CN112098397B

CN112098397B - Flexible PTEPB photoelectrode, preparation method and application

Info

Publication number: CN112098397B
Application number: CN202010984673.0A
Authority: CN
Inventors: 李曹龙; 王飞; 唐俊彦
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2022-10-14
Anticipated expiration: 2040-09-17
Also published as: CN112098397A

Abstract

The invention discloses a flexible PTEPB photoelectrode, a preparation method and application. The photoelectrode comprises an ITO polyester substrate and a PTEPB layer attached to the surface of the substrate. The flexible PTEPB photoelectrode with visible light response is obtained by synthesizing a conjugated organic polymer PTEPB containing a 1, 3-diyne bond structure on the surface of an ITO polyester substrate. The monoclonal antibody of the COVID-19S protein is fixed on the surface of the photoelectrode by a glutaraldehyde covalent cross-linking method, so that the photoelectrode can specifically recognize a target antigen. Because the formed antigen-antibody complex can block the transmission of electrons and the reduction of dissolved oxygen on the surface of the photoelectrode, the change of the induced photocurrent signal can realize the rapid detection of COVID-19. The flexible photoelectrode improves the detection accuracy, is easy to prepare and cut in batches, and is suitable for large-batch centralized preliminary diagnosis and screening.

Description

Flexible PTEPB photoelectrode, preparation method and application

Technical Field

The invention relates to a photoelectrode, a preparation method and application, in particular to a flexible PTEPB photoelectrode, a preparation method and application.

Background

Since the end of 2019, acute respiratory infectious diseases caused by a novel coronavirus (COVID-19) have spread widely in various national regions around the world. The prior research shows that the infection way of the COVID-19 mainly comprises droplet transmission and contact transmission, and has a strong popular form for people. Because the incubation period of the COVID-19 infected human body is long and the COVID-19 has the ability of human transmission in the incubation period, a large number of suspected cases and recessive infectors can become potential infection sources, and before the specific vaccine can be clinically applied, future epidemic prevention measures have the characteristics of normalization and persistence, which puts higher requirements on the prevention and control of the virus, especially on the early rapid detection, diagnosis and screening of the virus infection. Meanwhile, in order to ensure the sufficiency of gold standard detection reagent resources, diversified qualitative and quantitative analysis methods are developed, and a hierarchical detection and diagnosis system is also imperative to be constructed.

Epidemic outbreaks to date, various diagnostic reagents have been developed for the nucleic acid of COVID-19 and IgM antibody produced by human body after infection. The nucleic acid detection is mostly based on a fluorescent quantitative PCR technology, has high accuracy and is the current gold standard for diagnosis. However, nucleic acid detection requires expensive instruments and equipment, the operation process is relatively complicated, and the average time consumption is about several hours, so that the requirement of large-scale rapid detection and screening is difficult to meet. The existing colloidal gold test paper for IgM antibody can obtain a test result in about 10-15 minutes, is convenient and quick, but because IgM is generated later than viruses are replicated, the time window of the test method is relatively narrow. Compared with nucleic acid detection and antibody detection, the antigen detection method can effectively reduce the false negative problem of nucleic acid detection and the false positive problem of antibody detection, and has the advantages of rapid diagnosis and low requirement on hardware of equipment. The antigen detection is combined with the photoelectrochemistry immunoassay technology, and the advantages of the specific combination of the monoclonal antibody and the target antigen and the quick response of the photoelectrochemistry analysis method can be relied on. Realize the rapid identification and detection of the antigen protein.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide an electrode containing a 1, 3-conjugated diyne bond structure and a visible light response conjugated organic polymer PTEPB and a preparation method thereof, and the flexible PTEPB photoelectrode easy to cut and process can be obtained by in-situ polymerization on the surface of an ITO polyester electrode.

Another purpose of the invention is to realize photoelectrochemical immunoassay of the S protein by modifying and fixing a COVID-19 spike protein (S protein) antibody on the surface of a PTEPB photoelectrode by a glutaraldehyde covalent crosslinking method.

The technical scheme is as follows: the invention provides a flexible PTEPB photoelectrode, which comprises an ITO polyester substrate and a PTEPB layer attached to the surface of the substrate.

Further, the photovoltaic electrode with the surface attached with PTEPB is a p-type semiconductor material.

The preparation method of the flexible PTEPB photoelectrode comprises the steps of overlapping an ITO polyester substrate and a copper sheet, placing the overlapped ITO polyester substrate and the copper sheet into a pyridine solution of 1,3, 5-tri (4-ethynylphenyl) benzene, heating for polymerization reaction, taking out the polyester substrate after the reaction is finished, washing the polyester substrate by an organic solvent, and drying to obtain the photoelectrode with the PTEPB attached to the surface.

The concentration of the 1,3, 5-tri (4-ethynylphenyl) benzene is preferably 0.5-2.0 mg/mL, and as the reaction proceeds, terminal alkyne in the 1,3, 5-tri (4-ethynylphenyl) benzene is coupled under the catalysis of copper, the molecular weight gradually increases, and finally the high molecular polymer is formed. After the reaction time reaches 24h, PTEPB can be stably attached to the surface of the ITO polyester substrate and can generate cathode photocurrent signals under the irradiation of visible light, so that 24h is taken as the optimal parameter of the polymerization reaction time of the 1,3, 5-tri (4-ethynylphenyl) benzene.

The organic solvent used for washing the PTEPB photoelectrode in the step (1) is preferably any one or a combination of more of pyridine, N-dimethylformamide, dichloromethane, methanol, ethanol and acetone. Most preferred are pyridine, dichloromethane, methanol sequentially.

The PTEPB prepared by the method is attached to the surface of an ITO polyester substrate in the form of a film, can be stretched, bent or twisted along with the substrate, and is not easy to break or fall off. Based on the advantage of the flexible substrate, the PTEPB photoelectrode can be flexibly cut in size and shape according to the needs of those skilled in the art during the actual use. For conventional electrochemical analysis, the PTEPB photoelectrode designed by the invention is in a rectangular shape of 1cm multiplied by 2 cm.

The flexible PTEPB photoelectrode is used as an immunosensor for photoelectrochemical immunoassay.

The analysis method comprises the following steps:

(1) Dripping glutaraldehyde water solution on the surface of a PTEPB electrode, and standing at room temperature;

(2) Dripping the COVID-19S protein monoclonal antibody solution on the surface of the electrode, and crosslinking the antibody with glutaraldehyde to obtain a PTEPB/Ab electrode;

(3) Then, dripping neutral Tris-HCl buffer solution containing bovine serum albumin on the surface of the electrode, and blocking the free binding active site to obtain a PTEB/Ab/BSA electrode;

(4) Dripping COVID-19 spike protein antigen solutions with different concentrations on the surface of the electrode, incubating in a constant temperature incubator, and specifically combining the antigen and the antibody to form an antigen-antibody compound to obtain a PTEPB/Ab/BSA/Ag electrode;

(5) Taking a PTEPB/Ab/BSA/Ag electrode as a working electrode, a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, recording the change of a photocurrent signal of the antibody modified photoelectrode after combining with the antigen, and identifying and detecting the target antigen.

The ITO polyester substrate is a polyester film with a conductive layer composed of indium oxide and tin dioxide attached to the surface. Because the polyester substrate has good flexibility, the cutting processing can be flexibly carried out according to the required size in the using process. The ITO polyester substrate is soaked and cleaned in solvents such as acetone, ethanol, deionized water and the like in advance to remove surface impurity pollutants, and then is dried by argon for later use.

The copper sheet is preferably subjected to ultrasonic cleaning in 3M hydrochloric acid, methanol, ethanol and other solutions in advance to remove impurities such as surface oxides and the like, and then is dried by argon for later use.

The PETPB of the present invention is "poly (1, 3, 5-tris (4-ethynylphenyl) benzene)". The invention utilizes copper to catalyze the end alkyne of 1,3, 5-tri (4-ethynylphenyl) benzene to carry out coupling reaction in an alkaline pyridine solvent to generate the conjugated organic polymer PTEB containing a 1, 3-conjugated diyne bond structure. In the reaction process, univalent and bivalent copper ions generated in the copper sheet can be diffused to the surface of the ITO polyester substrate, so that the coupling reaction can be carried out on the ITO surface, and finally the photoelectrode with the PTEPB attached to the surface is obtained.

The concentration of the glutaraldehyde aqueous solution in the step (1) is preferably 2.5wt%. The operation provides covalent cross-linking binding sites for subsequent antibody modification by introducing aldehyde groups on the surface of PTEPB. The specific reaction between aldehyde groups in glutaraldehyde and amino groups in the antibody helps to more stably immobilize the antibody to the electrode surface.

The COVID-19S protein monoclonal antibody in the step (2) is diluted into antibody solutions with the concentrations of 1, 2, 4, 6, 8 mu g/mL and the like by Tris (hydroxymethyl) aminomethane-hydrochloric acid (Tris-HCl) buffer solution. The present invention preferably has 4. Mu.g/mL as the optimum concentration of the modified antibody.

The step (2) involves multiple coating modification operations, and after each operation, the surface of the electrode needs to be washed to remove unbound and firm free molecules on the surface. The washing solution used is preferably Tris-HCl buffer pH 7.0 containing 0.05vt% Tween-20.

Furthermore, the concentration of the COVID-19 spike protein monoclonal antibody solution is 1-8 mug/mL.

Further, the incubation time is 15 min-120 min.

Has the advantages that: the invention has the following advantages:

according to the invention, the PTEPB photoelectrode with good flexibility is prepared by in-situ polymerization on the surface of the ITO polyester substrate, is easy to cut and process, and is more flexible in application compared with the traditional solid rigid electrode. And the PTEPB serving as the photoresponse material is attached to the surface of the substrate in a film form, a high-molecular adhesive is not required to be introduced to fix the material, the PTEPB is not easy to fall off in the using process, and compared with a photoelectrode constructed by a powder material, the PTEPB has better stability and preparation reproducibility.

The PTEPB has abundant benzene rings and 1, 3-diyne bonds which are alternately connected to form a long-range delocalized conjugated structure, so that the polymer can absorb visible light, realize photoelectric analysis under the irradiation of the visible light, and effectively avoid the problem of functional failure caused by the damage of ultraviolet light to biomolecules on the surface of an electrode. In addition, as a p-type organic semiconductor material, the PTEPB is not easy to react with electron donating substances in a sample under illumination, so that the interference of reducing molecules in photoelectrochemical analysis can be better avoided, and the PTEPB has more advantages in detection application of actual biological samples.

The sensing electrode used by the photoelectrochemistry immunoassay method provided by the invention is easy to miniaturize and prepare in batches, and can be widely used as a portable detection platform, thereby providing help for large-scale screening and diagnosis work. Meanwhile, the antigen analysis method designed aiming at the COVID-19S protein can also supplement the existing nucleic acid detection method and antibody detection method so as to further improve the accuracy of the detection result and reduce the occurrence of false positive, false negative and other false detection results.

Drawings

FIG. 1 is the UV-VIS diffuse reflectance spectrum of the PTEPB photoelectrode prepared in example 1;

FIG. 2 is a schematic view of a flow chart for preparing the photoelectrochemical immunosensor in example 2;

FIG. 3 is the electrochemical AC impedance profile (antigen concentration 0.1 ng/mL) of the PTEPB (a), PTEPB/Ab (b), PTEPB/Ab/BSA (c), PTEPB/Ab/BSA/Ag, and (d) electrodes of example 5;

FIG. 4 shows photocurrent curves (antigen concentration: 0.1 ng/mL) of PTEPB (a), PTEPB/Ab (b), PTEPB/Ab/BSA (c), PTEPB/Ab/BSA/Ag, and (d) electrodes in example 5.

Detailed Description

Example 1 preparation of Flexible PTEPB photoelectrode

And (3) cleaning the ITO polyester electrode with the size of 1cm multiplied by 2cm in acetone, ethanol and deionized water in sequence, and drying under argon flow for later use after cleaning. And (3) ultrasonically cleaning a copper sheet with the area equal to that of the ITO polyester electrode in 3M hydrochloric acid, methanol and ethanol in sequence, drying the copper sheet under argon flow after cleaning is finished, and quickly transferring the copper sheet laminated on the surface of the ITO polyester electrode into a reaction bottle. Adding 1.0mg/mL pyridine solution of 1,3, 5-tri (4-ethynylphenyl) benzene into a reaction bottle, continuously reacting for 24h at 60 ℃, taking out an ITO polyester electrode with PTEPB attached to the surface after the reaction is finished, and sequentially washing with pyridine, dichloromethane and methanol to obtain the PTEPB photoelectrode. Fig. 1 is an ultraviolet-visible diffuse reflection spectrum of a PTEPB photoelectrode. The graph shows that PTEPB has obvious absorption in the visible region, and the absorption edge is at 556nm. The high visible light utilization rate enables the PTEPB to generate photoelectric signals without being excited by ultraviolet light in the photoelectric analysis process, and further avoids the damage of high-energy ultraviolet light to biological recognition units such as electrode surface modification antibodies and the like.

Example 2 construction of PTEPB photoelectrochemical immunosensor

The surface of the PTEPB photoelectrode obtained in example 1 was coated with 20. Mu.L of a 2.5wt% glutaraldehyde aqueous solution by drops, and left to stand at room temperature for 30min, followed by washing with Tris-HCl buffer (pH 7.0,0.05vt% Tween-20). And dripping 20 mu L of 2 mu g/mL COVID-19S protein monoclonal antibody solution on the surface of the electrode, then placing the electrode in a low-temperature environment at 4 ℃ for 16h to ensure that glutaraldehyde on the surface of the electrode is fully crosslinked with amino in the antibody, so that the antibody can be stably fixed on the electrode, and washing the electrode by using Tris-HCl buffer solution to obtain the PTEPB/Ab antibody modified electrode. mu.L of Tris-HCl blocking buffer (pH 7.0,3wt% BSA) was applied dropwise to the electrode surface and maintained at 4 ℃ for 2h in a low temperature environment to complete the blocking of the free binding sites by BSA, and after washing with Tris-HCl buffer, the PTEPB/Ab/BSA electrode was obtained and used as an immunosensor.

20 mu L of 1.0ng/mL COVID-19S protein antigen solution is dripped to the surface of a PTEPB/Ab/BSA electrode, and the electrode is placed in a constant temperature incubator at 37 ℃ for standing and incubation for 30min, so that the antibody and the antigen generate specific recognition and binding reaction, and a stable antigen-antibody compound is formed on the surface of the electrode. And after the incubation process is finished, taking out the electrode, washing unbound and firm free molecules by using Tris-HCl buffer solution, and carrying out photoelectrochemical analysis and detection by using the PTEPB/Ab/BSA/Ag electrode. The process flow of modification of the PTEPB photoelectrode and construction of the immunosensor can be referred to fig. 2.

Taking a PTEPB/Ab/BSA/Ag electrode as a working electrode, a platinum wire electrode and a silver/silver chloride electrode as a counter electrode and a reference electrode respectively, immersing the three electrodes into a pH 7.0Tris-HCl buffer solution, and irradiating the buffer solution with a 500W xenon lamp light source (lambda is more than 420nm, and the light intensity is 80 mW/cm) ² ) Next, the photocurrent signal change of the working electrode under-0.4V bias was recorded to determine the immunosensor response to the target antigen.

Example 3

The surface of the PTEPB photoelectrode obtained in example 1 was coated with 20. Mu.L of a 2.5wt% glutaraldehyde aqueous solution by drops, and left to stand at room temperature for 30min, followed by washing with Tris-HCl buffer (pH 7.0,0.05vt% Tween-20). 20 mu L of 4 mu g/mL COVID-19S protein monoclonal antibody solution is dripped on the surface of the electrode, and then the electrode is placed in a low-temperature environment at 4 ℃ for 16h. And (4) washing by using a Tris-HCl buffer solution to obtain the PTEPB/Ab antibody modified electrode. mu.L of Tris-HCl blocking buffer (pH 7.0,3wt% BSA) was applied dropwise to the electrode surface and maintained at 4 ℃ for 2h in a low temperature environment. After being washed by Tris-HCl buffer solution, the PTEPB/Ab/BSA electrode is obtained and used as an immunosensor.

20 mu L of 1.0ng/mL COVID-19S protein antigen solution is dripped to the surface of a PTEPB/Ab/BSA electrode, and the electrode is placed in a constant temperature incubator at 37 ℃ for standing and incubation for 30min. And after the incubation process is finished, taking out the electrode, washing unbound and firm free molecules by using Tris-HCl buffer solution to obtain the PTEPB/Ab/BSA/Ag electrode for photoelectrochemical analysis and detection.

Taking a PTEPB/Ab/BSA/Ag electrode as a working electrode, a platinum wire electrode and a silver/silver chloride electrode as a counter electrode and a reference electrode respectively, immersing the three electrodes into a Tris-HCl buffer solution with the pH value of 7.0, and irradiating by a 500W xenon lamp light source (the lambda is more than 420nm, and the light intensity is 80mW/cm ² ) Next, the photocurrent signal change of the working electrode under-0.4V bias was recorded to determine the immunosensor response to the target antigen.

Example 4

The surface of the PTEPB photoelectrode obtained in example 1 was coated with 20. Mu.L of a 2.5wt% glutaraldehyde aqueous solution by drops, and left to stand at room temperature for 30min, followed by washing with Tris-HCl buffer (pH 7.0,0.05vt% Tween-20). 20 mu L of 6 mu g/mL COVID-19S protein monoclonal antibody solution is dripped on the surface of the electrode, and then the electrode is placed in a low-temperature environment at 4 ℃ for 16h. And (4) washing by using a Tris-HCl buffer solution to obtain the PTEPB/Ab antibody modified electrode. mu.L of Tris-HCl blocking buffer (pH 7.0,3wt% BSA) was applied dropwise to the electrode surface and maintained at 4 ℃ for 2h in a low temperature environment. After being washed by Tris-HCl buffer solution, the PTEPB/Ab/BSA electrode is obtained and used as an immunosensor.

A PTEPB/Ab/BSA/Ag electrode is used as a working electrode, a platinum wire electrode and a silver/silver chloride electrode are respectively used as a counter electrode and a reference electrode, the three electrodes are immersed into a pH 7.0Tris-HCl buffer solution, and the change of a photocurrent signal of the working electrode under-0.4V bias voltage is recorded under the irradiation of a 500W xenon lamp light source (lambda is more than 420nm, and the light intensity is 80mW/cm & lt 2 & gt) so as to judge the response of the immunosensor to a target antigen.

Example 5 application of PTEPB photoelectrochemical immunosensor to detection of new coronavirus S protein

20 mu.L of 0.001,0.01,0.1,1.0, 10, 100ng/mL COVID-19S protein antigen solution with different concentrations is dripped on the surface of a PTEPB/Ab/BSA electrode, and the electrode is placed in a 37 ℃ constant temperature incubator for standing and incubation for 1h. And taking out the electrode after the incubation process is finished, and washing unbound and firm free molecules by using Tris-HCl buffer solution to obtain the PTEPB/Ab/BSA/Ag electrode for photoelectrochemical analysis and detection. Taking a PTEPB/Ab/BSA/Ag electrode as a working electrode, a platinum wire electrode and a silver/silver chloride electrode as a counter electrode and a reference electrode respectively, immersing the three electrodes into a Tris-HCl buffer solution with the pH value of 7.0, and irradiating by a 500W xenon lamp light source (the lambda is more than 420nm, and the light intensity is 80mW/cm ² ) Next, the photocurrent signal change of the working electrode under-0.4V bias was recorded.

In order to confirm the influence of each modification process on the surface properties of the electrode, the properties of the electrode interface are characterized by adopting an electrochemical alternating-current impedance spectrum, and the result is shown in fig. 3. Electrochemical AC impedance spectroscopy test in the presence of 5mMK ₃ [Fe(CN) ₆ ]/K ₄ [Fe(CN) ₆ ](1: 1) in a 0.1M KCl solution, and the diameter of a semi-circle area corresponding to a high-frequency area in a spectrogram reflects the size of the charge transfer resistance on the surface of the electrode. The four curves a, b, c and d in the figure correspond to the impedance spectra of PTEB, PTEB/Ab/BSA and PTEB/Ab/BSA/Ag electrodes (antigen concentration 0.1 ng/mL), respectively. After the antibody and the BSA are modified in sequence, the surface resistance of the electrode is increased, because the antibody and the BSA are biological macromolecules with poor conductivity, the modification on the surface of the electrode is not favorable for electron transmission, and the transmission of interface charges is blocked. The results also show thatThe antibody as a recognition unit is successfully modified on the surface of the electrode, so that the PTEPB photoelectrode can specifically recognize target antigens. The resistance of PTEPB/Ab/BSA/Ag is the greatest, indicating that the formation of antigen-antibody complexes further impedes electron transport.

The four curves a, b, c, d in FIG. 4 correspond to the photocurrent signals of PTEPB, PTEPB/Ab/BSA and PTEPB/Ab/BSA/Ag electrodes under-0.4V bias, respectively. Under the irradiation of light, the unmodified PTEPB electrode can generate a remarkable cathode photocurrent, which indicates that the PTEPB belongs to a p-type semiconductor, and the generation of the photocurrent is related to the reduction of photo-generated electrons received by dissolved oxygen on the surface of the electrode. After the antibody and BSA are modified, the photocurrent intensity is reduced along with the obstruction of electron transmission, and the photocurrent intensity corresponds to the change of an impedance spectrogram. When the antigen and the antibody are combined to form a compound, the steric hindrance of the electrode surface is increased, so that dissolved oxygen molecules in the electrolyte are not easily diffused to the PTEPB surface for reduction, and the photoelectric current of the electrode is further reduced.

The results show that the PTEPB photoelectrode provided by the invention can detect and identify the S protein on the surface of the new coronavirus after gradual modification. The immunosensor can respond to a target antigen within the concentration range of 0.001-100 ng/mL, the detection limit is 0.86pg/mL, the immunosensor can be used for rapid, large-batch and centralized primary diagnosis and screening, the existing nucleic acid detection and antibody detection methods are supplemented, and the accuracy of detection results is improved.

Claims

1. Use of a flexible PTEPB photoelectrode, characterized in that: the flexible PTEPB photoelectrode is used as an immunosensor for photoelectrochemical immunoassay,

the flexible PTEPB photoelectrode comprises an ITO polyester substrate and a PTEPB layer attached to the surface of the substrate;

the preparation method of the flexible PTEPB photoelectrode comprises the steps of overlapping an ITO polyester substrate and a copper sheet, placing the overlapped ITO polyester substrate and the copper sheet in pyridine solution of 1,3, 5-tri (4-ethynylphenyl) benzene, heating for polymerization reaction, taking out the polyester substrate after the reaction is finished, washing the polyester substrate by using an organic solvent, and drying to obtain the photoelectrode with the PTEPB attached to the surface, wherein the photoelectrode with the PTEPB attached to the surface is a p-type semiconductor material;

the analysis method comprises the following steps:

(2) Dripping the COVID-19 spike protein monoclonal antibody solution on the surface of the electrode, and crosslinking the antibody and glutaraldehyde to obtain a PTEB/Ab electrode;

(3) Then, dripping neutral Tris-HCl buffer solution containing bovine serum albumin on the surface of the electrode, and blocking the free binding active site to obtain a PTEPB/Ab/BSA electrode;

(5) And recording the change of a photocurrent signal of the antibody modified photoelectrode after combining with the antigen by taking the PTEPB/Ab/BSA/Ag electrode as a working electrode, the platinum wire electrode as a counter electrode and the silver/silver chloride electrode as a reference electrode, and identifying and detecting the target antigen.

2. Use according to claim 1, characterized in that: the concentration of the COVID-19 spike protein monoclonal antibody solution is 1-8 mug/mL.

3. Use according to claim 1, characterized in that: the incubation time is 15 min-120 min.