A photoelectrochemical biosensor based on porous membrane and its preparation method and application

Abstract

The present invention provides a photoelectrochemical biosensor based on a porous membrane, and a preparation method and application thereof, which belongs to the field of photoelectrochemical biosensors. The photoelectrochemical biosensor comprises: an electrode, the electrode comprises ITO conductive glass and a composite membrane coated on the ITO conductive glass: the composite membrane refers to modifying a photosensitive material in the nanopores of the porous membrane, and then connecting a capture probe in the nanopores to obtain a photoelectrochemical biosensor based on a porous membrane. The sensor has the following advantages: 1. It can detect other biological molecules by changing the type of capture probe, and has excellent practical application value; 2. The porous membrane loaded on the ITO conductive glass is used to replace the traditional metal electrode, and the detection is accurate, the detection time is short, the cost is low, and it has good reproducibility and stability.

Description

Technical Field

[0001] The invention belongs to the field of photoelectrochemical biosensors, and in particular relates to a photoelectrochemical biosensor based on a porous membrane and a preparation method and application thereof. Background Art

[0002] Photoelectrochemical biosensor is an emerging technology based on the photoelectric conversion characteristics of active materials such as semiconductors. It uses the physical or chemical changes produced by bioactive materials during the detection process to convert biological information into electrical signals to detect biochemical information in the body (such as the concentration of the target). Photoelectrochemical biosensor is an analytical device based on the principle of photoelectrochemistry and biorecognition technology. It achieves the purpose of analysis and detection by connecting the target biological molecules to the surface of the semiconductor photoelectrode to produce electrochemical signal changes. Photoelectrochemical biosensors have the characteristics of fast response speed, high sensitivity, good stability and easy miniaturization. They have irreplaceable broad application prospects in rapid screening of diseases, drug screening and life mechanism research. Photoelectrochemical biosensors show high detection sensitivity and rapid response and recognition capabilities for target molecules. They are suitable for real-time monitoring and rapid analysis in complex biological environments. However, since current biosensor technology still generally relies on biorecognition units such as antibodies, enzymes and aptamers, these biomaterials often show problems of insufficient stability and selectivity in complex in vivo detection environments, which greatly limits the feasibility of photoelectrochemical biosensors in practical applications. Therefore, the present invention selects the porous membrane as the carrier of the photosensitive material and the biorecognition unit, which greatly improves the stability and selectivity of the sensor and provides strong support for the further application of the photoelectrochemical biosensor in medical diagnosis and treatment technology. Summary of the invention

[0003] The purpose of the present invention is to provide a photoelectrochemical biosensor based on a porous membrane and its preparation method and application, which is a new technology for detection by placing a composite membrane on an ITO glass electrode as a working electrode to realize a classic three-electrode system.

[0004] The technical scheme of the present invention is as follows: a photoelectrochemical biosensor based on a porous membrane, with a nuclear pore membrane as the porous membrane, a Z-type heterojunction gC 3 N 4 @In 2 S 3 Taking photosensitive materials as an example, the specific preparation steps are as follows:

[0005] Z-type heterojunction gC 3 N 4 @In 2 S 3 Preparation: gC was prepared by hydrothermal method 3 N4 @In 2 S 3 Composite material; after the hydrothermal reaction is completed, the composite material is centrifuged and dried using ethanol and ultrapure water, and then sintered in a muffle furnace and then cooled;

[0006] Preparation of photosensitive material modified by porous membrane on ITO conductive glass: ultrasonic cleaning of ITO conductive glass in an ultrasonic cleaning machine and drying; coating the nucleopore membrane on the ITO substrate, and then forming a Z-type heterojunction gC 3 N 4 @In 2 S 3 Place in the aqueous solution, stir evenly, and immerse the ITO substrate coated with the nuclear pore membrane into the mixed solution;

[0007] Preparation of photoelectrochemical biosensor: ITO conductive glass loaded with porous membrane modified photosensitive material was immersed in 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) aqueous solution for 2 hours, and rinsed thoroughly with pure water. After rinsing, it was dried, and then the treated membrane was immersed in a capture probe solution with amino groups overnight. Finally, the modified membrane was washed three times with distilled water and dried at room temperature. Finally, the capture probe functionalized composite membrane was immersed in a bovine serum albumin (BSA) aqueous solution, then washed with deionized water and dried in the air to prepare a porous membrane-based photoelectrochemical biosensor.

[0008] The solution of the hydrothermal method of the technical solution is gC 3 N 4 and InCl 2 and TAA mixed solution.

[0009] The technical solution has an EDC concentration of 10 to 15 mg / L, an NHS concentration of 3 to 5 mg / L, a probe DNA aqueous solution concentration of (2 to 5 µmol / L, pH 7.0 to 8.0), a probe reaction time of 12 hours, a BSA aqueous solution concentration of (8 to 10 mg / mL, pH 7.0 to 8.0), an immersion time of 1 hour, and is carried out at room temperature.

[0010] In addition, the present invention also provides an application of a photoelectrochemical biosensor based on a porous membrane, the main steps of which are as follows: using an electrochemical workstation to perform a test with a three-electrode system, with a silver chloride electrode as a reference electrode, a platinum wire as a counter electrode, and the prepared ITO conductive glass loaded with a porous membrane modified photosensitive material as a working electrode, the voltage of the working electrode is set to 0.5 to 0.7 V, in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8, with a xenon lamp as a light source and a wavelength of 350 to 380 nm, using a chronoamperometry method to record the photocurrent response value; (2) performing a photocurrent test to obtain the photocurrent response value before incubation; (3) adding a target standard sample as an incubation solution to the capture probe functionalized nanopore of the electrochemical biosensor, and incubating at 35 to 40°C for 20 to 30 minutes. After 40 minutes, the incubation solution was aspirated, washed with deionized water, and the electrolyte solution with the same conditions was added. The test conditions were the same as before, and a photocurrent test was performed to obtain the photocurrent response value after incubation; (4) The changes in the photocurrent response value before and after incubation were compared to analyze whether the target and the probe were bound.

[0011] The target substances are nucleic acids and proteins, and the concentration of the target substances is 0.5 to 2 µmol / L. All tests are performed at room temperature.

[0012] The detection principle of the present invention is: firstly, a layer of nuclear pore membrane is fixed on the ITO conductive glass, and then a photosensitive material Z-type heterojunction gC is modified in the nanopore of the nuclear pore membrane. 3 N 4 @In 2 S 3 , and then the capture probe is fixed on the surface of the nanopore by covalent bond connection, and then the active site is blocked with bovine serum albumin, thus obtaining a photoelectrochemical biosensor based on a porous membrane. Under light, when the capture probe specifically recognizes and captures the target, the photocurrent signal will change accordingly. Since the concentration of the target is closely related to the change in the photocurrent signal, the electrochemical biosensor can achieve accurate identification and quantitative analysis of the target through the change in the output photocurrent signal.

[0013] Compared with the prior art, the beneficial effects of the present invention are at least as follows: the present invention successfully prepares a photoelectrochemical biosensor using a porous membrane and a photosensitive material, and can design specific recognition sequences for different analytes based on the same mechanism by changing the type of capture probe, and can also be used for the detection of other analytes, and has excellent practical application value; the porous membrane loaded on ITO conductive glass is used to replace the traditional metal electrode, and the detection is accurate, the detection time is short, the cost is low, and it has good reproducibility and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0015] Figure 1 Schematic diagram of the photoelectrochemical biosensor detection device of the present invention

[0016] Figure 2 This is an electron microscope image of the nuclear pore membrane in the embodiment of the present invention.

[0017] Figure 3 This is an electron microscope image of a Z-type heterojunction in an embodiment of the present invention.

[0018] Figure 4 Characterization diagram of materials used in the embodiments of the present invention

[0019] Figure 5 The photocurrent response diagram during the preparation process of the embodiment of the present invention is

[0020] Figure 6 The photocurrent response diagram of the target with different concentrations in the embodiment of the present invention is shown in FIG.

[0021] Figure 7 Specific recognition diagram and stability exploration diagram in the embodiment of the present invention DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present invention. In view of this, in one embodiment of the present invention, a photoelectrochemical biosensor is provided, the photoelectrochemical biosensor comprising: an electrode, the electrode comprising ITO conductive glass and a composite film coated on the ITO conductive glass: the composite film refers to modifying a photosensitive material in the nanopores of a porous film, and then connecting a capture probe in the nanopores to obtain a photoelectrochemical biosensor based on a porous film.

[0023] The composite membrane is a nuclear pore membrane, wherein the nanopores of the membrane have modified Z-shaped heterojunctions and capture probes;

[0024] The target can specifically bind to the capture probe, thereby being used to capture the analyte and then detect the analyte.

[0025] In another specific embodiment of the present invention, a method for preparing the above-mentioned photoelectrochemical biosensor is provided: ITO conductive glass loaded with porous membrane modified photosensitive material is immersed in an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2 hours, and thoroughly rinsed with pure water. After rinsing, it is dried, and then the treated membrane is immersed in a capture probe solution with an amino group overnight, and finally the modified membrane is washed three times with distilled water and dried at room temperature; finally, the capture probe functionalized composite membrane is immersed in an aqueous solution of bovine serum albumin (BSA), and then washed with deionized water and dried in air to prepare a photoelectrochemical biosensor based on a porous membrane.

[0026] Wherein, the solution of the hydrothermal method of the technical solution is gC 3 N 4 and InCl 2 and TAA mixed solution;

[0027] Among them, the concentration of EDC in the technical solution is 10 ~ 15 mg / L, the concentration of NHS is 3 ~ 5 mg / L, the concentration of the probe DNA aqueous solution is (2 ~ 5 µmol / L, pH 7.0 ~ 8.0), the probe reaction time is 12 hours, the concentration of the BSA aqueous solution is (8 ~ 10 mg / mL, pH 7.0 ~ 8.0), the immersion time is 1 hour, and it is carried out at room temperature.

[0028] In another specific embodiment of the present invention, an application of a photoelectrochemical biosensor based on a porous membrane is provided, and the main steps are as follows: using an electrochemical workstation to perform a test with a three-electrode system, with a silver chloride electrode as a reference electrode, a platinum wire as a counter electrode, and the prepared ITO conductive glass loaded with a porous membrane modified photosensitive material as a working electrode, the voltage of the working electrode is set to 0.5 ~ 0.7 V, in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 ~ 8, using a xenon lamp as a light source, and a wavelength of 350 ~ 380 nm, using a chronoamperometry method to record the photocurrent response value; (2) performing a photocurrent test to obtain the photocurrent response value before incubation; (3) adding a target standard sample as an incubation solution to the capture probe functionalized nanopore of the electrochemical biosensor, and incubating at 35 ~ 40 ° C for 20 ~ 30 minutes. After 40 minutes, the incubation solution was aspirated, washed with deionized water, and the electrolyte solution with the same conditions was added. The test conditions were the same as before, and a photocurrent test was performed to obtain the photocurrent response value after incubation; (4) The changes in the photocurrent response value before and after incubation were compared to analyze whether the target and the probe were bound.

[0029] Wherein, the target is nucleic acid or protein, the concentration of the target is 0.5 ~2 µmol / L and all tests are performed at room temperature;

[0030] Experimental Results

[0031] This example uses scanning electron microscopy (SEM) as a powerful technique to help observe the morphology and structure of the prepared materials. Figure 3 As shown, gC after solvothermal treatment 3 N 4 @In 2 S 3 The structure of the composite material can be clearly seen, with irregular layers unevenly covering the In 2 S 3 Surface, indicating gC 3 N 4 In the spherical 2 S 3 The surface was successfully coated. Although the two monomers are combined to make In 2 S 3 The surface is rough, but the single In 2 S 3 The spherical structure of gC remains almost unchanged, and good contact is established at the interface. This unique hierarchical structure makes gC 3 N 4 @In 2 S 3 It has a larger specific surface area and provides more active sites for the reaction.

[0032] Material characterization: In this example, X-ray diffraction (XRD) was used to determine gC 3 N 4 @In 2 S 3 By analyzing the phase structure of Figure 4 Shown gC 3 N 4 @In 2 S 3 The XRD spectrum shows that the diffraction peaks near 13.58° and 27.85° correspond to the two-dimensional layered structure gC 3 N 4 @In 2 S 3 The (002) lattice plane. 3 N 4 @In 2 S 3 The XRD spectrum of the sample has only one diffraction peak at 27.35°, corresponding to the flower-like spherical structure In 2 S 3The (102) lattice plane of the particles may be due to the gC 3 N 4 @In 2 S 3 The crystallinity of gC is weak and the content is low. 3 N 4 @In 2 S 3 The composite material does not lead to a significant loss of crystallinity and a significant loss of crystalline components. In summary, we have successfully prepared gC 3 N 4 @In 2 S 3 Composite materials.

[0033] This example uses Fourier transform infrared spectroscopy (FT-IR) to record Figure 4 The spectrum shown. 3 N 4 @In 2 S 3 In the composite material, gC 3 N 4 andIn 2 S 3 The characteristic peaks of gC 3 N 4 The center is 812 cm -1 The characteristic peak intensity at gC is significantly lower than that at gC 3 N 4 , which may be due to gC 3 N 4 Closely attached to In 2 S 3 The FTIR results are consistent with other characterization results, proving that gC 3 N 4 @In 2 S 3 Successful construction of heterojunction.

[0034] Experimental verification of the principle: In order to prove the feasibility of this technical solution, this implementation case used a method of preparing a photoelectrochemical biosensor using different modified electrodes. Figure 5 As shown, a Z-type heterojunction gC is coated on the ITO conductive glass. 3 N 4 @In 2 S 3 After the nucleopore membrane was formed, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8. A photocurrent value of 745 μA was observed, indicating that the Z-type heterojunction gC3 N 4 @In 2 S 3 The nuclear pore membrane has good photocurrent performance; after activation with EDC and NHS, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8, and a photocurrent value of 725 µA was observed; after modification of the capture probe, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8, and a photocurrent value of 702 µA was observed; after blocking with BSA, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8, and a photocurrent value of 674 µA was observed; after incubation with the target, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8, and a photocurrent value of 244 µA was observed, which was much smaller than the photocurrent value before incubation, indicating that when the target is present, it specifically recognizes the probe, thereby changing the transfer state of the photogenerated electron-hole pairs on the electrode surface, causing a change in the photocurrent.

[0035] Sensitivity experiment: In order to evaluate the sensitivity of the detection target of this technical solution, the photocurrent of the target with different concentrations was measured under the optimal experimental conditions. Figure 6 As shown, the photocurrent decreases as the concentration of the target increases. At the same time, there is a certain functional relationship between the change in photocurrent and the concentration of the target. Therefore, the target can be analyzed by photoelectrochemical biosensors. By combining different photoelectrically active substances and different biorecognition elements, photoelectrochemical biosensors with different detection functions can be constructed.

[0036] Specificity and stability experiments: In order to study the specificity of the photoelectrochemical biosensor, e.g. Figure 7 As shown, this embodiment uses four mismatches and one specific target as a control to perform a specific experiment; the results are shown in Figure 6 As shown, under the same reaction conditions, the photocurrent difference of the target object is much greater than that of the non-target object.

[0037] In order to study the stability of the photoelectrochemical biosensor, five parallel photocurrent responses were carried out in PBS buffer solution with a concentration of 0.1 mol / L and a pH of 6 to 8. The results showed that the photoelectrochemical biosensor has excellent stability.

[0038] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a photoelectrochemical biosensor based on a porous membrane, characterized in that: The nuclear pore membrane is used as the porous membrane, and the Z-type heterojunction g-C3N4@In2S3 is used as the photosensitive material. The specific preparation steps are as follows: Step 1: Preparation of Z-type heterojunction g-C3N4@In2S3: The g-C3N4@In2S3 composite material is prepared by a hydrothermal method; after the hydrothermal reaction, the composite material is centrifuged and dried using ethanol and ultrapure water, and then sintered in a muffle furnace and then cooled; Step 2: Preparation of photosensitive material modified by porous membrane loaded on ITO conductive glass: The ITO conductive glass is placed in an ultrasonic cleaning machine for ultrasonic cleaning and drying; The nuclear pore membrane is coated on the ITO substrate, and then the Z-type heterojunction g-C3N4@In2S3 is placed in an aqueous solution, stirred evenly, and the ITO substrate coated with the nuclear pore membrane is immersed in the mixed A nuclear pore membrane with photosensitive materials modified in the nanopores is obtained in the solution; Step three: Preparation of photoelectrochemical biosensor: ITO conductive glass loaded with nuclear pore membrane modified photosensitive materials is immersed in an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and rinsed thoroughly with pure water. After rinsing, it is dried, and then the treated membrane is immersed in a capture probe solution with amino groups overnight, and finally the modified membrane is washed three times with distilled water and dried at room temperature; finally, the composite membrane functionalized with capture probes is immersed in an aqueous solution of bovine serum albumin (BSA), and then rinsed with deionized water and dried in the air to prepare a photoelectrochemical biosensor based on a porous membrane.

2. The use of the photoelectrochemical biosensor prepared by the method for preparing a photoelectrochemical biosensor based on a porous membrane according to claim 1, characterized in that: The specific detection process is as follows: (1) an electrochemical workstation is used to perform the test using a three-electrode system, with a silver chloride electrode as the reference electrode, a platinum wire as the counter electrode, and the prepared ITO conductive glass loaded with a porous membrane modified with a photosensitive material as the working electrode. In a PBS buffer solution, a xenon lamp is used as the light source, and the chronoamperometry method is used to record the photocurrent response value; (2) a photocurrent test is performed to obtain the photocurrent response value before incubation; (3) a target standard sample is added as an incubation solution into the functionalized nanopore of the capture probe of the electrochemical biosensor. After incubation, the incubation solution is aspirated, washed with deionized water, and an electrolyte solution with the same conditions is added. The test conditions are the same as before, and a photocurrent test is performed to obtain the photocurrent response value after incubation; (4) the changes in the photocurrent response values ​​before and after incubation are compared to analyze whether the target and the probe are bound.

3. The use of a photoelectrochemical biosensor based on a porous membrane according to claim 2, characterized in that: The targets are nucleic acids and proteins, and all tests are performed at room temperature.

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