Preparation method of electrochemical biosensor based on molecular imprinting track etching film

Abstract

The invention provides a preparation method of an electrochemical biosensor based on a molecular imprinting track etching film, and belongs to the field of electrochemical biosensing analysis. The electrochemical biosensor based on the molecularly imprinted track etching film is formed by taking a track etching film modified working electrode as a substrate, taking graphene oxide as an electric signal amplification material and taking a molecularly imprinted polymer with a template molecularly imprinted cavity as a specific recognition element. When the mixed solution contains a target object, target molecules are captured by an imprinting cavity on the molecular imprinting layer according to specific interaction, so that a nano pore channel is blocked, the electron transfer rate in a system is reduced due to the increase of steric hindrance, and the ultrasensitive quantitative detection of the target molecules is realized. The method has very important theoretical and practical significance in the field of medical diagnosis, and a new direction is provided for application of the track etching film in the field of analysis and detection.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical biosensors, and in particular relates to a method for preparing an electrochemical biosensor based on a molecular imprinting track etching film. Background Art

[0002] Acute myocardial infarction is a serious cardiovascular disease that causes high morbidity and mortality worldwide, posing a serious threat to people's lives and health. Troponin (cTnI) is considered to be a reliable and important biomarker for accurate diagnosis and effective monitoring of myocardial infarction due to its sensitivity and specificity. Early detection of cTnI can effectively prevent myocardial infarction, prolong the life of patients and reduce related mortality. Therefore, it is of great significance to establish a reliable, selective and sensitive cTnI detection method.

[0003] Electrochemical biosensors are a type of analyte detection system that is integrated into an electronic sensor to convert biochemical signals into measurable electrical signals. The recognition elements of biosensors are usually natural receptors, such as enzymes, antibodies, and nucleic acids, thereby achieving high selectivity for the target analyte. However, natural receptors are often not easy to obtain, which is not only expensive and time-consuming, but also has the disadvantages of poor stability and reproducibility. Molecularly imprinted polymers are a special type of polymer material that is easy to produce, inexpensive, has good stability and reproducibility, and is considered to be the best alternative to natural receptors. Molecular imprinting technology is based on the formation of a complex between analyte / template / guest molecules and functional monomers / host molecules. The resulting analyte-functional monomer complex forms a polymer structure through a polymerization reaction initiated by temperature or irradiation, and then the template molecule is removed, leaving an imprinted cavity, so that the polymer acquires its spatial characteristics (size, shape, functional group positioning), and exhibits strong selectivity, sensitivity, and the ability to bind molecules identical to the template molecule due to the interaction of the recognition sites. Track-etched membranes can effectively and selectively transfer ions due to their unique pore size and arrangement structure, while molecular imprinting technology can form selective recognition sites on polymer substrates, which greatly improves the detection sensitivity of biosensors to specific molecules and reduces the response to non-target markers. Summary of the invention

[0004] The present invention aims to provide a method for preparing an electrochemical biosensor based on a molecular imprinting track-etched membrane, which belongs to the field of electrochemical biosensor analysis. The electrochemical biosensor based on a molecular imprinting track-etched membrane is composed of a working electrode modified with a track-etched membrane as a substrate, graphene oxide as an electrical signal amplification material, and a molecular imprinting polymer with a template molecular imprinting cavity as a specific recognition element. After the imprinting membrane removes troponin, according to the imprinting sites left on the MIP with the same shape and size as the template molecule spatial structure, when the mixed solution contains a target, the target molecule will be captured by the imprinting cavity recognition site on the molecular imprinting layer according to a specific interaction, thereby causing the nanopore to be blocked, and the electron transfer rate in the system drops sharply due to the increase in steric hindrance, thereby achieving ultra-sensitive quantitative detection of the target molecule.

[0005] The method for preparing the electrochemical biosensor based on molecular imprinting track etching film comprises the following steps:

[0006] (1) The track-etched membrane is placed on the working electrode, and then the electrical signal amplification material is fixed in its nanopores to increase the electron transfer rate in the system;

[0007] (2) mixing the template molecule with the functional monomer and allowing the mixture to stand for a period of time, and placing the working electrode prepared above in the mixed solution to prepare a molecular imprinting layer;

[0008] (3) The template molecules in the molecular imprinting layer synthesized in step (2) are removed by using an elution solution, thereby successfully preparing an electrochemical biosensor based on the molecular imprinting track etching membrane.

[0009] The molecular imprinted track etching membrane consists of a recognition layer with an imprinted cavity and an ion transport layer with nanopores.

[0010] The electrical signal amplifying material in step 1) includes one or more of two-dimensional nanomaterials such as graphene, graphene oxide, black phosphorus, MXene, molybdenum disulfide or tungsten disulfide.

[0011] Step 2) The template molecule is a detection target, including proteins, ions, small molecules, nucleic acids or pathogens; the functional monomer is one or more of o-phenylenediamine, hydroquinone, methacrylic acid, acrylamide, methyl methacrylate, dopamine, aniline or pyrrole; the preparation conditions of the molecular imprinting layer include but are not limited to light, electricity, heat or radiation means.

[0012] The elution solution in step 3) is one or more of sodium dodecyl sulfate, formic acid, methanol, hydrochloric acid, acetic acid, acetonitrile, water or sodium hydroxide.

[0013] An analytical method for establishing an electrochemical biosensor for detecting troponin as described above comprises the following steps:

[0014] 1) A working electrode with a track-etched membrane as a substrate, graphene oxide as a signal amplification material, and a molecular imprinted layer as a specific recognition element is placed in an electrolytic cell, an electrolyte solution is injected into the electrolytic cell, and an auxiliary electrode and a reference electrode are also placed in the solution; then an electrochemical workstation is used to perform an electrochemical test on the system, and the response current is recorded, and the current at this time is recorded as I0;

[0015] 2) The electrochemical biosensor based on molecular imprinting track-etched membrane was placed in different concentrations of troponin solution for incubation, and then rinsed with phosphate buffer for multiple times to remove troponin that was not specifically bound to the imprinted cavity. The current value at this time was recorded as I. The relative change rate of the current before and after the addition of troponin antigen was calculated by the electrochemical workstation to achieve quantitative detection of troponin, and a standard curve was drawn with the horizontal and vertical coordinates being troponin concentration and the current relative change rate (I0-I) / I0, respectively.

[0016] The technical principle of the present invention is: since the track-etched membrane pores carry carboxyl groups after etching, its surface is negatively charged, and positively charged two-dimensional nanomaterials can be introduced through electrostatic adsorption to prepare an effective electrochemical biosensor substrate, and a molecular imprinting layer with a template molecular imprinting cavity is prepared on the modified working electrode, thereby constructing an electrochemical biosensor based on a molecular imprinting track-etched membrane for efficient and sensitive detection of biomarkers. When the mixed solution contains a target, the target molecule will be captured by the imprinting cavity recognition site on the molecular imprinting layer according to a specific interaction, thereby causing the nanopore to be blocked, and the electron transfer rate in the system drops sharply due to the increase in steric hindrance, thereby achieving ultra-sensitive quantitative detection of the target molecule. The present invention mainly uses the electrochemical signal amplification effect of two-dimensional nanomaterials to improve sensitivity, and improves selectivity through the specific capture of the molecular imprinting layer, thereby achieving ultra-sensitive rapid quantitative detection of troponin.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] 1. The electrochemical biosensor for troponin antigen detection provided by the present invention adopts two-dimensional nanomaterials as a signal amplification strategy and uses a molecular imprinting layer to selectively capture the molecules to be detected, so that the sensor has high sensitivity.

[0019] 2. The electrochemical biosensor for troponin antigen detection provided by the present invention has high specificity and has no response to other non-targeted proteins.

[0020] 3. The electrochemical biosensor based on molecular imprinting track etching membrane provided by the present invention is easy to use and has fast analysis, and is suitable for the actual needs in the industrialization of electrochemical biosensors.

[0021] 4. The present invention can be expanded to the detection of other targets by replacing the template molecule to prepare the molecular imprinting layer, providing valuable insights for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 Schematic diagram of the electrochemical sensing principle for detecting target substances using an electrochemical biosensor based on molecular imprinting track-etched membrane

[0024] Figure 2 DPV curves of cTnI responses at different concentrations

[0025] Figure 3 Standard curve of the current change rate curve in response to different concentrations of cTnI

[0026] Figure 4 Repeatability, reproducibility and stability bar graph (current relative rate of change bar graph)

[0027] Figure 5 Specificity bar graph (bar graph of relative current change rate corresponding to different antigens) DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following embodiments.

[0029] Example 1

[0030] A method for preparing an electrochemical biosensor based on molecular imprinting track etching film, wherein the method is as follows Figure 1 As shown, the following steps are included:

[0031] (1) Placing the PET track-etched membrane on the working electrode, and then fixing the electrical signal amplification material graphene oxide in its nanopores to increase the electron transfer rate in the system;

[0032] (2) The template molecule troponin is mixed with the functional monomer o-phenylenediamine (OPD) and allowed to stand for a period of time, the functionalized working electrode is placed in the mixed solution, ethylene glycol dimethacrylate (EDGMA) and azobisisobutyronitrile (AIBN) are added, and the mixture is irradiated with ultraviolet light for 2 hours to prepare a molecular imprinting layer;

[0033] (3) The troponin in the molecular imprinting layer synthesized in step (2) was removed using an elution solution (sodium dodecyl sulfate: acetic acid = 1:5; V / V), thereby successfully preparing an electrochemical biosensor based on molecular imprinting track etching membrane.

[0034] Example 2

[0035] An analytical method for establishing an electrochemical biosensor for detecting troponin as described above comprises the following steps:

[0036] The working electrode with track-etched membrane as the base plate, graphene oxide as the signal amplification material, and molecular imprinting layer as the specific recognition element was placed in an electrolytic cell, and 5 mmol / L [Fe(CN)6] 3- / 4- solution, and the auxiliary electrode and the reference electrode are also placed in the electrolyte solution at the same time; then the system is electrochemically tested using a workstation, and the electrochemical response current is recorded, and the current at this time is recorded as I0;

[0037] The electrochemical biosensor based on molecular imprinting track-etched membrane was incubated in different concentrations of troponin test solution, then rinsed with phosphate buffer several times to remove troponin that was not specifically bound to the imprinted cavity, and tested. The current value at this time was recorded as I ( Figure 2 ), the relative rate of change of current before and after adding troponin antigen was calculated by electrochemical workstation to achieve quantitative detection of troponin, and a standard curve with troponin concentration and relative rate of change of current (I0-I) / I0 was drawn. Figure 3 ).

[0038] Example 3 Under the same conditions, the current value of the same electrochemical biosensor was measured six times in succession, and its relative standard deviation (RSD) was about 1.13%; the current value of six electrochemical biosensors based on molecular imprinting track etching membranes prepared from the same batch was monitored, and its RSD was 1.78%; after storage at 4°C for 5 weeks, 91.3% of the response current value was retained compared with the first day ( Figure 4 ).

[0039] Example 4

[0040] The specificity of the electrochemical biosensor based on molecularly imprinted track-etched membrane for troponin Figure 5As shown in the figure, other types of proteins at the same concentration (500 ng / mL), such as cardiac troponin C (cTnC), prostate antigen (PSA) and human serum albumin (HSA), were selected as interfering substances. The sensor was incubated in different solutions, and the current values ​​before and after incubation were recorded to compare the relative change rates of the sensor current caused by different solutions. For the interfering substances cTnC, PSA and HSA, the relative change rates of the current of the sensing platform were approximately 5.55±0.52%, 3.85±0.61% and 2.17±1.01%, respectively, while the relative change rate of the current of the entire system was approximately 37.6±1.12% when the target was cTnI.

[0041] The above description is only the best embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent substitutions, improvements, etc. made should be included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical biosensor based on molecular imprinting track etching film, characterized in that: The method comprises the following steps: (1) Placing the track-etched membrane on the working electrode and then fixing the electrical signal amplification material in its nanopores; (2) mixing the template molecule with the functional monomer and allowing the mixture to stand for a period of time, and placing the working electrode prepared above in the mixed solution to prepare a molecular imprinting layer; (3) The template molecules in the molecular imprinting layer synthesized in step (2) are removed by using an elution solution, thereby successfully preparing an electrochemical biosensor based on the molecular imprinting track etching membrane.

2. The method for preparing an electrochemical biosensor based on molecular imprinting track etching film according to claim 1, characterized in that: The molecular imprinted track etching membrane consists of a recognition layer with an imprinted cavity and an ion transport layer with nanopores.

3. The method for preparing an electrochemical biosensor based on molecular imprinting track etching film according to claim 1, characterized in that: The electrical signal amplifying material in step 1) includes one or more of two-dimensional nanomaterials such as graphene, graphene oxide, black phosphorus, MXene, molybdenum disulfide or tungsten disulfide.

4. The method for preparing an electrochemical biosensor based on molecular imprinting track etching film according to claim 1, characterized in that: Step 2) The template molecule is a detection target, including proteins, ions, small molecules, nucleic acids or pathogens; the functional monomer is one or more of o-phenylenediamine, hydroquinone, methacrylic acid, acrylamide, methyl methacrylate, dopamine, aniline or pyrrole; the preparation method of the molecular imprinting layer includes but is not limited to light, electricity, heat or radiation.

5. The method for preparing an electrochemical biosensor based on molecular imprinting track etching film according to claim 1, characterized in that: The elution solution in step 3) is one or more of sodium dodecyl sulfate, formic acid, methanol, hydrochloric acid, acetic acid, acetonitrile, water or sodium hydroxide.

6. The method for preparing an electrochemical biosensor based on molecular imprinting track etching film according to claim 1, characterized in that: The analytical method for establishing an electrochemical biosensor for detecting troponin, using graphene oxide as an electrical signal amplification material and troponin as a template molecule, comprises the following steps: (1) A working electrode with a track-etched membrane as a substrate, graphene oxide as a signal amplification material, and a molecular imprinting layer as a specific recognition element is placed in an electrolytic cell, an electrolyte solution is injected into the electrolytic cell, and an auxiliary electrode and a reference electrode are also placed in the solution; then, an electrochemical test is performed on the system using an electrochemical workstation, and the response current is recorded, and the current at this time is recorded as I0; (2) The electrochemical biosensor based on molecular imprinting track-etched membrane was placed in a solution of troponin with different concentrations for incubation, and then rinsed with phosphate buffer for multiple times to remove the troponin that was not specifically bound to the imprinted cavity. The current value at this time was recorded as I. The relative change rate of the current before and after the addition of troponin antigen was calculated by the electrochemical workstation test to achieve quantitative detection of troponin, and a standard curve was drawn with the horizontal and vertical coordinates being the troponin concentration and the current relative change rate (I0-I) / I0, respectively.

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