therefore, in this application, the glassy carbon electrode is used as the electrode for preparing the electrochemical biosensor. The glassy carbon electrode was polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm to make the surface of the glassy carbon electrode smooth, which is convenient for preparing the electrochemical biosensor. After polishing, the glassy carbon electrode was sonicated and then rinsed with ethanol and water for 3 minutes respectively.

[0011] S02: Recombinant human renin and methyldopa were uniformly dispersed in PBS solution and sonicated to form a homogeneous solution.

[0012] Recombinant human renin and methyldopa were uniformly dispersed in PBS (English name: phosphate buffered saline; Chinese name: phosphate buffered saline) solution according to a certain molar ratio and sonicated to form a homogeneous solution. In this application, the pH value of the PBS solution is 7.4 and the concentration is 0.2 M.

[0013] The ratio of recombinant human renin to methyldopa determines the sensing performance of the electrochemical biosensor, such as the detection range and the lowest detection limit. In this application, the molar ratio of recombinant human renin to methyldopa is preferably 1:(2 - 6), and most preferably 1:5.

[0014] S03: The polished glassy carbon electrode was placed into the homogeneous solution and cyclic voltammetry polymerization was used to obtain the molecularly imprinted polymer.

[0015] The polished glassy carbon electrode was placed into the homogeneous solution. Cyclic voltammetry technology was used to electrochemically polymerize and prepare the molecularly imprinted polymer on the surface of the glassy carbon electrode. The conditions for cyclic voltammetry polymerization were -0.1 V to 0.9 V, and the number of polymerization cycles was 21.

[0016] S04: The molecularly imprinted polymer was eluted with an eluent to obtain the electrochemical biosensor.

[0017] The molecularly imprinted polymer was eluted with an eluent with a concentration of 10% for 20 minutes to wash off the template molecule recombinant human renin on the surface of the molecularly imprinted polymer, forming an electrochemical biosensor with a three-dimensional cavity structure. In this application, the eluent includes one of SDS (English name: Sodium dodecyl sulfate; Chinese name: sodium dodecyl sulfate), acetic acid, a mixture of acetic acid and SDS, and acetone. Among them, SDS has the highest elution efficiency.

[0018] In addition, the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the present application can specifically recognize and detect the hypertension disease marker recombinant human renin. At the same time, it can functionally treat hypertension diseases, achieving the integration of diagnosis and treatment.

[0019] The present invention has the following beneficial effects:

[0020] 1) The present invention provides an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, a preparation method and an application thereof. The electrochemical biosensor has a recombinant human renin molecularly imprinted polymer loaded on the electrode surface. Among them, the molecularly imprinted polymer is prepared by an electrochemical polymerization method using recombinant human renin as a template and methyldopa as a functional monomer.

[0021] 2) The electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the present invention can specifically detect the target molecule recombinant human renin, and at the same time can also treat hypertension, achieving the purpose of integrating detection and treatment.

[0022] 3) The preparation method of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the present invention is simple, convenient to operate, and has a low cost. Description of the Drawings

[0023] Figure 1 It is a TEM (English name: Transmission Electron Microscope; Chinese name: Transmission Electron Microscope) image of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the embodiment of the present application;

[0024] Figure 2 It is a specific detection image of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the embodiment of the present application;

[0025] Figure 3 It is an electrochemical alternating current impedance signal detection image of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the embodiment of the present application;

[0026] Figure 4 It is a linear detection concentration image of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the embodiment of the present application;

[0027] Figure 5 It is a treatment efficacy image of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided by the embodiment of the present application on spontaneously hypertensive rats. Detailed Embodiments

[0028] The technical solutions of the present invention will be further explained and illustrated below through specific embodiments.

[0029] Example 1

[0030] An embodiment of the present application provides a method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, the method comprising:

[0031] S101: The glassy carbon electrode is polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm, sonicated, and rinsed with ethanol and water for 3 minutes respectively.

[0032] S102: After mixing recombinant human renin and methyldopa in a molar ratio of 1:5, they are uniformly dispersed in a PBS solution with a concentration of 0.2 M and a pH value of 7.4, and sonicated to form a homogeneous solution.

[0033] S103: The polished glassy carbon electrode is placed in the homogeneous solution, and cyclic voltammetry polymerization is carried out under the conditions of 0.5 V and 21 polymerization cycles to obtain a molecularly imprinted polymer.

[0034] S104: The molecularly imprinted polymer is eluted with a 10% SDS solution to obtain an electrochemical biosensor.

[0035] Example 2

[0036] An embodiment of the present application provides a method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, the method comprising:

[0037] S201: The glassy carbon electrode is polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm, sonicated, and rinsed with ethanol and water for 3 minutes respectively.

[0038] S202: After mixing recombinant human renin and methyldopa in a molar ratio of 1:2, they are uniformly dispersed in a PBS solution with a concentration of 0.2 M and a pH value of 7.4, and sonicated to form a homogeneous solution.

[0039] S203: The polished glassy carbon electrode is placed in the homogeneous solution, and cyclic voltammetry polymerization is carried out under the conditions of -0.1 V and 21 polymerization cycles to obtain a molecularly imprinted polymer.

[0040] S204: The molecularly imprinted polymer is eluted with 10% acetic acid to obtain an electrochemical biosensor.

[0041] Example 3

[0042] An embodiment of the present application provides a method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, the method comprising:

[0043] S301: The glassy carbon electrode was polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm, sonicated, and rinsed with ethanol and water for 3 minutes respectively.

[0044] S302: After mixing recombinant human renin and methyldopa in a molar ratio of 1:4, they were uniformly dispersed in a PBS solution with a concentration of 0.2 M and a pH value of 7.4, and sonicated to form a homogeneous solution.

[0045] S303: The polished glassy carbon electrode was placed into the homogeneous solution, and cyclic voltammetry polymerization was carried out under the conditions of 0.3 V and 21 polymerization cycles to obtain a molecularly imprinted polymer.

[0046] S304: The molecularly imprinted polymer was eluted with a mixed solution of 10% acetic acid and SDS to obtain an electrochemical biosensor.

[0047] Example 4

[0048] The embodiment of the present application provides a preparation method of an electrochemical biosensor based on a molecularly imprinted polymer of recombinant human renin. The method includes:

[0049] S401: The glassy carbon electrode was polished successively in alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm, sonicated, and rinsed with ethanol and water for 3 minutes respectively.

[0050] S402: After mixing recombinant human renin and methyldopa in a molar ratio of 1:6, they were uniformly dispersed in a PBS solution with a concentration of 0.2 M and a pH value of 7.4, and sonicated to form a homogeneous solution.

[0051] S403: The polished glassy carbon electrode was placed into the homogeneous solution, and cyclic voltammetry polymerization was carried out under the conditions of 0.9 V and 21 polymerization cycles to obtain a molecularly imprinted polymer.

[0052] S404: The molecularly imprinted polymer was eluted with 10% acetone to obtain an electrochemical biosensor.

[0053] The embodiment of the present application also carried out transmission electron microscope inspection and performance tests such as specificity, sensing performance, and disease treatment efficacy on the prepared electrochemical biosensor based on the molecularly imprinted polymer of recombinant human renin. The following takes the electrochemical biosensor based on the molecularly imprinted polymer of recombinant human renin prepared in Example 1 as an example for specific description.

[0054] 1. Transmission electron microscope inspection

[0055] The electrochemical biosensor prepared in Example 1 was subjected to transmission electron microscope inspection to obtain the attachment Figure 1 . From the attachment Figure 1It can be seen that there are holes on the interface surface of the electrochemical biosensor prepared in Example 1. These holes are three-dimensional holes formed after the recombinant human renin is eluted.

[0056] 2. Specific detection

[0057] Equal amounts of the electrochemical biosensor were added to the buffer solutions prepared from the mixed solutions of recombinant human renin, alpha-fetoprotein AFP, carcinoembryonic antigen CEA, human immunoglobulin E, human serum albumin, and the mixed solution containing recombinant human renin. After soaking for 20 minutes, the change rate (%) of the impedance of the electrochemical biosensor was detected, and the detection results are shown in the appendix. Figure 2 As shown in the appendix Figure 2 It can be seen that the response ability of the electrochemical biosensor prepared in Example 1 to alpha-fetoprotein AFP, carcinoembryonic antigen CEA, human immunoglobulin E, and human serum albumin is negligible, while the response ability to recombinant human renin and the mixed solution containing recombinant human renin is very strong and extremely close. This indicates that the electrochemical biosensor prepared in Example 1 has high recognition and selection specificity for recombinant human renin and almost no recognition performance for interfering components such as alpha-fetoprotein AFP. This is because the three-dimensional imprinted hole recognition sites on the sensing interface of the electrochemical biosensor are completely matched with the size, shape, and other structures of the target molecule recombinant human renin, so there is no recognition performance for other components.

[0058] 3. Sensing performance detection

[0059] Target molecule recombinant human renin solutions with concentrations of 0, 5*10 -6 ng / mL, 5*10 -5 ng / mL, 5*10 -4 ng / mL, 5*10 -3 ng / mL, 5*10 -2 ng / mL, 5*10 -1 ng / mL, and 5*10 Figure 3 , 4 were prepared using sterile water. The electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer prepared in Example 1 was placed in the prepared recombinant human renin solution and incubated for 20 minutes. The electrochemical impedance spectroscopy (abbreviation in English: EIS) was used to test the electrochemical impedance signal, and the detection results are shown in the appendix Figure 3 . Among them, a-e in the appendix -6 correspond to concentrations of 0, 5*10 -5 ng / mL, 5*10 -4 ng / mL, 5*10 -3 ng / mL, 5*10 -2 ng / mL, 5*10 -1Target molecule recombinant human renin solution at ng / mL.

[0060] Attached Figure 3 It can be seen that as the concentration of the target molecule recombinant human renin solution increases, the EIS signal of the electrochemical biosensor prepared in Example 1 of this application gradually increases until it reaches saturation. This indicates that the interface of the electrochemical biosensor has gradually achieved effective binding with the target molecule recombinant human renin, and the sensing interface of the electrochemical biosensor can effectively detect the target molecule recombinant human renin. When the concentration of the target molecule recombinant human renin solution is 5*10 -1 ng / mL, the target binding state of the electrochemical biosensor has reached a stable state, as shown by curve g in the attachment Figure 3 , which indicates that the adsorption of the electrochemical biosensor to recombinant human renin has reached a saturated state.

[0061] Taking the concentration of the target molecule recombinant human renin solution as the abscissa and the impedance change rate of the electrochemical biosensor in the attachment Figure 3 as the ordinate to plot a graph, a linear detection concentration graph as shown in the attachment Figure 4 is obtained. It can be seen from the attachment Figure 4 that the change in the EIS signal of the electrochemical biosensor is linearly correlated with the Log value of the concentration of the target molecule recombinant human renin solution.

[0062] 3. Detection of disease treatment efficacy

[0063] After the spontaneously hypertensive rats were adaptively fed for seven days, a non-invasive tail artery blood pressure measurement system was used to measure the systolic blood pressure and diastolic blood pressure of the rats at rest, and the average value was taken after measuring three times. The molecularly imprinted polymer with a concentration of 6.75 mg / kg was intravenously injected into the spontaneously hypertensive rats respectively. The systolic blood pressure and diastolic blood pressure of the rats at rest were measured at 30 min, 1 h, 2 h, and 4 h after injection, and the average value was taken after measuring three times. A graph was plotted based on the measured systolic blood pressure and diastolic blood pressure to obtain the attachment Figure 5 . It can be seen from the attachment Figure 5 that after injecting the molecularly imprinted polymer, the systolic blood pressure and diastolic blood pressure of the spontaneously hypertensive rats both decreased significantly. Especially at 1 h after injection, the systolic blood pressure and diastolic blood pressure decreased the most, reaching the best treatment effect.

[0064] The above detections show that the electrochemical biosensor prepared in the examples of this application has high specificity for the target molecule recombinant human renin and can specifically detect recombinant human renin. In addition, this electrochemical biosensor can also treat hypertension, achieving the purpose of integrating detection and treatment. The preparation process of the electrochemical biosensor based on the recombinant human renin molecularly imprinted polymer provided in the examples of this application is simple, convenient to operate, and has a low cost.

[0065] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. An electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, characterized in that, the electrochemical biosensor has a recombinant human renin molecularly imprinted polymer loaded on the electrode surface, and the recombinant human renin molecularly imprinted polymer is prepared by electrochemical polymerization using recombinant human renin as a template and methyldopa as a functional monomer.

2. A method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer, characterized in that, it includes: The glassy carbon electrode is polished in an alumina suspension, sonicated, and rinsed with ethanol and water respectively; Recombinant human renin and methyldopa are uniformly dispersed in a PBS solution and sonicated to form a homogeneous solution; The polished glassy carbon electrode is placed in the homogeneous solution and subjected to cyclic voltammetry polymerization to obtain a molecularly imprinted polymer; The molecularly imprinted polymer is eluted with an eluent to obtain an electrochemical biosensor.

3. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the molar ratio of the recombinant human renin to the methyldopa is 1: (2 - 6).

4. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the molar ratio of the recombinant human renin to the methyldopa is 1:

5.

5. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the pH value of the PBS solution is 7.4 and the concentration is 0.2 M.

6. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the conditions for the cyclic voltammetry polymerization are -0.1 V to 0.9 V, and the number of polymerization cycles is 21.

7. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the eluent includes one of SDS, acetic acid, a mixture of acetic acid and SDS, and acetone.

8. The method for preparing an electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 2, characterized in that, the glassy carbon electrode is polished in alumina suspensions with alumina particle sizes of 1.0, 0.3, and 0.05 µm in sequence.

9. The electrochemical biosensor based on a recombinant human renin molecularly imprinted polymer according to claim 1 or the electrochemical biosensor prepared by the preparation method according to any one of claims 2 - 8 is used for specifically recognizing and detecting recombinant human renin.

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