Photoelectrochemical sensor, preparation method thereof and use thereof

Abstract

An optoelectrochemical sensor, a preparation method thereof, and uses thereof. The sensor uses p-type semiconductor Cu₂O modified on an ITO electrode and can generate a cathodic photocurrent signal. Further, the electrode interface is modified with a capture antibody Ab1 and BSA. When the target phosphorylated tau181 protein is present, through antigen-antibody specific recognition, the probe MZIS-Ab2 is introduced. And due to the energy level matching between MZIS and Cu₂O, the photocurrent electron transfer path is changed, so that the photocurrent polarity is reversed to generate an anodic photocurrent. The developed PEC sensor with target-induced photocurrent polarity reversal has excellent anti-interference ability and low background signal, and can avoid false positive and / or negative signals in the highly sensitive analysis of phosphorylated tau181 protein.

Description

Technical Field

[0001] This article relates to the technical field of optoelectrochemical biosensors, and particularly to an optoelectrochemical sensor for detecting phosphorylated tau181 protein, a preparation method thereof, and its uses. 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 rate shows an upward trend year by year. Amyloid plaque deposition and neurofibrillary tangles (NFT) in the brain are the main pathological features of AD. NFT is an aggregate formed by the hyperphosphorylation and misfolding of tau protein in neurons. Tau protein is a microtubule-associated protein that exists in high abundance in neuronal cells, and its binding to microtubules is mainly regulated by serine S / threonine T-directed phosphorylation. However, abnormal hyperphosphorylation at tau sites results in the loss of the ability to bind to microtubules. High abundance of phosphorylated tau181 protein (p-tau181) is a typical pathological marker in the brains of AD patients. Therefore, designing and creating a highly accurate detection strategy for phosphorylated tau181 protein is of great significance for the diagnosis of Alzheimer's disease.

[0003] Currently, the commonly used diagnostic methods include cerebrospinal fluid (CSF) analysis, positron emission tomography (PET), and magnetic resonance imaging (MRI), etc. However, they are invasive, expensive, time-consuming, and when the disease is detected based on these traditional imaging methods, patients are often in the middle and late stages. In recent years, the research on developing body fluid-based AD biomarker analysis for early diagnosis of AD has developed rapidly. Detecting biomarkers using biological body fluids such as blood, urine, and saliva for early diagnosis and treatment of AD provides an opportunity to reduce the pain of patients and social and economic costs. However, since the content of AD biomarkers such as phosphorylated tau181 protein in blood is extremely low, developing highly sensitive and highly selective detection and analysis methods is crucial for non-invasive early body fluid detection of AD.

[0004] Due to the complete separation of the excitation light source and the detected current signal, photoelectrochemical (PEC) analysis technology has the advantages of low background signal, high sensitivity, low cost, and easy operation, and has attracted more and more attention in the field of biological analysis. PEC immunosensing mainly consists of two parts: a photoactive material and a biorecognition probe. The photoactive material is the basis of the PEC sensor and the source of the photocurrent signal. Its photoelectric conversion efficiency directly affects the magnitude of the photocurrent, and thus affects the detection sensitivity of the sensor. Photoactive materials include p-type and n-type semiconductors, which generate cathodic photocurrent and anodic photocurrent respectively. Commonly used biorecognition probes include modifying appropriate photoactive materials and enzyme labeling; selecting appropriate energy-level-matched optoelectronic materials 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 an electron donor ascorbic acid, the photocurrent signal is enhanced. In practical applications, the "signal-on" or "signal-off" strategy often has difficulty avoiding the false positive or false negative signal problems caused by the coexistence of various interfering factors in real samples. Therefore, it is necessary to develop a PEC analysis and sensing method that can eliminate false positive and / or negative signals and consider 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 jointly used with the photocathode for detecting phosphorylated tau181 protein in a sample to be tested;

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

[0009] After the photocathode binds phosphorylated tau181 protein, it can bind to the probe anode, thereby enabling a polarity conversion of the photocurrent and realizing sensitive detection of phosphorylated tau181 protein in the sample to be tested.

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

[0011] 1) Prepare the photocathode:

[0012] After pretreating the ITO electrode, Cu2O was dropped onto the surface of the ITO electrode to obtain the Cu2O-ITO electrode.

[0013] 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 the first antibody-chitosan-Cu2O-ITO electrode.

[0014] A BSA solution was dropped onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain the BSA-first antibody-chitosan-Cu2O-ITO electrode, which is the photocathode.

[0015] 2) Preparation of the probe anode:

[0016] 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 the MXene-ZnIn2S4 to obtain the second antibody-MXene-ZnIn2S4, which is the probe anode.

[0017] On the other hand, the present application also provides the use of the photoelectrochemical sensor described herein in the detection of 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) Different concentrations of phosphorylated tau181 protein solutions were dropped onto the photocathode described herein, incubated at 37 °C for 1 hour, and then the probe anode was dropped to obtain the MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode.

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

[0021] 3) Detection was performed by the current-time method, the set voltage was 0 V, and a 300 W xenon lamp was used as the irradiation source with a power density of 200 mW / cm 2 ;

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

[0023] 5) Replace the phosphorylated tau181 protein solution in step 1) with the test sample solution, and detect the photocurrent signal of the sample according to steps 1)-4), and the concentration of phosphorylated tau181 protein in the sample can be obtained.

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

[0025] The present application has developed a photoelectrochemical sensing method based on target-induced photocurrent polarity conversion, constructed an indium tin oxide (ITO) electrode functionalized with p-type Cu2O modified with a phosphorylated tau181 capture antibody (i.e., the first antibody, also called Ab1). When phosphorylated tau181 exists in the test solution, due to antigen-antibody recognition, the phosphorylated tau181 protein is specifically adsorbed on the electrode surface. After adding a probe modified with a labeled antibody (i.e., the second antibody, also called Ab2) of MXene-ZnIn2S4 (MZIS-Ab2), a strong anodic photocurrent signal is generated under light illumination. Cu2O and MZIS are p-type semiconductor and n-type semiconductor respectively. When the target phosphorylated tau181 is absent, the electrode interface modified with Cu2O generates a cathodic photocurrent; while when phosphorylated tau181 exists, due to the energy level matching 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 the highly sensitive and highly selective analysis of phosphorylated tau181 in serum.

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

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

[0028] 2. The electrode interface of the present application is modified with cuprous oxide to generate a cathodic photocurrent signal. After the antibody recognizes the antigen, the probe is modified, thus generating an anodic photocurrent signal. Compared with the co-directional background photocurrent of traditional photoelectrochemical sensing, the background signal of this analysis method with photocurrent polarity change caused by the presence of the target is lower, thus improving the sensitivity of the sensor.

[0029] 3. When the PEC immunosensor for photocurrent polarity conversion 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 detection linear range of phosphorylated tau181 protein in this application is 1 pg / mL - 5000 pg / mL, and the detection limit reaches 1.11 pg / mL.

[0030] Other features and advantages of this application will be described in the subsequent specification, and, in part, will become apparent from the specification, or will be understood by implementing this application. Other advantages of this application can be achieved and obtained through the solutions described in the specification and the accompanying drawings. Brief Description of the Drawings

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

[0032] Figure 1 It is a flowchart for the preparation of the photoelectrochemical sensor in an embodiment of this application.

[0033] Figure 2 Shows the EIS and PEC photocurrent responses of different modification steps of the counter electrode in an embodiment of this application: (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 Shows the reasons for the change in photocurrent polarity and the mechanism of photogenerated electron transfer in an embodiment of this application.

[0035] Figure 4 Shows the photocurrent responses and logarithmic calibration curves of the photoelectrochemical immunosensor to different concentrations of phosphorylated tau181 in an embodiment of this application. Detailed Description of the Embodiments

[0036] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. When a quantity, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper limit of a value and a preferred lower limit of a value, it should be understood that it is equivalent to specifically disclosing any range obtained by combining any pair of the upper limit of the range or the preferred value with any lower limit of the range or the preferred value, regardless of whether the range is specifically disclosed. Unless otherwise specified, 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" or "approximately", when used in combination with a numerical variable, generally refer to the value of the variable and all values of the variable within the experimental error (e.g., within a 95% confidence interval for the average value) or within ±10% of the specified value, or within a wider range.

[0038] The expressions "comprising" or similar expressions synonymous therewith, such as "including", "containing", and "having", are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of" excludes any unstated element, step, or component. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optionally existing elements, steps, or components that do not substantially affect the basic and novel features 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 serving as a signal conversion element, and

[0042] A probe anode combined with the photocathode for detecting phosphorylated tau181 protein in a sample to be tested;

[0043] Wherein, the photocathode uses a Cu2O-ITO electrode as the photocathode substrate, the surface is modified with a first antibody capable of binding phosphorylated tau181 protein, and BSA is added dropwise to prevent non-specific adsorption; the probe anode uses MXene-ZnIn2S4 as the probe anode substrate, and the surface is modified with a second antibody capable of binding phosphorylated tau181 protein;

[0044] After the photocathode binds phosphorylated tau181 protein, it can bind to the probe anode, thereby enabling a polarity conversion of the photocurrent to achieve sensitive detection of 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 phosphorylated tau181 protein, respectively.

[0046] In some embodiments, chitosan is used to link the first antibody to the surface of the Cu2O-ITO electrode.

[0047] In some embodiments, other electrodes that can be used for electrochemistry can be used to replace 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) Prepare a photoanode:

[0050] After pre-treating the ITO electrode, drop Cu2O onto the surface of the ITO electrode to obtain a Cu2O-ITO electrode.

[0051] Modify chitosan (CS) onto the surface of the Cu2O-ITO electrode and link the first antibody capable of binding to phosphorylated tau181 protein to obtain a first antibody-chitosan-Cu2O-ITO electrode.

[0052] Drop a BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain a BSA-first antibody-chitosan-Cu2O-ITO electrode, which is the photoanode.

[0053] 2) Prepare a probe anode:

[0054] Prepare MXene-ZnIn2S4 (referred to as MZIS herein) and link the second antibody capable of binding to phosphorylated tau181 protein to the surface of MXene-ZnIn2S4 to obtain a second antibody-MXene-ZnIn2S4, which is the probe anode.

[0055] In some embodiments, the pre-treatment of the ITO electrode is as follows: Immerse the ITO electrode successively in acetone, an ethanol solution of 1M NaOH (V 乙醇 :V 水 = 1:1) and water, ultrasonically treat for 30 minutes, and then dry for use.

[0056] The preparation method of Cu2O is as follows: Add copper sulfate pentahydrate and sodium citrate dihydrate to water, stir and dissolve, add sodium hydroxide, and then add ascorbic acid for reduction. After aging at room temperature, centrifuge, wash, and dry.

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

[0058] Modify the Cu2O-ITO electrode with 0.1 wt% chitosan to provide amino groups, and connect the first antibody through 0.5% glutaraldehyde (GLD). Modify the electrode with 25 μg / mL of the first antibody to obtain the first antibody-chitosan-Cu2O-ITO electrode;

[0059] Drop 1% BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to avoid non-specific adsorption during detection, and obtain the 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 condition is room temperature; and after each step of modification, the surface of the electrode 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 this application can be adjusted according to specific needs as long as BSA can well avoid non-specific adsorption during detection.

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

[0063] Prepare MXene: Slowly add LiF to the HCl solution, continuously stir until LiF is completely dissolved, then add Ti3AlC2, gently stir, and stir and react at 35 °C for 24 hours. The obtained product is centrifugally washed several times with deionized water (DI) until the pH reaches about 6. Finally, exfoliate the flaky MXene by ultrasonic treatment;

[0064] Prepare MXene-ZnIn2S4: Disperse MXene in water by ultrasonic treatment, add zinc chloride, indium chloride tetrahydrate, and thioacetamide and stir to dissolve, and generate MXene-ZnIn2S4 through hydrothermal reaction. After cooling, centrifuge, wash, and dry to collect.

[0065] In some embodiments, the method for preparing the second antibody-MXene-ZnIn2S4 is as follows: MXene-ZnIn2S4 is added to a thioglycolic acid solution and ultrasonically dispersed evenly. It is stirred at room temperature to provide carboxyl groups to MXene-ZnIn2S4. After centrifugation and washing, it is redispersed in deionized water. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the above solution and reacted with oscillation, and then the supernatant is removed by centrifugation; the second antibody is added to the obtained precipitate, stirred at room temperature, and then the supernatant is removed by centrifugation; 1% BSA is added and incubated at room temperature for 1 hour and then centrifuged to obtain the product.

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

[0067] 1) Prepare the photocathode:

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

[0069] Prepare Cu2O as follows: 1.5 mmol of copper sulfate pentahydrate and 0.5 mmol of sodium citrate dihydrate are added to 80 mL of water and stirred for 15 minutes, then 20 mL of 1.25M sodium hydroxide is added, stirred for 15 minutes, and then 50 mL of 0.03M ascorbic acid is added and stirred for 3 minutes, aged at room temperature for 1 hour, and centrifuged, washed, and dried;

[0070] Drop Cu2O onto the surface of the ITO electrode to obtain a Cu2O-ITO electrode,

[0071] Modify 0.1 wt% chitosan on the Cu2O-ITO electrode to provide amino groups, and connect the first antibody through 0.5% glutaraldehyde. Add 25 μg / mL of the first antibody and incubate at room temperature for 1 hour to obtain a first antibody-chitosan-Cu2O-ITO electrode;

[0072] Drop 1% BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode and incubate at room temperature for 1 hour to avoid non-specific adsorption during detection, and obtain a BSA-first antibody-chitosan-Cu2O-ITO electrode; wherein, after each step of modification, the electrode surface is rinsed with PBS buffer solution to remove unbound reactants and dried with N2;

[0073] 2) Prepare the probe anode:

[0074] Preparation of MXene: 1 g of LiF was added to 10 mL of 9 M HCl, and continuously stirred until completely dissolved. Then, 0.5 g of Ti3AlC2 powder was slowly added, and the reaction was continued at 35 °C with a rotation speed of 300 revolutions per minute for 24 hours. The obtained product was centrifuged several times with deionized water until the pH reached about 6. The centrifuged solid was dispersed in 100 mL of deionized water, and nitrogen was purged for 20 minutes to remove the oxygen in the solution and avoid the oxidation of MXene. After ultrasonic treatment for 1 hour, the solution was centrifuged at 3500 revolutions per minute for 1 hour, and 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 vigorously stirred for 30 min. 1.8 mmol of thioacetamide was added to the above solution and stirred for another 30 min. Then the solution was heated to 95 °C and reacted with vigorous stirring for 5 h. The obtained precipitate was collected by centrifugation and washing;

[0076] Preparation of Second Antibody-MXene-ZnIn2S4: 10 mg of MXene-ZnIn2S4 was added to 10 mL of 2 mM mercaptoacetic acid solution and ultrasonically dispersed evenly. The reaction was stirred at room temperature for 4 hours, centrifuged and washed, and then redispersed in 10 mL of deionized water. The reaction of MXene-ZnIn2S4 with TGA provided carboxyl groups for subsequent antibody modification. 1 mL of the above was taken, 100 μL of 40 mM EDC and 20 mM NHS were added, and the reaction was oscillated for 30 min. After centrifugation, the supernatant was removed. 400 μL of 25 μg / mL second antibody was added to the obtained precipitate, stirred at room temperature for 2 h or incubated at 37 °C for 1 h. After centrifugation, the supernatant was removed. 400 μL of 1% BSA was added and incubated at room temperature for 1 hour, then centrifuged and redispersed in 400 μL of PBS and stored 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 step of modification, the electrode surface was 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 by the method described herein.

[0079] On the other hand, the present application also provides the use of the photoelectrochemical sensor described herein in the detection of phosphorylated tau181 protein. In some embodiments, the present application also provides the use of the photoelectrochemical sensor described herein for the 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) Drop different concentrations of phosphorylated tau181 protein solution onto the photocathode described herein, incubate at 37 °C for 1 hour, and then drop the probe anode to obtain a MXene-ZnIn2S4-secondary antibody-phosphorylated tau181 protein-BSA-primary antibody-chitosan-Cu2O-ITO electrode;

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

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

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

[0085] 5) Replace the phosphorylated tau181 protein solution in step 1) with the test sample solution, and detect the photocurrent signal of the sample according to steps 1)-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 h, and the incubation condition is 37 °C

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

[0088] The mechanism of the photocurrent polarity change is as follows: When Cu2O is modified on the electrode interface and irradiated with visible light, Cu2O is excited to form corresponding photo-generated electrons (e - ) / holes (h + ) in the conduction band (CB) / valence band (VB). The photo-generated e - on the CB of Cu2O transfers to the electrolyte and reduces O2 as an electron acceptor to generate O2 ·- , producing a cathodic photocurrent. After modifying the probe MZIS, since the CB of MZIS is more negative than that of Cu2O, the photo-generated e - transfers from the CB of MZIS to the CB of Cu2O and then to the ITO electrode, and the direction of the photocurrent changes from the cathodic photocurrent to the anodic photocurrent.

[0089] One of the objectives 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 photo-generated carriers in ZnIn2S4 are improved; the energy levels of Cu2O and MZIS match, and the photocurrent polarity changes before and after modification, avoiding false positive and / or negative signals during actual detection.

[0090] Another objective of this application is based on target recognition-induced photocurrent polarity change. The electrode interface is modified with Cu2O to generate a cathodic photocurrent signal. After the antibody recognizes the antigen, the probe is modified, thereby generating an anodic photocurrent signal, and the photocurrent polarity changes, featuring a negative background signal, which improves the sensitivity of the sensor.

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

[0092] This application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to those of ordinary skill in the art that there can be more embodiments and implementation schemes within the scope of the embodiments described in this application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature of any embodiment can be combined with any other feature in any other embodiment, or can replace any other feature in any other embodiment.

[0093] This application includes and contemplates combinations with features known to those of ordinary skill in the art. The disclosed embodiments and features of this application may also be combined with any conventional features to form unique inventive solutions defined by the claims. Any feature of any embodiment may also be combined with features from other inventive solutions to form another unique inventive solution defined by the claims. Accordingly, it should be understood that any feature shown and / or discussed in this application may be implemented alone or in any suitable combination. Thus, the embodiments are not limited except as defined by the appended claims and their equivalents. In addition, various modifications and changes can be made within the scope of the appended claims.

[0094] In addition, in describing representative embodiments, the specification may have presented methods and / or processes as a particular sequence of steps. However, to the extent that the method or process does not depend on a particular order of the steps described herein, the method or process should not be limited to the particular order of steps described. As will be understood by those of ordinary skill in the art, other sequences of steps are possible. Accordingly, the particular order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, as those skilled in the art can readily appreciate that such orders may vary and still remain within the spirit and scope of the embodiments of this application.

[0095] For the experimental methods without specific conditions noted in the following examples, they are generally determined according to national standards. The experimental materials not indicated the source in the following examples are all commercially available raw materials. The equipment used in each step of the following examples are all conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise defined or stated, 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 methods and materials similar or equivalent to the described content can be applied to the methods of this application.

[0096] Examples

[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 ultrasonic treatment. The mixture was stirred at room temperature for 4 hours, centrifuged, washed, and then redispersed in 10 mL of deionized water. The reaction between MZIS and TGA was to provide carboxyl groups for subsequent antibody modification. 1 mL of the above solution was taken and added with 100 μL of 40 mM EDC and 20 mM NHS, and the mixture was oscillated for 30 min and then centrifuged to remove the supernatant. 400 μL of 25 μg / mL phosphorylated tau181 secondary antibody (Hangzhou Jingjie Biotech Co., Ltd., catalog number ABT-112009) (Ab2) was added to the obtained precipitate, and the mixture was stirred at room temperature for 2 h and then centrifuged to remove the supernatant. 400 μL of 1% BSA was added and incubated at room temperature for 1 h and then centrifuged. It was redispersed in 400 μL of PBS and stored at 4 °C until use. The preparation process was as shown in Figure 1 shown in a of

[0099] 2. Modification of the electrode and preparation of the photoelectrochemical immunosensor interface

[0100] The preparation process was as shown in Figure 1 shown in b of

[0101] (1) Pretreatment of the ITO electrode

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

[0103] (2) Assembly of the photochemical immunosensor

[0104] After the pretreatment of the ITO electrode, 1 mg / mL of Cu2O was dropped on the electrode surface and dried to obtain the Cu2O-ITO electrode. Subsequently, 0.1 wt% chitosan was modified to provide amino groups, and the primary antibody (Ab1) was linked through 0.5% glutaraldehyde. 25 μg / mL of phosphorylated tau181 primary antibody (Nanjing Zhonghongtai Biotech Co., Ltd., catalog number: Tau181102) (Ab1) was modified to obtain the Ab1-CS-Cu2O-ITO electrode. 1% BSA solution was dropped to avoid non-specific adsorption during the detection process to obtain the BSA-Ab1-CS-Cu2O-ITO electrode. Different concentrations of phosphorylated tau181 protein solution were dropped and incubated at 37 °C for 1 h, and then the synthesized probe solution MZIS-Ab2 was dropped to obtain the MZIS-Ab2-phosphorylated tau181-BSA-Ab1-CS-Cu2O-ITO electrode, that is, the photoelectrochemical immunosensor for the detection of phosphorylated tau181 was prepared. After each modification, it must be washed with buffer and dried with nitrogen.

[0105] 3. Method for Detecting p-tau181 Using a Photoelectrochemical Immunosensor with Target-Induced Photocurrent Polarity Conversion

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

[0107] Example 1. Performance Test of the Photoelectrochemical Sensor

[0108] The photoelectrochemical immunosensor constructed in this application was used to characterize the electrode assembly by the photocurrent-time curve (i-t curve) and electrochemical impedance spectroscopy (EIS). The i-t curve was tested at 0 V voltage in a 10 mM Tris-HCl (pH = 7.4) electrolyte solution (see Figure 2 a), and EIS was carried out in a 0.2 M KC1 electrolyte solution containing 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (see Figure 2 b). Figure 2 a shows the change in photocurrent signal during the stepwise modification of the electrode. The photocurrent response of the bare ITO electrode was almost 0 (curve a); when Cu2O was modified, a cathodic photocurrent was obtained (curve b). After introducing CS, Ab1, BSA, and phosphorylated tau181 protein onto the Cu2O / ITO electrode, the photocurrent gradually decreased due to the non-conductivity of proteins and the increase in steric hindrance (curves c-f). When the PEC immunosensor was incubated with MZIS-Ab2, an obvious anodic photocurrent was generated (curve e), which was due to the change in the transfer path of photoelectrons / holes through the matching energy levels between Cu2O and MZIS. Similarly, Figure 2 b shows the change in EIS during the stepwise modification of the electrode. The impedance increased after Cu2O was modified on the ITO surface. Since Cu2O has poor conductivity, the impedance gradually increased after Ab1, BSA, and phosphorylated tau181 were successively immobilized on the electrode surface, which was due to the non-conductive protein molecules hindering the electron transfer rate. When the sensor was incubated with MIZ-Ab2 in the presence of the target phosphorylated tau181, the impedance value decreased, indicating that MZIS has good electrochemical performance. The above shows that the construction of this photoelectrochemical immunosensor is successful and effective.

[0109] Example 2. Detection of phosphorylated tau181 protein with 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 the photocurrent-time curve (i-t). The anodic photocurrent intensity increased with the increase in the concentration of phosphorylated tau181 (as shown in a of Figure 4 ), and a linear relationship was obtained between the photocurrent intensity and the logarithm of the phosphorylated tau181 protein concentration (as shown in b of Figure 4 , y = 0.2286lgC - 0.03176, R 2 = 0.957).

[0111] In summary, the photoelectrochemical immunosensor with target-induced photocurrent polarity conversion was used to detect the Alzheimer's disease biomarker phosphorylated tau181 protein. The electrode interface was modified with cuprous oxide to generate a cathodic photocurrent signal. After the antibody recognized the antigen, the probe MZIS-Ab2 was modified, and the photocurrent polarity changed to generate an anodic photocurrent signal, constructing a highly sensitive photoelectrochemical immunosensor. Under the optimal conditions, the measurement results showed that the detection range for phosphorylated tau181 was 1 pg / mL - 5000 pg / mL, and the detection limit was 1.11 pg / mL (S / N = 3).

Claims

1. An optoelectrochemical sensor for detecting phosphorylated tau181 protein, comprising: a photocathode serving as a signal conversion element, and a probe anode combined with the photocathode for detecting phosphorylated tau181 protein in a sample to be measured; wherein, the photocathode uses a Cu2O-ITO electrode as the photocathode substrate, and chitosan is used to connect the first antibody capable of binding phosphorylated tau181 protein to the surface of the Cu2O-ITO electrode, and a BSA solution is dropped to prevent non-specific adsorption, thus obtaining the photocathode; the probe anode uses MXene-ZnIn2S4 as the probe anode substrate, and the surface is modified with a second antibody capable of binding phosphorylated tau181 protein; The preparation method of the MXene-ZnIn2S4 is as follows: Prepare MXene: Slowly add LiF to the HCl solution, continuously stir until LiF is completely dissolved, then add Ti3AlC2, gently stir, and stir and react at 35 °C for 24 hours. The obtained product is centrifuged and washed several times with deionized water until the pH value reaches 6. Finally, exfoliate the flaky MXene by ultrasonic treatment; Prepare the MXene-ZnIn2S4: Disperse MXene in water by ultrasonic dispersion, add zinc chloride, indium chloride tetrahydrate, and thioacetamide and stir to dissolve, and generate MXene-ZnIn2S4 by hydrothermal reaction. After cooling, centrifuge, wash, and dry to collect; After the photocathode binds phosphorylated tau181 protein, it can bind to the probe anode, thereby realizing the polarity conversion of the photocurrent and achieving the sensitive detection of phosphorylated tau181 protein in the sample to be measured; The first antibody and the second antibody bind to different epitopes of phosphorylated tau181 protein respectively.

2. The preparation method of the optoelectrochemical sensor according to claim 1, comprising the following steps: 1) Prepare the photocathode: After pre-treating the ITO electrode, drop Cu2O onto the surface of the ITO electrode to obtain the Cu2O-ITO electrode, Modify chitosan on the surface of the Cu2O-ITO electrode and connect the first antibody capable of binding phosphorylated tau181 protein to obtain the first antibody-chitosan-Cu2O-ITO electrode, Drop a BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to obtain the BSA-first antibody-chitosan-Cu2O-ITO electrode, which is the photocathode; 2) Prepare the probe anode: Prepare MXene-ZnIn2S4, and connect the second antibody capable of binding phosphorylated tau181 protein to the surface of the MXene-ZnIn2S4 to obtain the second antibody-MXene-ZnIn2S4, which is the probe anode.

3. According to the preparation method of claim 2, wherein, The pre-treatment of the ITO electrode is: Immerse the ITO electrode in acetone, an ethanol solution of 1 M NaOH, and water in sequence, ultrasonically treat for 30 minutes, and then dry for use; The preparation method of the Cu2O is as follows: Dissolve copper sulfate pentahydrate and sodium citrate dihydrate in water with stirring, add sodium hydroxide, and then add ascorbic acid for reduction. After aging at room temperature, centrifuge, wash, and dry.

4. According to the preparation method described in claim 2 or 3, wherein The preparation method of the photocathode is as follows: Modify 0.1 wt% chitosan on the Cu2O-ITO electrode to provide amino groups, and connect the first antibody through 0.5% glutaraldehyde, and modify 25 µg / mL of the first antibody to obtain the first antibody-chitosan-Cu2O-ITO electrode; Drop 1% BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode to avoid non-specific adsorption during detection, and obtain the BSA-first antibody-chitosan-Cu2O-ITO electrode, that is, the photocathode.

5. According to the preparation method described in claim 2, wherein The preparation method of the probe anode is as follows: Add MXene-ZnIn2S4 to a mercaptoacetic acid solution and disperse it evenly by ultrasonic treatment. Stir and react at room temperature to provide carboxyl groups for MXene-ZnIn2S4. After centrifuging and washing, redisperse it in deionized water. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the above solution and react by oscillation, then centrifuge to remove the supernatant; add the second antibody to the obtained precipitate, stir and react at room temperature, and then centrifuge to remove the supernatant; add 1% BSA and incubate at room temperature for 1 hour, and then centrifuge to obtain the probe anode.

6. According to the preparation method described in claim 2, the method includes the following steps: 1) Prepare the photocathode: Immerse the ITO electrode successively in acetone, 1 M NaOH / ethanol mixture and water, ultrasonically treat for 30 minutes, and then dry for use; The preparation method of the Cu2O is as follows: Add 1.5 mmol of copper sulfate pentahydrate and 0.5 mmol of sodium citrate dihydrate to 80 mL of water and stir for 15 minutes, then add 20 mL of 1.25 M sodium hydroxide, stir for 15 min, and then add 50 mL of 0.03 M ascorbic acid and stir for 3 min. Age at room temperature for 1 h, centrifuge, wash, and dry; Drop the Cu2O onto the surface of the ITO electrode to obtain the Cu2O-ITO electrode, Modify 0.1 wt% chitosan on the Cu2O-ITO electrode to provide amino groups, connect the first antibody through 0.5% glutaraldehyde, add 25 µg / mL of the first antibody, and incubate at room temperature for 1 h to obtain the first antibody-chitosan-Cu2O-ITO electrode; Drop 1% BSA solution onto the surface of the first antibody-chitosan-Cu2O-ITO electrode, incubate at room temperature for 1 h, avoid non-specific adsorption during detection, and obtain the BSA-first antibody-chitosan-Cu2O-ITO electrode; wherein, after each modification, rinse the electrode surface with PBS buffer to remove unbound reactants, and dry with N2; 2) Prepare the probe anode: Preparation of MXene: Add 1 g of LiF to 10 mL of 9 M HCl, continuously stir until completely dissolved, then slowly add 0.5 g of Ti3AlC2 powder, continue the reaction at 35 °C with a rotation speed of 300 revolutions per minute for 24 hours. The obtained product is centrifuged several times with deionized water until the pH value reaches 6. The centrifuged solid is dispersed in 100 mL of deionized water, purged with nitrogen for 20 minutes to remove the oxygen in the solution and avoid the oxidation of MXene, ultrasonically treated for 1 hour, and after centrifuging the solution at 3500 revolutions per minute for 1 hour, the supernatant is collected to obtain a dark green MXene solution; Preparation of the MXene-ZnIn2S4: Disperse 8 mg of MXene in 30 mL of deionized water, add 0.3 mmol of zinc chloride and 0.6 mmol of indium chloride tetrahydrate, stir vigorously for 30 min, add 1.8 mmol of thioacetamide to the above solution and stir for another 30 min, then heat the solution to 95 °C and react under vigorous stirring for 5 h. Centrifuge and wash to collect the obtained precipitate to obtain the MXene-ZnIn2S4; Preparation of the second antibody-MXene-ZnIn2S4: Add 10 mg of the MXene-ZnIn2S4 to 10 mL of 2 mM mercaptoacetic acid solution and ultrasonically disperse evenly. Stir and react at room temperature for 4 hours, centrifuge and wash, and then redisperse in 10 mL of deionized water. Take 1 mL of the above solution, add 100 μL of 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 20 mM N-hydroxysuccinimide, oscillate and react for 30 min, then centrifuge to remove the supernatant. Add 400 μL of 25 μg / mL of the second antibody to the obtained precipitate, stir at room temperature for 2 h or incubate at 37 °C for 1 h, centrifuge to remove the supernatant, add 400 μL of 1% BSA, incubate at room temperature for 1 hour and then centrifuge, and redisperse in 400 μL of PBS and store at 4 °C until use.

7. Use of the photoelectrochemical sensor according to claim 1 for non-diagnostic purposes in the detection of phosphorylated tau181 protein.

8. A method for detecting phosphorylated tau181 protein for non-diagnostic purposes, using the photoelectrochemical sensor according to claim 1, the method comprising the following steps: 1) Drop different concentrations of phosphorylated tau181 protein solution on the photocathode, incubate at 37 °C for 1 hour, and then drop the probe anode to obtain a MXene-ZnIn2S4-second antibody-phosphorylated tau181 protein-BSA-first antibody-chitosan-Cu2O-ITO electrode; 2) Detection was carried out using an electrochemical workstation with a three - electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum electrode was used as the counter electrode, and the MXene - ZnIn2S4 - secondary antibody - phosphorylated tau181 protein - BSA - primary antibody - chitosan - Cu2O - ITO electrode was used as the working electrode. The detection was carried out in a solution containing 10 mM Tris - HCl at pH = 7.

4. 3) Detection is carried out by the current-time method, the set voltage is 0 V, and a 300 W xenon lamp is used as the irradiation source with a power density of 200 mW / cm 2 ; 4) The light source was turned on for continuous irradiation for 10 s every 10 s, and then the photocurrent was recorded. The photocurrents corresponding to different concentrations of phosphorylated tau181 protein were recorded to obtain the corresponding regression equation and correlation coefficient. 5) The phosphorylated tau181 protein solution in step 1) was replaced with the test sample solution, and the photocurrent signal of the sample was detected according to steps 1) - 4) to obtain the concentration of phosphorylated tau181 protein in the sample.

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