Photoelectrochemical sensor as well as preparation method and application thereof

Abstract

The invention discloses a photoelectrochemical sensor and a preparation method and application thereof. The sensor adopts a p-type semiconductor Cu2O modified on an ITO (Indium Tin Oxide) electrode, and can generate a cathode photocurrent signal. Furthermore, an electrode interface is modified to capture antibodies Ab1 and BSA, when a target phosphorylated tau181 protein exists, a probe MZIS-Ab2 is introduced through antigen-antibody specific recognition, and due to energy level matching between MZIS and Cu2O, a photo-induced electron transfer path is changed, so that the polarity of photocurrent is converted to generate anode photocurrent. The developed target-induced photocurrent polarity conversion PEC sensor has excellent anti-interference capability and low background signal, and can avoid false positive and / or negative signals in high-sensitivity analysis of phosphorylated tau181 protein.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical biosensors, and in particular to a photoelectrochemical sensor for detecting phosphorylated tau181 protein, a preparation method thereof, and uses thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive and irreversible cognitive decline. Due to the aging of the global population, AD has become an increasingly serious problem, and its incidence has been increasing year by year. Amyloid plaque deposition and neurofibrillary tangles (NFTs) in the brain are the main pathological features of AD. NFTs are aggregates formed by hyperphosphorylated and misfolded tau proteins in neurons. Tau protein is a microtubule-associated protein that exists in high abundance in neuronal cells. Its binding to microtubules is mainly regulated by serine S / threonine T-directed phosphorylation. However, tau sites are abnormally hyperphosphorylated and lose their ability to bind to microtubules. High abundance of phosphorylated tau181 protein (p-tau181) is a typical pathological hallmark in the brains of AD patients. Therefore, designing and creating a highly accurate phosphorylated tau181 protein detection strategy is of great significance for the diagnosis of Alzheimer's disease.

[0003] Currently, the commonly used diagnostic methods are through cerebrospinal fluid (CSF) analysis, positron emission tomography (PET) and magnetic resonance imaging (MRI), but they are invasive, expensive and time-consuming, and when the disease is discovered based on these traditional imaging methods, the patient is often in the middle or late stage. In recent years, the development of AD marker analysis based on body fluids for early diagnosis of AD has developed rapidly. The use of biological fluids such as blood, urine and saliva to detect biomarkers for early diagnosis and treatment of AD provides an opportunity to alleviate patient suffering and reduce socioeconomic costs. However, since the content of AD markers such as phosphorylated tau181 protein in the blood is extremely low, the development of highly sensitive and highly selective detection and analysis methods is crucial for non-invasive early body fluid detection of AD.

[0004] Photoelectrochemical (PEC) analysis technology has attracted more and more attention in the field of bioanalysis due to the complete separation of the excitation light source and the detection current signal, and has the advantages of low background signal, high sensitivity, low cost and easy operation. PEC immunosensor mainly consists of two parts: photoelectric active material and biorecognition probe. Photoelectric active material is the basis of PEC sensor and the source of photocurrent signal. Its photoelectric conversion efficiency will directly affect the size of photocurrent, and then affect the detection sensitivity of the sensor. Photoelectric active materials are p-type and n-type semiconductors, which generate cathode photocurrent and anode photocurrent respectively. Commonly used biorecognition probes include modified photoelectric active materials and enzyme labels; select photoelectric materials with appropriate energy level matching to improve the separation efficiency of photogenerated carriers, thereby enhancing the photoelectric response. For example, by modifying alkaline phosphatase (ALP) as an enzyme label to catalyze the substrate to generate electron donor ascorbic acid, the photocurrent signal is enhanced. In practical applications, the "signal-on" or "signal-off" strategy is often difficult to avoid the false positive or false negative signal problem caused by the coexistence of various interference factors in real samples. Therefore, there is a need to develop PEC analytical sensing formats that can eliminate false positive and / or negative signals while taking into account high background signals. Summary of the invention

[0005] Based on the above background, the present application provides a photoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising:

[0006] A photocathode used as a signal conversion element, and

[0007] A probe anode used in conjunction with the photocathode to detect phosphorylated tau181 protein in a sample to be tested;

[0008] The photocathode uses a Cu2O-ITO electrode as a photocathode substrate, the surface of which is modified with a first antibody capable of binding to phosphorylated tau181 protein, and BSA is added dropwise to prevent nonspecific adsorption; the probe anode uses MXene-ZnIn2S4 as a probe anode substrate, the surface of which is modified with a second antibody capable of binding to phosphorylated tau181 protein;

[0009] After the photocathode is combined with the phosphorylated tau181 protein, it can be combined with the probe anode, thereby generating a polarity conversion of the photocurrent, thereby realizing sensitive detection of the phosphorylated tau181 protein in the sample to be tested.

[0010] On the other hand, the present application also provides a method for preparing a photoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising the following steps:

[0011] 1) Preparing the photocathode:

[0012] After pre-treating the ITO electrode, Cu2O is added dropwise to the surface of the ITO electrode to obtain the Cu2O-ITO electrode.

[0013] Chitosan (CS) is modified onto the surface of the Cu2O-ITO electrode, and a first antibody capable of binding to phosphorylated tau181 protein is connected to obtain a first antibody-chitosan-Cu2O-ITO electrode.

[0014] Adding a BSA solution dropwise onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain a BSA-first antibody-chitosan-Cu2O-ITO electrode, i.e., the photocathode;

[0015] 2) Preparation of probe anode:

[0016] MXene-ZnIn2S4 (referred to herein as MZIS) is prepared, and a second antibody capable of binding to phosphorylated tau181 protein is connected to the surface of the MXene-ZnIn2S4 to obtain a second antibody-MXene-ZnIn2S4, i.e., the probe anode.

[0017] In another aspect, the present application also provides use of the photoelectrochemical sensor described herein in detecting phosphorylated tau181 protein.

[0018] On the other hand, the present application also provides a method for detecting phosphorylated tau181 protein, the method comprising the following steps:

[0019] 1) Add different concentrations of phosphorylated tau181 protein solutions to the photocathode described in this article, incubate at 37° C. for 1 hour, and then add the probe anode to obtain a MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode;

[0020] 2) using an electrochemical workstation to perform detection using a three-electrode system, with an Ag-AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and the MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode as a working electrode, and performing detection in a solution containing 10 mM Tris-HCl at pH = 7.4;

[0021] 3) The current-time method was used for detection, the voltage was set to 0V, a 300W xenon lamp was used as the irradiation source, and the power density was 200mW / cm 2 ;

[0022] 4) Turn on the light source every 10 seconds for 10 seconds, then record the photocurrent, and record the photocurrent corresponding to different concentrations of phosphorylated tau181 protein to obtain the corresponding regression equation and correlation coefficient;

[0023] 5) replacing the phosphorylated tau181 protein solution in step 1) with the sample solution to be tested, and detecting the photocurrent signal of the sample according to steps 1) to 4) to obtain the concentration of phosphorylated tau181 protein in the sample.

[0024] On the other hand, the present application also provides a photoelectrochemical sensor for detecting phosphorylated tau181 protein prepared using the method described herein.

[0025] This application develops a photoelectrochemical sensing method based on target-induced photocurrent polarity conversion, constructs a p-type Cu2O functionalized indium tin oxide (ITO) electrode modified with phosphorylated tau181 capture antibody (i.e., first antibody, also known as Ab1), and when phosphorylated tau181 is present in the detection solution, due to the antigen-antibody recognition effect, the phosphorylated tau181 protein is specifically adsorbed on the electrode surface, and after adding the MXene-ZnIn2S4 (MZIS-Ab2) probe modified with the labeled antibody (i.e., second antibody, also known as Ab2), a strong anodic photocurrent signal is generated under light conditions. Cu2O and MZIS are p-type semiconductors and n-type semiconductors, respectively. When there is no target phosphorylated tau181, the electrode interface is modified with Cu2O to generate a cathode photocurrent; and when phosphorylated tau181 is present, due to the matching of the energy levels of Cu2O and MZIS, the photocurrent polarity is reversed, generating an anodic photocurrent. This PEC sensing platform has the characteristics of low background signal, high sensitivity and good selectivity, and successfully realizes highly sensitive and selective analysis of phosphorylated tau181 in serum.

[0026] Compared with the existing technology, the beneficial effects of this application are as follows:

[0027] 1. This application is based on the target-induced photocurrent polarity change, which avoids false positive and / or negative signals in the actual detection process. When the target is present, the photocurrent polarity changes after antigen-antibody immune recognition, which is beneficial to improve selectivity.

[0028] 2. The electrode interface of the present application is modified with cuprous oxide to generate a cathode photocurrent signal. After the antibody recognizes the antigen, the probe is modified to generate an anode photocurrent signal. Compared with the unidirectional background photocurrent of traditional photoelectrochemical sensing, the background signal of this analysis method in which the polarity of the photocurrent changes due to the presence of the target is lower, thereby improving the sensitivity of the sensor.

[0029] 3. When the photocurrent polarity conversion PEC immunosensor prepared in this application is used for the detection of phosphorylated tau181 protein, it has a short response time, a wide linear range, and a low detection limit, and can realize the detection of phosphorylated tau181 protein in biological media. The linear range of detection of phosphorylated tau181 protein in this application is 1pg / mL-5000pg / mL, and the detection limit is 1.11pg / mL.

[0030] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0032] Figure 1 The present invention is a flow chart of the preparation of a photoelectrochemical sensor according to one embodiment of the present application.

[0033] Figure 2 The EIS and PEC photoresponses of different electrode modification steps in one embodiment of the present application are shown: (a) bare ITO, (b) Cu2O-ITO, (c) CS-Cu2O-ITO, (d) Ab1-CS-Cu2O-ITO, (e) BSA-Ab1-CS-Cu2O, (f) phosphorylated tau181-BSA-Ab1-CS-Cu2O-ITO (phosphorylated tau181 = 100 pg / mL) and (g) MZIS-Ab2-phosphorylated tau181-BSA-Ab1-CS-Cu2O-ITO.

[0034] Figure 3 The reasons for the change in the polarity of the photocurrent and the photogenerated electron transfer mechanism in one embodiment of the present application are shown.

[0035] Figure 4 The photocurrent response and logarithmic calibration curve of the photoelectrochemical immunosensor to different concentrations of phosphorylated tau181 in one embodiment of the present application are shown. DETAILED DESCRIPTION

[0036] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0037] The terms "about" and "approximately" when used with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0038] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0039] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0040] In one aspect, the present application provides a photoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising:

[0041] A photocathode used as a signal conversion element, and

[0042] A probe anode used in conjunction with a photocathode to detect phosphorylated tau181 protein in a sample to be tested;

[0043] The photocathode uses a Cu2O-ITO electrode as the photocathode substrate, the surface of which is modified with a first antibody that can bind to the phosphorylated tau181 protein, and BSA is added to prevent nonspecific adsorption; the probe anode uses MXene-ZnIn2S4 as the probe anode substrate, and the surface of which is modified with a second antibody that can bind to the phosphorylated tau181 protein;

[0044] After the photocathode binds to the phosphorylated tau181 protein, it can bind to the probe anode, thereby generating a polarity conversion of the photocurrent, thereby achieving sensitive detection of the phosphorylated tau181 protein in the sample to be tested.

[0045] In some embodiments, the first antibody and the second antibody bind to different epitopes of the phosphorylated tau181 protein, respectively.

[0046] In some embodiments, chitosan is used to attach the primary antibody to the Cu2O-ITO electrode surface.

[0047] In some embodiments, other electrodes useful for electrochemistry may be used in place of the ITO electrode.

[0048] On the other hand, the present application also provides a method for preparing a photoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising the following steps:

[0049] 1) Preparation of photocathode:

[0050] After pre-treating the ITO electrode, Cu2O was added dropwise to the surface of the ITO electrode to obtain a Cu2O-ITO electrode.

[0051] Chitosan (CS) was modified onto the surface of the Cu2O-ITO electrode, and a first antibody capable of binding to phosphorylated tau181 protein was connected to obtain a first antibody-chitosan-Cu2O-ITO electrode.

[0052] Adding BSA solution dropwise onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain a BSA-first antibody-chitosan-Cu2O-ITO electrode, i.e., a photocathode;

[0053] 2) Preparation of probe anode:

[0054] MXene-ZnIn2S4 (referred to as MZIS in this article) was prepared, and a second antibody capable of binding to phosphorylated tau181 protein was connected to the surface of MXene-ZnIn2S4 to obtain a second antibody-MXene-ZnIn2S4, i.e., a probe anode.

[0055] In some embodiments, the ITO electrode is pretreated by sequentially immersing the ITO electrode in acetone and 1M NaOH ethanol solution (V 乙醇 :V 水 =1:1) and water, ultrasonically treated for 30 minutes, and then dried for use;

[0056] The preparation method of Cu2O is as follows: copper sulfate pentahydrate and sodium citrate dihydrate are added into water and stirred to dissolve, sodium hydroxide is added, and then ascorbic acid is added for reduction, and after aging at room temperature, centrifugation washing and drying are performed.

[0057] In some embodiments, the preparation method of the BSA-first antibody-chitosan-Cu2O-ITO electrode is:

[0058] The Cu2O-ITO electrode was modified with 0.1wt% chitosan to provide amino groups, and the first antibody was connected by 0.5% glutaraldehyde (GLD) to modify 25 μg / mL of the first antibody to obtain the first antibody-chitosan-Cu2O-ITO electrode;

[0059] 1% BSA solution was added dropwise to the surface of the first antibody-chitosan-Cu2O-ITO electrode to avoid nonspecific adsorption during the detection process, thereby obtaining a BSA-first antibody-chitosan-Cu2O-ITO electrode.

[0060] In some embodiments, the concentration of chitosan is 0.1% (wt%), the concentration of the first antibody is 25 μg / mL, the concentration of BSA is 1%, the incubation time of the first antibody and BSA is 1 h, and the incubation conditions are room temperature; and after each modification step, the electrode surface is rinsed with PBS buffer to remove unbound reactants and dried with N2.

[0061] In some embodiments, the concentration of chitosan can be adjusted according to the selected electrode. In some embodiments, the concentration of BSA and the incubation time used in the present application can be adjusted according to specific needs, as long as BSA can well avoid non-specific adsorption during the detection process.

[0062] In some embodiments, the preparation method of MXene-ZnIn2S4 is:

[0063] Preparation of MXene: LiF was slowly added to the HCl solution and stirred continuously until LiF was completely dissolved, then Ti3AlC2 was added and stirred gently. The reaction was stirred at 35 °C for 24 h. The obtained product was washed several times with deionized water (DI) by centrifugation until the pH reached about 6. Finally, the flake MXene was exfoliated by ultrasound.

[0064] Preparation of MXene-ZnIn2S4: Disperse MXene in water by ultrasonic dispersion, add zinc chloride, indium chloride tetrahydrate, and thioacetamide and stir to dissolve, generate MXene-ZnIn2S4 by hydrothermal reaction, cool, wash, dry and collect by centrifugation.

[0065] In some embodiments, the preparation method of the second antibody-MXene-ZnIn2S4 is: add MXene-ZnIn2S4 to a thioglycolic acid solution and ultrasonically disperse it evenly, stir the reaction at room temperature to provide carboxyl groups to MXene-ZnIn2S4, centrifuge and wash, and then redisperse it in deionized water, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the above solution, oscillate the reaction, and then centrifuge to remove the supernatant; add the second antibody to the obtained precipitate, stir the reaction at room temperature, and then centrifuge to remove the supernatant; add 1% BSA, incubate at room temperature for 1 hour, and then centrifuge to obtain.

[0066] In some embodiments, the method for preparing a photoelectrochemical sensor for detecting phosphorylated tau181 protein described herein comprises the following steps:

[0067] 1) Preparation of photocathode:

[0068] The ITO electrode was immersed in acetone and 1M NaOH ethanol solution (V 乙醇 :V 水 =1:1) and water, ultrasonically treated for 30 minutes, and then dried for use;

[0069] Cu2O was prepared as follows: 1.5mmol copper sulfate pentahydrate and 0.5mmol sodium citrate dihydrate were added to 80mL water and stirred for 15 minutes, then 20mL 1.25M sodium hydroxide was added and stirred for 15min, then 50mL 0.03M ascorbic acid was added and stirred for 3min, aged at room temperature for 1h, and centrifuged to wash and dry;

[0070] Cu2O was added dropwise onto the surface of the ITO electrode to obtain a Cu2O-ITO electrode.

[0071] The Cu2O-ITO electrode was modified with 0.1wt% chitosan to provide amino groups, and the first antibody was connected with 0.5% glutaraldehyde. 25μg / mL of the first antibody was added and incubated at room temperature for 1h to obtain the first antibody-chitosan-Cu2O-ITO electrode;

[0072] 1% BSA solution was added dropwise to the surface of the first antibody-chitosan-Cu2O-ITO electrode and incubated at room temperature for 1 hour to avoid nonspecific adsorption during the detection process, thereby obtaining a BSA-first antibody-chitosan-Cu2O-ITO electrode; wherein, after each modification step, the electrode surface was rinsed with PBS buffer to remove unbound reactants, and then dried with N2;

[0073] 2) Preparation of probe anode:

[0074] Preparation of MXene: 1 g LiF was added to 10 mL 9 M HCl and stirred continuously until completely dissolved. Then 0.5 g Ti3AlC2 powder was slowly added and the reaction was continued at 35 °C and 300 rpm for 24 hours. The obtained product was centrifuged several times with deionized water until the pH reached about 6. The solid obtained by centrifugation was dispersed in 100 mL deionized water and filled with nitrogen for 20 minutes to remove oxygen in the solution to prevent MXene from being oxidized. Ultrasonic treatment was performed for 1 hour. After the solution was centrifuged at 3500 rpm for 1 hour, the supernatant was collected to obtain a dark green MXene solution.

[0075] Preparation of MXene-ZnIn2S4: 8 mg of MXene was dispersed in 30 mL of deionized water, 0.3 mmol of zinc chloride and 0.6 mmol of indium chloride tetrahydrate were added, and the mixture was stirred vigorously for 30 min. 1.8 mmol of thioacetamide was added to the above solution and stirred for another 30 min. The solution was then heated to 95 °C and reacted for 5 h under vigorous stirring. The resulting precipitate was collected by centrifugation and washing.

[0076] Preparation of the second antibody - MXene-ZnIn2S4: Add 10 mg MXene-ZnIn2S4 to 10 mL 2 mM thioglycolic acid solution and disperse it evenly by ultrasonication. Stir the reaction at room temperature for 4 hours, wash it by centrifugation and then redisperse it in 10 mL deionized water. MXene-ZnIn2S4 reacts with TGA to provide carboxyl groups for subsequent antibody modification. Take 1 mL of the above and add 100 μL 40 mM EDC and 20 mM NHS to oscillate for 30 minutes. Centrifuge to remove the supernatant, add 400 μL 25 μg / mL of the second antibody to the resulting precipitate, stir at room temperature for 2 hours or incubate at 37°C for 1 hour, centrifuge to remove the supernatant, add 400 μL 1% BSA, incubate at room temperature for 1 hour, and then centrifuge. Re-disperse it in 400 μL PBS and store it at 4°C until use.

[0077] In some embodiments, the incubation time of MZIS-Ab2 is 60 min, and the incubation condition is 37° C. After each modification step, the electrode surface is rinsed with PBS buffer to remove unbound reactants, and dried with N2.

[0078] On the other hand, the present application also provides a photoelectrochemical sensor for detecting phosphorylated tau181 protein prepared using the method described herein.

[0079] In another aspect, the present application also provides the use of the photoelectrochemical sensor described herein in detecting phosphorylated tau181 protein. In some embodiments, the present application also provides the use of the photoelectrochemical sensor described herein for ultrasensitive detection of phosphorylated tau181 protein.

[0080] On the other hand, the present application also provides a method for detecting phosphorylated tau181 protein, the method comprising:

[0081] 1) Add different concentrations of phosphorylated tau181 protein solutions to the photocathode described in this article, incubate at 37°C for 1 hour, and then add the probe anode to obtain a MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode;

[0082] 2) The detection was performed using an electrochemical workstation with a three-electrode system, with an Ag / AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and a MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode as the working electrode, and the detection was performed in a solution containing 10 mM Tris-HCl at pH = 7.4;

[0083] 3) The current-time method was used for detection, the voltage was set to 0V, a 300W xenon lamp was used as the irradiation source, and the power density was 200mW / cm 2 ;

[0084] 4) Turn on the light source every 10 seconds for 10 seconds, then record the photocurrent, and record the photocurrent corresponding to different concentrations of phosphorylated tau181 protein to obtain the corresponding regression equation and correlation coefficient;

[0085] 5) replacing the phosphorylated tau181 protein solution in step 1) with the sample solution to be tested, and detecting the photocurrent signal of the sample according to steps 1) to 4) to obtain the concentration of phosphorylated tau181 protein in the sample.

[0086] In some embodiments, the incubation time of phosphorylated tau181 protein is 1 hour, and the incubation condition is 37°C.

[0087] The present application uses p-type semiconductor Cu2O as a substrate to generate a cathode photocurrent signal, and connects antibodies and BSA through chitosan glutaraldehyde modification. According to the specific recognition of the antigen and antibody, the antigen phosphorylated tau181 and the MZIS-modified secondary antibody (MZIS-Ab2) are further modified. Due to the relationship between the electronic energy levels of MZIS and Cu2O, an anode photocurrent signal is generated after the probe is modified. After target recognition and modification with MZIS-Ab2, the polarity of the photocurrent changes, thereby avoiding the generation of false positive and / or negative signals during the detection process, improving the selectivity of the detection, and realizing super-selective and sensitive detection of phosphorylated tau181 protein.

[0088] The mechanism of the change in photocurrent polarity is that when Cu2O is modified on the electrode interface, it is excited under visible light irradiation, and corresponding photogenerated electrons (e - ) / hole(h + ). Photogenerated electrons on Cu2O CB - Transferred to the electrolyte as an electron acceptor, O2 is reduced to generate O2 ·- After the modified probe MZIS, the CB of MZIS is more negative than that of Cu2O, and the photogenerated e - The photocurrent direction changes from cathode photocurrent to anode photocurrent when it is transferred from the CB of MZIS to the CB of Cu2O and then to the ITO electrode.

[0089] One of the purposes of this application is to synthesize Cu2O and MXene-ZnIn2S4 (MZIS). Due to the excellent conductivity of two-dimensional MXene, the separation and transport of photogenerated carriers in ZnIn2S4 are improved; the energy levels of Cu2O and MZIS are matched, and the polarity of the photocurrent is converted before and after modification, avoiding false positive and / or negative signals in the actual detection process.

[0090] The second purpose of the present application is to induce a change in the polarity of the photocurrent based on target recognition, modify Cu2O at the electrode interface to produce a cathode photocurrent signal, and modify the probe after the antibody recognizes the antigen to generate an anode photocurrent signal. A photocurrent polarity change occurs, which has the characteristics of a negative background signal, thereby improving the sensitivity of the sensor.

[0091] In some embodiments, the present application can also be used for the detection of other targets. The specific method is to replace the phosphorylated tau181 protein with other targets, replace the first antibody and the second antibody with two antibodies that can respectively bind to different epitopes of the target, and construct a photoelectrochemical sensor capable of detecting the target.

[0092] The present application describes a plurality of embodiments, but the description is exemplary rather than restrictive, and it is apparent to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0093] The present application includes and contemplates combinations of features known to those of ordinary skill in the art. The embodiments and features disclosed in the present application may also be combined with any conventional features to form a unique invention scheme defined by the claims. Any feature of any embodiment may also be combined with features from other invention schemes to form another unique invention scheme defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0094] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0095] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0096] Example

[0097] 1. Preparation of MZIS-Ab2

[0098] 10 mg of MZIS was added to 10 mL of 2 mM thioglycolic acid (TGA) solution and dispersed evenly by ultrasonication. The mixture was stirred at room temperature for 4 hours, washed by centrifugation, and then redispersed in 10 mL of deionized water. MZIS reacted with TGA to provide carboxyl groups for subsequent antibody modification. 1 mL of the above mixture was added with 100 μL of 40 mM EDC and 20 mM NHS and oscillated for 30 minutes, then centrifuged to remove the supernatant. 400 μL of 25 μg / mL phosphorylated tau181 secondary antibody (Hangzhou Jingjie Biotechnology Co., Ltd., catalog number ABT-112009) (Ab2) was added to the resulting precipitate, stirred at room temperature for 2 hours, and the supernatant was removed by centrifugation. 400 μL of 1% BSA was added, incubated at room temperature for 1 hour, and then centrifuged. The mixture was redispersed in 400 μL of PBS and stored at 4°C until use. The preparation process is as follows: Figure 1 As shown in a in .

[0099] 2. Electrode modification and preparation of photoelectrochemical immunosensor interface

[0100] Preparation process Figure 1 As shown in b, it specifically includes the following steps:

[0101] (1) Pretreatment of ITO electrodes

[0102] The ITO electrode was immersed in acetone, 1 M NaOH / ethanol mixture (1:1, v / v), and water in turn, ultrasonically treated for 30 min, and then dried for use;

[0103] (2) Assembly of photochemical immunosensor

[0104] After the ITO electrode was pretreated, 1 mg / mL Cu2O was dripped on the electrode surface and dried to obtain a Cu2O-ITO electrode; subsequently, 0.1 wt% chitosan was modified to provide amino groups, and the first antibody (Ab1) was connected by 0.5% glutaraldehyde to modify 25 μg / mL of phosphorylated tau181 first antibody (Nanjing Zhonghongtai Biotechnology Co., Ltd., product number: Tau181102) (Ab1) to obtain an Ab1-CS-Cu2O-ITO electrode; 1% BSA solution was dripped to avoid nonspecific adsorption during the detection process to obtain a BSA-Ab1-CS-Cu2O-ITO electrode; different concentrations of phosphorylated tau181 protein solutions were dripped and incubated at 37°C for 1 hour, and then the synthesized probe solution MZIS-Ab2 was dripped to obtain an MZIS-Ab2-phosphorylated tau181-BSA-Ab1-CS-Cu2O-ITO electrode, thereby preparing a photoelectrochemical immunosensor for phosphorylated tau181 detection. After each modification step, the sample must be washed with buffer and dried with nitrogen.

[0105] 3. Method for detecting p-tau181 using a photoelectrochemical immunosensor based on target-induced photocurrent polarity conversion

[0106] The detection was carried out using an electrochemical workstation with a three-electrode system. The working electrode was MZIS-Ab2-phosphorylated tau181-BSA-Ab1-CS-Cu2O-ITO electrode, the Ag / AgCl electrode was the reference electrode, and the platinum electrode was the counter electrode. The detection was carried out in a 10 mM Tris-HCl solution at pH = 7.4. A 300 W xenon lamp was used as the irradiation source with a power density of 200 mW / cm 2 , set the voltage to 0V; turn on the light source every 10s for 10s, then record the photocurrent, and record the photocurrent corresponding to different concentrations of antigen to obtain the corresponding regression equation and correlation coefficient.

[0107] Example 1. Performance test of photoelectrochemical sensor

[0108] The photoelectrochemical immunosensor constructed in this application was characterized by using photocurrent-time curve (it curve) and electrochemical impedance spectroscopy (EIS) to characterize the electrode assembly. The it curve was tested at 0V voltage in 10mM Tris-HCl (pH=7.4) electrolyte solution (see Figure 2 a), EIS was performed in 0.2M KC1 electrolyte solution containing 5mM K3[Fe(CN)6] / K4[Fe(CN)6] (see Figure 2 b) in the above. Figure 2 Figure a shows the change in the photocurrent signal during the gradual modification of the electrode. The photocurrent response of the bare ITO electrode is almost 0 (curve a); when modified with Cu2O, a cathode photocurrent is obtained (curve b). After the introduction of CS, Ab1, BSA and phosphorylated tau181 protein on the Cu2O / ITO electrode, the photocurrent gradually decreases due to the non-conductivity of the protein and increased steric hindrance (curve cf). When the PEC immunosensor is incubated with MZIS-Ab2, an obvious anodic photocurrent is generated (curve e), which is due to the change in the transmission path of photoelectrons / holes through the matching energy levels between Cu2O and MZIS. Similarly, Figure 2 Figure b shows the changes in EIS during the gradual modification of the electrode. The impedance increased after the ITO surface was modified with Cu2O, and the conductivity of Cu2O was poor. After Ab1, BSA, and phosphorylated tau181 were fixed on the electrode surface in sequence, the impedance gradually increased. This is because the non-conductive protein molecules hindered the electron transfer rate. When the sensor was incubated with MIZ-Ab2 in the presence of target phosphorylated tau181, the impedance value decreased, indicating that MZIS has good electrochemical properties. The above shows that the construction of the photoelectrochemical immunosensor is successful and effective.

[0109] Example 2. Detection of phosphorylated tau181 protein at a series of concentrations

[0110] The photoelectrochemical immunosensor constructed in this application was used to detect phosphorylated tau181 at different concentrations (1, 2.5, 5, 10, 50, 100, 500, 1000, 5000 pg / mL), and the detection results were recorded using a photocurrent-time curve (it). The intensity of the anode photocurrent increased with the increase in the concentration of phosphorylated tau181 (e.g. Figure 4 The photocurrent intensity is linearly related to the logarithm of the phosphorylated tau181 protein concentration (as shown in a Figure 4 As shown in b, y = 0.2286lgC-0.03176, R 2 =0.957).

[0111] In summary, the target-induced photocurrent polarity conversion photoelectrochemical immunosensor is used to detect the phosphorylated tau181 protein, a marker of Alzheimer's disease. The electrode interface is modified with cuprous oxide to generate a cathode photocurrent signal. After the antibody recognizes the antigen, the probe MZIS-Ab2 is modified, and the photocurrent polarity is converted to generate an anode photocurrent signal. A highly sensitive photoelectrochemical immunosensor is constructed. Under the optimal conditions, the measurement results show that the detection range of phosphorylated tau181 is 1pg / mL-5000pg / mL, and the detection limit is 1.11pg / mL (S / N=3).

Claims

1. A photoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising: A photocathode used as a signal conversion element, and A probe anode used in conjunction with the photocathode to detect phosphorylated tau181 protein in a sample to be tested; The photocathode uses a Cu2O-ITO electrode as a photocathode substrate, the surface of which is modified with a first antibody capable of binding to phosphorylated tau181 protein, and BSA is added dropwise to prevent nonspecific adsorption; the probe anode uses MXene-ZnIn2S4 as a probe anode substrate, the surface of which is modified with a second antibody capable of binding to phosphorylated tau181 protein; After the photocathode is combined with the phosphorylated tau181 protein, it can be combined with the probe anode, thereby generating a polarity conversion of the photocurrent, thereby realizing sensitive detection of the phosphorylated tau181 protein in the sample to be tested.

2. The photoelectrochemical sensor according to claim 1, wherein The first antibody and the second antibody respectively bind to different epitopes of the phosphorylated tau181 protein.

3. The photoelectrochemical sensor according to claim 1 or 2, wherein: The first antibody was attached to the surface of the Cu2O-ITO electrode using chitosan.

4. The method for preparing the photoelectrochemical sensor according to any one of claims 1 to 3, comprising the following steps: 1) Preparing the photocathode: After pre-treating the ITO electrode, Cu2O is added dropwise to the surface of the ITO electrode to obtain the Cu2O-ITO electrode. Chitosan is modified onto the surface of the Cu2O-ITO electrode, and a first antibody capable of binding to phosphorylated tau181 protein is connected to obtain a first antibody-chitosan-Cu2O-ITO electrode, Adding a BSA solution dropwise onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain a BSA-first antibody-chitosan-Cu2O-ITO electrode, i.e., the photocathode; 2) Preparation of probe anode: MXene-ZnIn2S4 is prepared, and a second antibody capable of binding to phosphorylated tau181 protein is connected to the surface of the MXene-ZnIn2S4 to obtain a second antibody-MXene-ZnIn2S4, i.e., the probe anode.

5. The preparation method according to claim 4, wherein: The ITO electrode was pretreated by sequentially immersing the ITO electrode in acetone and 1M NaOH ethanol solution (V 乙醇 :V 水 =1:1) and water, ultrasonically treated for 30 minutes, and then dried for use; The preparation method of Cu2O is as follows: adding copper sulfate pentahydrate and sodium citrate dihydrate into water, stirring and dissolving, adding sodium hydroxide, and then adding ascorbic acid for reduction, aging at room temperature, and centrifuging and washing to dry.

6. The preparation method according to claim 4 or 5, wherein: The preparation method of the photocathode is: Modifying the Cu2O-ITO electrode with 0.1 wt% chitosan to provide amino groups, connecting the first antibody with 0.5% glutaraldehyde, modifying 25 μg / mL of the first antibody, and obtaining the first antibody-chitosan-Cu2O-ITO electrode; 1% BSA solution was dripped onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to avoid nonspecific adsorption during the detection process, thereby obtaining the BSA-first antibody-chitosan-Cu2O-ITO electrode, namely the photocathode.

7. The preparation method according to any one of claims 4 to 6, wherein The preparation method of the MXene-ZnIn2S4 is: Preparation of MXene: LiF was slowly added to the HCl solution and stirred continuously until LiF was completely dissolved, then Ti3AlC2 was added and stirred gently. The reaction was stirred at 35 °C for 24 h. The obtained product was washed several times with deionized water by centrifugation until the pH reached about 6. Finally, the flake MXene was peeled off by ultrasound. Preparation of the MXene-ZnIn2S4: Disperse MXene in water by ultrasonic dispersion, add zinc chloride, indium chloride tetrahydrate, and thioacetamide, stir and dissolve, generate MXene-ZnIn2S4 by hydrothermal reaction, cool, centrifuge, wash, dry and collect.

8. The preparation method according to any one of claims 4 to 7, wherein The preparation method of the probe anode is as follows: adding MXene-ZnIn2S4 to a thioglycolic acid solution for ultrasonic dispersion, stirring the reaction at room temperature to provide carboxyl groups to MXene-ZnIn2S4, centrifuging and washing, and then re-dispersing it in deionized water; adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the above solution for oscillation reaction, and then centrifuging to remove the supernatant; adding the second antibody to the obtained precipitate, stirring the reaction at room temperature, and then centrifuging to remove the supernatant; adding 1% BSA, incubating at room temperature for 1 hour, and then centrifuging to obtain the probe anode.

9. The preparation method according to any one of claims 4 to 8, comprising the following steps: 1) Preparing the photocathode: The ITO electrode was immersed in acetone, 1M NaOH / ethanol mixture and water in turn, ultrasonically treated for 30 minutes, and then dried for use; The Cu2O was prepared as follows: 1.5 mmol of copper sulfate pentahydrate and 0.5 mmol of sodium citrate dihydrate were added to 80 mL of water and stirred for 15 minutes, then 20 mL of 1.25 M sodium hydroxide was added and stirred for 15 minutes, then 50 mL of 0.03 M ascorbic acid was added and stirred for 3 minutes, aged at room temperature for 1 hour, and centrifuged for washing and drying; The Cu2O is added dropwise onto the surface of the ITO electrode to obtain a Cu2O-ITO electrode. Modifying the Cu2O-ITO electrode with 0.1 wt% chitosan to provide amino groups, connecting the first antibody with 0.5% glutaraldehyde, adding 25 μg / mL of the first antibody, and incubating at room temperature for 1 hour to obtain the first antibody-chitosan-Cu2O-ITO electrode; 1% BSA solution was added dropwise to the surface of the first antibody-chitosan-Cu2O-ITO electrode, and incubated at room temperature for 1 hour to avoid nonspecific adsorption during the detection process, thereby obtaining the BSA-first antibody-chitosan-Cu2O-ITO electrode; wherein, after each modification step, the electrode surface was rinsed with PBS buffer to remove unbound reactants, and dried with N2; 2) Preparing the probe anode: Preparation of MXene: 1 g LiF was added to 10 mL 9 M HCl and stirred continuously until completely dissolved. Then 0.5 g Ti3AlC2 powder was slowly added and the reaction was continued at 35 °C and 300 rpm for 24 hours. The obtained product was centrifuged several times with deionized water until the pH reached about 6. The solid obtained by centrifugation was dispersed in 100 mL deionized water and filled with nitrogen for 20 minutes to remove oxygen in the solution to prevent MXene from being oxidized. Ultrasonic treatment was performed for 1 hour. After the solution was centrifuged at 3500 rpm for 1 hour, the supernatant was collected to obtain a dark green MXene solution. Preparation of the MXene-ZnIn2S4: 8 mg of MXene was dispersed in 30 mL of deionized water, 0.3 mmol of zinc chloride and 0.6 mmol of indium chloride tetrahydrate were added, and the mixture was stirred vigorously for 30 min. 1.8 mmol of thioacetamide was added to the above solution and stirred for another 30 min. The solution was then heated to 95° C. and reacted for 5 h under vigorous stirring. The resulting precipitate was collected by centrifugation and washing. Preparation of the second antibody - MXene-ZnIn2S4: 10 mg of the MXene-ZnIn2S4 was added to 10 mL of 2 mM thioglycolic acid solution and ultrasonically dispersed evenly, stirred for reaction at room temperature for 4 hours, centrifuged and washed, and then redispersed in 10 mL of deionized water. The MXene-ZnIn2S4 reacted with TGA to provide carboxyl groups for subsequent antibody modification. 1 mL of the above was added with 100 μL of 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 20 mM N-hydroxysuccinimide for oscillation reaction for 30 minutes, and then the supernatant was removed by centrifugation. 400 μL of 25 μg / mL of the second antibody was added to the resulting precipitate, stirred for 2 hours at room temperature or incubated at 37°C for 1 hour, and the supernatant was removed by centrifugation. 400 μL of 1% BSA was added, incubated at room temperature for 1 hour, and then centrifuged. Re-dispersed in 400 μL PBS was stored at 4°C until use.

10. Use of the photoelectrochemical sensor according to any one of claims 1 to 3 in detecting phosphorylated tau181 protein.

11. A method for detecting phosphorylated tau181 protein, the method comprising the following steps: 1) Add different concentrations of phosphorylated tau181 protein solution to the photocathode according to any one of claims 1 to 3, incubate at 37° C. for 1 hour, and then add the probe anode to obtain a MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode; 2) using an electrochemical workstation to perform detection using a three-electrode system, with an Ag / AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and the MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode as a working electrode, and performing detection in a solution containing 10 mM Tris-HCl at pH = 7.4; 3) The current-time method was used for detection, the voltage was set to 0V, a 300W xenon lamp was used as the irradiation source, and the power density was 200mW / cm 2 ; 4) Turn on the light source every 10 seconds for 10 seconds, then record the photocurrent, and record the photocurrent corresponding to different concentrations of phosphorylated tau181 protein to obtain the corresponding regression equation and correlation coefficient; 5) replacing the phosphorylated tau181 protein solution in step 1) with the sample solution to be tested, and detecting the photocurrent signal of the sample according to steps 1) to 4) to obtain the concentration of phosphorylated tau181 protein in the sample.

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