Electrochemiluminescence aptamer sensor for tau protein and preparation method and application thereof

Abstract

This invention belongs to the field of biosensing and detection technology, specifically disclosing a Tau protein electrochemiluminescent aptamer sensor, its preparation method, and its application. The sensor uses a CsPbBr₃@PVP / Au composite material as the electrochemiluminescent substrate. A Tau protein-specific aptamer is immobilized on the substrate surface via Au-S bonds, and non-specific adsorption sites are blocked with 6-mercapto-1-hexanol. The CsPbBr₃@PVP / Au composite material is prepared by encapsulating CsPbBr₃ with PVP and then combining it with Au NPs. This invention utilizes the coordination passivation effect of PVP and CsPbBr₃ to significantly improve the aqueous stability and electrochemiluminescence efficiency of perovskite nanocrystals. Combined with the anchoring effect of AuNPs and the specific recognition effect of aptamers, it achieves ultrasensitive detection of Tau protein with a detection limit as low as 0.80 fg / mL. Furthermore, it exhibits excellent selectivity, stability, and detection accuracy in complex biological samples such as cerebrospinal fluid, providing a novel and efficient detection platform for the early diagnosis of Alzheimer's disease and showing promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, specifically to a Tau protein electrochemiluminescence aptamer sensor, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a progressive, irreversible neurodegenerative disease. Its pathogenesis is closely related to the abnormal aggregation of Tau protein and β-amyloid protein. The amyloid plaques and neurofibrillary tangles formed by these protein aggregations disrupt the normal function of neurons, leading to progressive memory decline and cognitive impairment in patients. Early and accurate diagnosis of Alzheimer's disease is crucial for timely intervention.

[0003] Among biomarkers for Alzheimer's disease, total Tau protein in cerebrospinal fluid (CSF) is a crucial indicator reflecting the degree of neuronal damage and degeneration. Its concentration changes are closely related to the disease progression stage, making it a core target for early diagnosis. Currently, neuroimaging and CSF biomarker detection are the two most reliable methods for diagnosing Alzheimer's disease. However, neuroimaging lacks specificity for Alzheimer's disease, leading to fluctuations in the repeatability and accuracy of diagnostic results. CSF biomarker detection primarily relies on enzyme-linked immunosorbent assay (ELISA) for total Tau protein in CSF. However, this method suffers from drawbacks such as time-consuming operation, high testing costs, poor specificity, and limited sensitivity, making it difficult to meet the needs of early detection of trace amounts of Tau protein and significantly limiting its widespread clinical application and patient compliance. Therefore, developing a sensitive, efficient, low-cost, and easy-to-operate Tau protein detection method is an urgent need to address the challenges of early Alzheimer's disease diagnosis and promote its clinical application.

[0004] Electrochemiluminescence (ECL) technology, as a novel luminescent analysis technique, originates from a high-energy electron transfer reaction between active intermediates generated on the electrode surface via electrochemical methods. ECL sensors constructed based on this technology possess significant advantages such as high signal-to-noise ratio, excellent stability, high detection sensitivity, and ease of operation. They can achieve sensitive and accurate detection of various analytes and are currently widely used for the rapid determination of various biological targets, providing an ideal technological platform for the highly sensitive detection of Tau protein. Despite these advantages, the detection performance of ECL sensors is still determined by the luminescence efficiency of the luminescent material. Therefore, developing efficient ECL luminescent materials is the core key to improving sensor performance.

[0005] All-inorganic lead halide perovskite CsPbX3 (X=Cl, Br, I) nanocrystals possess excellent photoelectric properties and are potential high-efficiency ECL emitters. However, the ionic composition of this material makes it extremely susceptible to damage in aqueous media, and surface defects can also lead to a reduction in luminescence efficiency, severely limiting its application in the field of biosensing.

[0006] Therefore, developing an electrochemiluminescence sensor for Tau protein that combines high aqueous stability, high luminescence efficiency, and specific recognition capability is of great significance for achieving ultrasensitive detection of Tau protein and early diagnosis of Alzheimer's disease. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a Tau protein electrochemiluminescence aptamer sensor, its preparation method and application. This sensor has the advantages of good aqueous phase stability, high luminescence efficiency, high detection sensitivity and strong specificity, and can realize ultrasensitive detection of Tau protein, providing an efficient detection platform for the early diagnosis of Alzheimer's disease.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a Tau protein electrochemiluminescence aptamer sensor, comprising a working electrode, a perovskite nanocomposite material modified on the surface of the working electrode, and a Tau protein-specific aptamer immobilized on the surface of the perovskite nanocomposite material. The perovskite nanocomposite material is a CsPbBr3@PVP / Au composite material, composed of polyvinylpyrrolidone-encapsulated cesium lead bromide nanocrystals and gold nanoparticles. The polyvinylpyrrolidone achieves coating and passivation of the cesium lead bromide nanocrystals through coordination between its carbonyl groups and lead ions on the surface of the cesium lead bromide. The gold nanoparticles, modified on the surface of the polyvinylpyrrolidone-encapsulated cesium lead bromide nanocrystals, are zero-valent gold. The Tau protein-specific aptamer is a thiol-modified aptamer with a 5'-3' base sequence: CGG ACA CCA ACA ACC CCG CCC ACG C-C6-SH, immobilized on the surface of the gold nanoparticles via gold-sulfur bonds for specific recognition of Tau protein.

[0010] Furthermore, the working electrode is a glassy carbon electrode, which has good biocompatibility and electron transfer performance, making it suitable for Tau protein biosensing scenarios; the CsPbBr3 nanocrystals have a cubic structure with an average particle size of 7.87±1.73nm; the CsPbBr3@PVP / Au lattice fringe spacing is 0.41nm, corresponding to the (110) crystal plane, and the X-ray diffraction pattern of the water-dispersed CsPbBr3@PVP / Au composite material shows characteristic diffraction peaks of cesium lead bromide and gold.

[0011] Furthermore, the CsPbBr3@PVP / Au forms a complete, continuous, and stable superlattice thin film on the surface of the working electrode.

[0012] Furthermore, in the polyvinylpyrrolidone-encapsulated cesium lead bromide nanocrystals, the mass fraction of polyvinylpyrrolidone is 6%. This ratio achieves optimal surface defect passivation and ECL performance improvement; too high or too low a ratio will affect sensor performance.

[0013] Furthermore, it also includes a 6-mercapto-1-hexanol blocking layer, which is modified on the surface of the aptamer-immobilized CsPbBr3@PVP / Au composite material to suppress non-specific adsorption and improve the specificity and detection accuracy of the sensor.

[0014] This invention also provides a method for preparing the above-mentioned Tau protein electrochemiluminescent aptamer sensor, comprising the following steps:

[0015] S1. Preparation of CsPbBr3@PVP composite material: Lead bromide and cesium bromide were dissolved in N,N-dimethylacetamide and heated and stirred to obtain a mixed solution; oleic acid, oleylamine and polyvinylpyrrolidone solution were heated and added to the above mixed solution, and the reaction was carried out by stirring and controlling the temperature to obtain a precursor solution; the precursor solution was added to ethyl acetate, stirred and mixed, precipitated under controlled temperature, and dried by centrifugation to obtain CsPbBr3@PVP powder;

[0016] S2. Preparation of CsPbBr3@PVP / Au composite material: CsPbBr3@PVP was dispersed in deionized water, chloroauric acid solution was added and stirred, sodium borohydride solution was added dropwise under ice bath conditions to carry out reduction reaction, centrifugation purification and freeze drying were performed to obtain CsPbBr3@PVP / Au powder;

[0017] S3. Sensor Assembly: Sensor assembly includes the following steps:

[0018] S301. Pretreatment of working electrode: The glassy carbon electrode is successively polished, cleaned and activated;

[0019] S302. Composite material modified working electrode: CsPbBr3@PVP / Au solution is drop-coated onto the surface of the pretreated glassy carbon electrode and dried to form a uniform sensing film;

[0020] S303. Immobilization of aptamers: Tau protein-specific aptamers activated by tris(2-carboxyethyl)phosphine were drop-coated onto the surface of the working electrode modified with composite material. Temperature-controlled incubation was used to immobilize the aptamers on the surface of the working electrode modified with composite material. After incubation, non-specifically adsorbed aptamers were washed away.

[0021] S304. Sealing treatment: Drop 6-mercapto-1-hexanol solution onto the sensor, incubate to seal non-specific adsorption sites, and rinse after incubation to obtain the sensor.

[0022] Furthermore, in S1, the feed ratio of cesium bromide, lead bromide, and N,N-dimethylacetamide was 0.0851 g: 0.1468 g: 10 ml, the mixing temperature was 60 °C, and the mixing time was 60 minutes; the polyvinylpyrrolidone solution concentration was 6%, and the feed ratio of cesium bromide, oleic acid, oleylamine, and polyvinylpyrrolidone was 0.0851 g: 200 µL: 500 µL: 200 µL, the reaction temperature was 60 °C, and the reaction time was 30 minutes; the feed ratio of the precursor solution to ethyl acetate was 1 ml: 10 ml, the precipitation temperature was 60 °C, and the precipitation time was 60 minutes.

[0023] Furthermore, in S2, the concentration of chloroauric acid solution is 10 mM, the concentration of sodium borohydride solution is 10 mM, and the feed ratio of CsPbBr3@PVP, deionized water, chloroauric acid solution, and sodium borohydride solution is 10 mg: 10 ml: 20 µL: 100 µL.

[0024] Further, in S301, the glassy carbon electrode was polished using 0.3µm and 0.05µm aluminum oxide powders; the cleaning method was ultrasonic cleaning with deionized water and ethanol; the activation method was activation in 0.5M sulfuric acid solution, characterized by a redox potential difference of less than 120mV using 5mM potassium ferricyanide / potassium ferrocyanide solution; in S302, the concentration of CsPbBr3@PVP / Au solution was 1 mg / mL, the drop volume was 6µL, the drying temperature was 37℃, and the drying time was 4 hours; in S303, the Tau protein-specific aptamer solution was activated with 3 mg / mL tris(2-carboxyethyl)phosphine at a concentration of 10µM, the drop volume was 6µL, the incubation temperature was 4℃, and the incubation time was 12 hours; after incubation, it was treated with 0.1 M solution at pH 7.4. Rinse with PBS buffer; in S304, the concentration of 6-mercapto-1-hexanol solution is 10 mM, the drop volume is 6 µL, the incubation temperature is room temperature, the incubation time is 1 hour, and after incubation, rinse with 0.1 M PBS buffer at pH 7.4.

[0025] The present invention also provides a method for detecting Tau protein, comprising the following steps: dropping the sample solution to be tested onto the working electrode surface of the above-mentioned Tau protein electrochemiluminescence aptamer sensor, incubating at 37°C for 1 h, and rinsing with PBS buffer; placing the rinsed sensor in PBS buffer containing K2S2O8, performing electrochemiluminescence detection using a three-electrode system, and realizing quantitative detection of Tau protein based on the change in electrochemiluminescence signal intensity.

[0026] Furthermore, the counter electrode of the three-electrode system is a platinum wire electrode, and the reference electrode is an Ag / AgCl electrode.

[0027] Furthermore, the concentration of K2S2O8 was 200mM. During the test, the photomultiplier tube (PMT) voltage was set to 1200 V, the potential range was 0 to -2.0 V, and the cyclic voltammetry (CV) scan rate was 0.1 V / s.

[0028] Furthermore, the detection concentration range of the Tau protein is 10 fg / ml to 10 fg / ml. 8 The detection limit is 0.80 fg / ml.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention uses PVP to encapsulate CsPbBr3 nanocrystals. On the one hand, the long molecular chain of PVP forms a dense hydrophobic protective shell, which significantly improves the stability of CsPbBr3 nanocrystals in aqueous medium and solves the technical bottleneck of easy degradation of perovskite nanocrystals. On the other hand, the lone pair electrons of carbonyl oxygen atoms in PVP form a strong coordination with Pb²⁺ on the surface of CsPbBr3, passivating surface defects and enabling more charge carriers to generate photons through radiative recombination, thereby increasing the ECL intensity by four times and significantly improving the luminescence performance.

[0031] (2) The present invention introduces gold nanoparticles to modify CsPbBr3@PVP composite material, which can not only serve as anchoring sites for Tau protein-specific aptamers and achieve stable fixation of aptamers through Au-S bonds to ensure the specificity of the sensor; but also accelerate the electron transfer process in the ECL reaction through the electrocatalytic effect of metal ions, further improving the electrochemical response performance of the sensor.

[0032] (3) The ECL aptamer sensor constructed in this invention forms an aptamer-target complex based on the specific recognition of aptamers and Tau proteins, which hinders the efficiency of the ECL reaction and generates an "off-off" state ECL sensing response, thus achieving ultrasensitive detection of Tau proteins with a detection limit as low as 0.80 fg / mL and a wide detection range (10 fg / mL to 10 fg / mL). 8 The linear relationship was good (R²=0.996).

[0033] (3) The sensor of the present invention has excellent selectivity and stability, and has a very small response to interfering substances such as alpha-fetoprotein (AFP) and bovine serum albumin (BSA). The RSD of 10 consecutive cyclic scans is only 1.09%, and the ECL intensity still maintains more than 80% of the initial intensity in the 120-hour long-term stability test. At the same time, it shows good detection performance in complex biological samples such as mouse cerebrospinal fluid, with high spike recovery rate and good reproducibility, and is suitable for detection of actual samples.

[0034] (4) The sensor preparation method of the present invention is simple to operate, low in cost, and the reagents used are easy to obtain. It does not require complicated instruments and equipment, which facilitates large-scale production and clinical application. It not only broadens the application of CsPbBr3 perovskite material in ECL biosensing, but also provides a novel and efficient detection platform for the early diagnosis of AD. Attached Figure Description

[0035] Figure 1 The results of TEM, HRTEM and HAADF-STEM EDS mappings of the composite material are shown below. Among them, (A) TEM image of CsPbBr3; (B) TEM image of CsPbBr3@PVP / Au; (C) HRTEM image of CsPbBr3@PVP / Au; (D) magnified morphology of the (110) crystal plane of single CsPbBr3@PVP / Au, with a calculated average lattice spacing of 0.41 nm; (E) HAADF-STEM EDS mappings of CsPbBr3@PVP / Au.

[0036] Figure 2 The figure shows the particle size statistics of CsPbBr3.

[0037] Figure 3 The results show the FT-IR, XRD, and XPS characterization of the composite materials. Among them, (A): (i) FT-IR spectra of CsPbBr3@PVP / Au, (ii) CsPbBr3@PVP, (iii) CsPbBr3 and (iv) PVP; (B) XRD spectra of (i) CsPbBr3@PVP / Au, (ii) CsPbBr3@PVP and (iii) CsPbBr3; (C) XPS full spectrum of CsPbBr3@PVP / Au; (D) XPS high-resolution spectra of Au 4f, (E) C 1s, and (F) Pb4f.

[0038] Figure 4 For high-resolution X-ray photoelectron spectra: (A) Nitrogen 1s (N 1s); (B) Cesium 3d (Cs 3d); (C) Bromine 3d (Br 3d); (D) Oxygen 1s.

[0039] Figure 5 The excitation (EX) and emission (Em) spectra of CsPbBr3, as well as the fluorescence spectra at different excitation wavelengths, are shown below; (A) excitation (EX) and emission (Em) spectra of CsPbBr3; (B) fluorescence spectra of CsPbBr3 at different excitation wavelengths.

[0040] Figure 6 The images show the excitation (EX) and emission (Em) spectra of CsPbBr3@PVP, as well as the fluorescence spectra at different excitation wavelengths; where (A) is the excitation (EX) and emission (Em) spectra of CsPbBr3@PVP; and (B) is the fluorescence spectra of CsPbBr3@PVP at different excitation wavelengths.

[0041] Figure 7 The images show the excitation (EX) and emission (Em) spectra of CsPbBr3@PVP / Au, as well as the fluorescence spectra at different excitation wavelengths. Among them, (A) the excitation (EX) and emission (Em) spectra of CsPbBr3@PVP / Au; and (B) the fluorescence spectra of CsPbBr3@PVP / Au at different excitation wavelengths.

[0042] Figure 8 The UV-Vis absorption spectra of CsPbBr3, CsPbBr3@PVP, and CsPbBr3@PVP / Au are shown.

[0043] Figure 9 The results show the ECL performance of the composite materials; (A) ECL intensity-potential curves of bare glassy carbon electrode, CsPbBr3, CsPbBr3@PVP and CsPbBr3@PVP / Au in pH 7.4 and 0.1 M PBS containing 100 mM K2S2O8; (B) cyclic voltammetry curves; (C) cathode ECL and PL spectra of CsPbBr3@PVP / Au; (D) ECL-time curves of CsPbBr3@PVP / Au under 15 consecutive scans; (E) and (F) long-term stability of CsPbBr3 and CsPbBr3@PVP / Au.

[0044] Figure 10 EIS curves and cyclic voltammetry curves for the process of gradually modifying the surface of a glassy carbon electrode to construct a Tau aptamer sensor; where (A) is the EIS curve and (B) is the cyclic voltammetry curve.

[0045] Figure 11The experimental results were investigated to examine the influencing factors, including (A) the content of polyvinylpyrrolidone (PVP); (B) the concentration of potassium persulfate (K2S2O8) as a co-reactant; (C) the scanning potential range; and (D) the loading of CsPbBr3@PVP / Au on the glassy carbon electrode.

[0046] Figure 12 ECL curves and calibration curves of the sensor at different concentrations of Tau protein; wherein, (A) ECL curves of the sensor at different concentrations of Tau protein (ah: 10, 10², 10³, 10, respectively) are shown. 4 10 5 10 6 10 7 and 10 8 (a) ECL curve at fg / mL; (b) Calibration curve of ECL intensity versus logarithm of Tau concentration.

[0047] Figure 13 The results of stability test for Tau protein (10 fg / mL) detected by the sensor.

[0048] Figure 14 For the sensor to detect Tau protein (10 8 fg / mL) and interfering substances (10 fg / mL) 8 ECL response results (fg / mL).

[0049] Figure 15 ECL curves and calibration curves of the sensor for different concentrations of Tau protein in 1‰ mouse cerebrospinal fluid are shown; where (A) the sensor for different concentrations of Tau protein (ah: 10, 10², 10³, 10, etc.) are shown. 4 10 5 10 6 10 7 and 10 8 (a) ECL curve at fg / mL; (b) Calibration curve of ECL intensity versus logarithm of Tau concentration. Detailed Implementation

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that experimental materials whose source is not specified in the embodiments of the present invention are all commercially available. Experimental methods whose specific conditions are not specified in the embodiments of the present invention are generally performed according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers. All solutions whose solvent type is not specifically named in the present invention are aqueous solutions. The main experimental reagents and instruments involved in the present invention are as follows:

[0051] 1. Experimental reagents

[0052] Lead bromide (PbBr2, 99% purity), cesium bromide (CsBr, 99.5% purity), N,N-dimethylacetamide (DMA, 99% purity), oleic acid (OA, 85% purity), oleylamine (OAm), polyvinylpyrrolidone (PVP), ethyl acetate (EA, 99.5% purity), potassium ferricyanide (K3Fe(CN)6·H2O), potassium chloride (KCl, 99.5% purity), tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP, 98% purity), and 6-mercapto-1-hexanol (MCH) were purchased from Shanghai Aladdin Reagent Co., Ltd. Disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Shanghai Sigma-Aldrich Co., Ltd.

[0053] Ethanol (C2H5OH, 95% purity) and sulfuric acid (H2SO4, analytical grade) were purchased from Shanghai Shushi Reagent Co., Ltd. All other reagents were of analytical grade and were used directly without further purification.

[0054] All experimental aqueous solutions were prepared using ultrapure water (18.2 MΩ・cm, Milli-Q, USA).

[0055] Tau protein aptamer base sequence (5'-3'): CGG ACA CCA ACA ACC CCG CCC ACG C-C6-SH.

[0056] 2. Experimental apparatus

[0057] Transmission electron microscopy (TEM) images were acquired using a JEM-200CX transmission electron microscope (Nippon Electron Ltd., Osaka, Japan).

[0058] The ultraviolet-visible (UV-vis) spectra were recorded using a JASCO V-750 spectrophotometer (Toshiba Corporation, Tokyo, Japan).

[0059] Fourier transform infrared (FT-IR) spectra were measured using a Thermo Fisher Scientific Nicolet iS50 instrument (Waltham, Massachusetts, USA).

[0060] X-ray powder diffraction (XRD) patterns were obtained using a Bruker AXS D2 Phaser diffractometer.

[0061] X-ray photoelectron spectroscopy (XPS) was performed using the Shimadzu Kratos axis ultra DLD (Kyoto, Japan).

[0062] Electrochemiluminescence (ECL) signals were detected using a BPCL-Q-GP21-TGC ultra-weak fluorescence analyzer equipped with a conventional three-electrode system, which consists of a platinum wire counter electrode, an Ag / AgCl (3M KCl) reference electrode, and a modified glassy carbon electrode (GCE) as the working electrode.

[0063] The operating parameters of BPCL are: photomultiplier tube (PMT) voltage 1200 V, potential scan range 0~-2.0 V, scan rate 0.1 V / s.

[0064] Electrochemical tests were performed using a multichannel potentiostat (CHI1000C, Shanghai Chenhua Instrument Co., Ltd.).

[0065] The glassy carbon electrodes used in the embodiments of this invention are all XR303 glassy carbon electrodes from Shanghai Xianren Instrument Co., Ltd., with a diameter of 3mm.

[0066] Example 1: Preparation of composite materials

[0067] (1) Preparation of CsPbBr3@PVP composite material

[0068] 0.1468 g lead bromide, 0.0851 g cesium bromide, and 10 ml N,N-dimethylacetamide were mixed and stirred at 60 °C for 60 minutes. 200 µL oleic acid, 500 µL oleylamine, and 200 µL 6% polyvinylpyrrolidone solution were heated to 60 °C and then rapidly added sequentially to the above mixed solution. The mixture was stirred at 60 °C for another 30 minutes. Under vigorous stirring, 1 ml of the above precursor solution was rapidly added to 10 ml ethyl acetate, stirred for 2 minutes, and then incubated at 60 °C for 60 minutes. The mixed solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was removed, and the resulting precipitate was dried under vacuum at 60 °C overnight to obtain CsPbBr3@PVP powder.

[0069] (2) Preparation of CsPbBr3@PVP / Au composite material

[0070] 10 mg of cesium lead bromide@polyvinylpyrrolidone was dispersed in 10 ml of deionized water, and 20 µL of 10 mM chloroauric acid solution (HAuCl4) was added. The mixture was stirred for 5 minutes. Under ice bath conditions, 100 µL of 10 mM sodium borohydride aqueous solution was slowly added dropwise, and stirring was continued for 10 minutes. The reaction product was centrifuged, purified, and freeze-dried to obtain the final CsPbBr3@PVP / Au powder.

[0071] Example 2: Structural characterization and ECL performance testing of composite materials

[0072] (1) Characterization of composite material structure

[0073] Following the method described in step (1) of Example 1, CsPbBr3 nanocrystals were prepared directly without adding PVP solution.

[0074] CsPbBr3@PVP composite material was prepared according to the method described in step (1) of Example 1.

[0075] CsPbBr3@PVP / Au composite material was prepared by referring to the methods described in steps (1) and (2) of Example 1.

[0076] The structures of the prepared CsPbBr3 nanocrystals, CsPbBr3@PVP composites, and CsPbBr3@PVP / Au composites were characterized.

[0077] TEM characterization showed that CsPbBr3 exhibited a typical cubic structure with uniform size. Figure 1 A), with an average particle size of approximately 7.87 ± 1.73 nm. Figure 2 Au NPs modification can be clearly observed on the surface of CsPbBr3@PVP / Au composite material. Figure 1 (B, 1C) indicates that Au NPs were successfully composited on the CsPbBr3@PVP surface.

[0078] HRTEM characterization showed that the lattice fringe spacing of the CsPbBr3@PVP / Au composite material was 0.41 nm. Figure 1 D), corresponding to the (110) crystal plane of CsPbBr3, indicates that the crystal structure of CsPbBr3 has not been destroyed.

[0079] HAADF-STEM-EDS mapping analysis showed that the CsPbBr3@PVP / Au composite material contains four elements: Cs, Pb, Br, and Au, and they are evenly distributed, further confirming the successful synthesis of the CsPbBr3@PVP / Au composite material. Figure 1 E).

[0080] FT-IR characterization showed that the CsPbBr3@PVP / Au composite material exhibited an Au-O vibration peak at 1009 cm⁻¹, a characteristic CN bond peak in PVP at 1250 cm⁻¹, and a C=O stretching vibration peak in PVP at 1673 cm⁻¹. Furthermore, the C=O absorption peak of CsPbBr3@PVP was significantly weaker than that of pure PVP, indicating that the C=O in PVP forms a strong coordination relationship with the Pb²⁺ on the CsPbBr3 surface. Figure 3 A).

[0081] XRD characterization showed that the XRD peaks of the synthesized CsPbBr3 and CsPbBr3@PVP were consistent with the CsPbBr3 standard card (PDF number 18-0364), indicating that they conform to the standard 3D crystal phase of CsPbBr3. In contrast, the XRD pattern of the water-dispersed CsPbBr3@PVP / Au showed characteristic peaks of both CsPbBr3 (PDF number 18-0364) and Au (PDF number 00-004-0784), indicating that CsPbBr3 and Au NPs coexisted in the composite material. Figure 3 B).

[0082] XPS characterization revealed the elemental composition of CsPbBr3@PVP / Au ( Figure 3 The results show that the binding energies of 83.80 eV and 87.70 eV in the Au 4f spectrum correspond to the 4f5 / 2 and 4f7 / 2 orbitals of Au NPs, respectively. Figure 3 D); The peaks at 284.80 eV, 285.95 eV, and 287.69 eV in the C 1s spectrum are attributed to the characteristic peaks of CC, CN, and C=O in PVP, respectively. Figure 3 E); the peaks at 142.71 eV and 137.85 eV in the Pb 4f spectrum are attributed to spin-orbit splitting of Pb 4f5 / 2 and Pb 4f7 / 2 (E); Figure 3 F), corresponding to the Pb-Br bond. The absence of peaks at ~141.0 eV and ~136.0 eV in the Pb 4f spectrum indicates the absence of zero-valence Pb metal in CsPbBr3@PVP / Au, thus reducing the non-radiative recombination process caused by Pb metal. High-resolution XPS spectra of N 1s, Cs 3d, Br 3d, and O 1s also verified the successful synthesis of the CsPbBr3@PVP / Au composite material. Figure 4 ).

[0083] CsPbBr3 can be stably dispersed in toluene, appears pale yellow under white light, and emits green fluorescence under 365 nm ultraviolet light. At an excitation wavelength (Ex) of 365 nm, the emission wavelength (Em) is 522 nm. Figure 5A); Scanning the excitation wavelength revealed that the emission wavelength of CsPbBr3 did not change ( Figure 5 B), indicating good crystal quality. After being combined with PVP, CsPbBr3@PVP can be stably dispersed in water, exhibits a light green color under sunlight, and emits green fluorescence under 365 nm ultraviolet light. The excitation and emission wavelengths of CsPbBr3@PVP are consistent with those of CsPbBr3, both being 365 nm and 522 nm (B). Figure 6 A), and the emission wavelength does not change with the excitation wavelength ( Figure 6 B). After recombination with Au NPs, the resulting CsPbBr3@PVP / Au has an excitation wavelength of 343 nm and an emission wavelength of 520 nm. Figure 7 A), excitation-independent luminescence properties ( Figure 7 B), compared to before recombination, exhibits a slight blue shift in emission wavelength and a decrease in fluorescence intensity. Furthermore, the ultraviolet absorption peak shifts from 520 nm to 509 nm, and the absorption intensity also decreases. Figure 8 This is attributed to the exchange of Br⁻ in CsPbBr3 with Cl⁻ in HAuCl4, as well as the energy transfer effect between CsPbBr3 and Au NPs.

[0084] (2) ECL performance test analysis

[0085] Referring to the method described in step (1) of Example 1, without adding PVP solution, CsPbBr3 nanocrystals were directly prepared, and the glassy carbon electrode was modified with CsPbBr3 nanocrystals according to the methods described in steps (1) and (2) of Example 2.

[0086] CsPbBr3@PVP composite material was prepared according to the method described in step (1) of Example 1, and glassy carbon electrode was modified with CsPbBr3@PVP composite material according to the methods described in steps (1) and (2) of Example 2.

[0087] CsPbBr3@PVP / Au composite material was prepared according to the method described in steps (1) and (2) of Example 1, and the glassy carbon electrode was modified with CsPbBr3@PVP / Au composite material according to the method described in steps (1) and (2) of Example 2.

[0088] The ECL performance of bare glassy carbon electrode, CsPbBr3-modified glassy carbon electrode, CsPbBr3@PVP-modified glassy carbon electrode, and CsPbBr3@PVP / Au-modified glassy carbon electrode were tested in 0.1 M PBS containing 0.1 M K2S2O8 at pH 7.4. The results showed that the ECL strength of CsPbBr3@PVP was approximately four times higher than that of CsPbBr3. Figure 9A), which is attributed to the lone pair electrons of the carbonyl oxygen in PVP forming a strong coordination interaction with the uncoordinated Pb²⁺ ions on the surface of CsPbBr3 nanocrystals. This coordination interaction passivates surface defects, allowing more charge carriers to generate photons through radiative recombination, thereby significantly enhancing the ECL intensity and efficiency. When Au NPs are modified on the surface of CsPbBr3@PVP to synthesize CsPbBr3@PVP / Au, a slight decrease in ECL intensity is observed due to the energy transfer effect between the two. Figure 9 A).

[0089] like Figure 9 As shown in Figure B, the cyclic voltammetry curves indicate that the cathode current response of the composite material CsPbBr3@PVP is approximately twice that of CsPbBr3, demonstrating that PVP significantly improves the stability of CsPbBr3 and substantially increases the number of nanocrystals effectively participating in the electrochemical reaction on the electrode surface. Simultaneously, the PVP insulating layer increases the kinetic barrier for electron transport, requiring a more negative potential to drive electron injection, resulting in a slight negative shift of the reduction peak. After loading Au NPs, the electrocatalytic effect of metal ions accelerates the electron transfer process in the ECL reaction, further improving the cathode current response of CsPbBr3@PVP / Au. Furthermore, compared to the fluorescence (FL) emission peak, the ECL emission peak shows a red shift of approximately 20 nm and an increase in full width at half maximum (FWHM) of approximately 19 nm. Figure 9 (C) This change can be attributed to the strong correlation between ECL and the surface chemistry of the luminescent material, where unavoidable surface defects lead to the relaxation of electrons and holes, thereby extending and redshifting the ECL emission.

[0090] The possible ECL reaction equations involved in CsPbBr3@PVP / Au are as follows:

[0091] (1)

[0092] (2)

[0093] (3)

[0094] (4)

[0095] (5)

[0096] The possible reaction mechanism is speculated to be as follows: First, the electrochemical reduction of S2O8²⁻ generates sulfate anion radicals ( ); strong oxidizing intermediate Electrons are extracted from CsPbBr3@PVP, and then holes are injected into the highest occupied molecular orbital to generate... Subsequently, CsPbBr3@PVP / Au is electrochemically reduced to a negatively charged state by injecting electrons into the lowest unoccupied molecular orbital. ); Oxidizing free radicals ( ) and electrogenerated reducing free radicals ( Electron transfer and annihilation between the two states generate excited states of CsPbBr3*@PVP / Au, which ultimately emit photons through the radiation path, resulting in strong ECL emission.

[0097] The stability of the composite material was investigated by performing continuous scanning ECL tests over 15 cycles. The results showed that the ECL strength of the CsPbBr3@PVP / Au composite material exhibited good stability over 15 cycles. Figure 9 D).

[0098] To further investigate the long-term stability of the composite material, a 120-hour continuous ECL test was conducted. The results showed that after 120 hours, the ECL strength of the CsPbBr3@PVP / Au composite material still maintained more than 80% of its initial strength. Figure 9 (E, 9F), demonstrating its excellent long-term stability, making it suitable for ECL biosensors.

[0099] Example 3: Fabrication of a Tau electrochemiluminescence aptamer sensor

[0100] (1) Pretreatment of glassy carbon electrode (GCE): The glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powder in sequence, then rinsed with deionized water, then rinsed with ethanol, and then placed in an ultrasonic cleaner for ultrasonic cleaning for 2 minutes to remove the residual Al2O3 powder on the electrode surface; the cleaned glassy carbon electrode was placed in 0.5 M H2SO4 solution and activated by cyclic voltammetry with a scanning range of -0.2 V to 1.0 V and a scanning rate of 0.1 V / s until the redox potential difference was less than 120 mV when characterized by 5 mM potassium ferricyanide / potassium ferrocyanide solution. After removal, it was rinsed with deionized water and dried for later use.

[0101] (2) CsPbBr3@PVP / Au modified electrode: Weigh 1 mg of CsPbBr3@PVP / Au powder prepared in Example 1, disperse it in 1 mL of deionized water, and sonicate it for 10 minutes to obtain a 1 mg / mL CsPbBr3@PVP / Au dispersion; use a pipette to take 6 μL of the dispersion and drop it onto the center of the pretreated glassy carbon electrode, and dry it at 37°C for 4 hours to make CsPbBr3@PVP / Au uniformly adhere to the electrode surface to obtain a CsPbBr3@PVP / Au modified electrode.

[0102] (3) Aptamer immobilization: Tau protein-specific aptamers were diluted to 10 μM with 0.1 M, pH 7.4 PBS buffer, and TCEP solution (final concentration 3 mg / mL) was added. The aptamers were incubated at room temperature for 30 minutes to activate the thiol groups at the ends of the aptamers. 6 μL of the activated aptamer solution was pipetted onto the surface of the CsPbBr3@PVP / Au modified electrode and incubated at 4 °C for 12 hours to allow the aptamers to bind to Au NPs on the electrode surface through Au-S bonds. After incubation, the electrode surface was rinsed with 0.1 M, pH 7.4 PBS buffer to remove non-specifically adsorbed aptamers.

[0103] (4) Blocking treatment: Use a pipette to draw 6 μL of 10 mM MCH solution and drop it onto the electrode surface. Incubate at room temperature for 1 hour to block the active sites of Au NPs that are not bound to aptamers on the electrode surface and reduce non-specific adsorption. After incubation, rinse the electrode surface with 0.1M, pH 7.4 PBS buffer and air dry to obtain the Tau protein electrochemiluminescent aptamer sensor.

[0104] Example 4: Sensor Feasibility Study

[0105] The sensor prepared in Example 3 was tested and analyzed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. Figure 10 As shown in A and 10B, the electrode conductivity decreased due to the modification of the electrode surface with the composite material CsPbBr3@PVP / Au, resulting in increased electrode surface resistance and a reduced current response. After incubating the aptamer on the electrode surface, the aptamer chain binds to the electrode through Au-S bonds. Due to the increased steric hindrance, the resistance further increases, and the current response continues to decrease. Adding MCH to the electrode surface reduces non-specific interference. When the Tau protein standard solution is dropped onto the ECL biosensor, the Tau protein aptamer can accurately capture the protein. The aptamer-target complex formed on the glassy carbon electrode surface generates significant steric hindrance, hindering the diffusion of redox substances to the interface, thereby causing a significant decrease in current response and a corresponding increase in charge transfer resistance.

[0106] Example 5: Investigation of Influencing Factors

[0107] (1) Effect of PVP content: CsPbBr3@PVP composite materials with PVP mass fractions of 2%, 4%, 6%, 8%, and 10% were prepared according to the method in Example 1, and then CsPbBr3@PVP / Au composite materials were prepared. Sensors were prepared according to the method in Example 3. The ECL intensity of the sensors under different PVP contents was tested in 0.1 M PBS buffer containing 0.1 M K2S2O8 at pH 7.4. The results showed that the ECL intensity reached its maximum value when the PVP content was 6%. Figure 11A) If the PVP content is too low, defect passivation is insufficient, resulting in low ECL strength; if the PVP content is too high, the excessively thick insulation layer hinders charge transfer, further reducing ECL strength. Therefore, the optimal PVP content is 6%.

[0108] (2) Effect of K2S2O8 concentration: K2S2O8 was added to 0.1 M, pH 7.4 PBS buffer at concentrations of 10 mM, 50 mM, 100 mM, 150 mM, 180 mM, 200 mM, and 300 mM, respectively, and the ECL intensity of the sensor was tested at different K2S2O8 concentrations. The results showed that the ECL intensity reached its maximum value when the K2S2O8 concentration was 200 mM. Figure 11 (B) If the concentration is too low, there will be insufficient active intermediates, resulting in a weak ECL signal; if the concentration is too high, excessive co-reactants will interfere with the interfacial reaction, leading to a decrease in ECL intensity. Therefore, the optimal K2S2O8 concentration is 200 mM.

[0109] (3) Effect of potential scanning range: The potential scanning ranges were set to 0 to -1.8 V, 0 to -1.9 V, 0 to -2.0 V, 0 to -2.1 V, and 0 to -2.2 V, and the ECL intensity of the sensor was tested in different scanning ranges. The results showed that the ECL intensity reached its maximum value when the scanning range was 0 to -2.0 V. Figure 11 (C) If the scan range is too narrow, the voltage will be insufficient to fully reduce the co-reactants and luminescent material, resulting in a weak ECL signal; if the scan range is too negative, it will trigger severe side reactions, causing a decrease in ECL intensity. Therefore, the optimal potential scan range is 0 to -2.0 V.

[0110] (4) Effect of CsPbBr3@PVP / Au Dosage: 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of 1 mg / mL CsPbBr3@PVP / Au dispersion were respectively added to the electrode surface. The sensor was prepared according to the method in Example 3, and its ECL intensity was tested. The results showed that the ECL intensity reached its maximum value when the dosage was 6 μL. Figure 11 (D) Insufficient addition will prevent the formation of a complete superlattice film, resulting in low ECL strength; excessive addition will hinder electron transfer, further reducing ECL strength. Therefore, the optimal addition amount of CsPbBr3@PVP / Au is 6 μL.

[0111] Example 6: Quantitative Detection of Tau Protein

[0112] Under optimal experimental conditions—namely, a PVP content of 6% in the CsPbBr3@PVP / Au composite material, a K2S2O8 concentration of 200 mM, a positional scan range of 0 to -2.0 V, and a CsPbBr3@PVP / Au drop volume of 6 μL—a sensor was prepared according to the method in Example 3 to quantitatively detect different concentrations of Tau protein. The specific steps are as follows:

[0113] Construction of standard curves: Tau protein standards were diluted with 0.1 M, pH 7.4 PBS buffer to concentrations of 10 fg / mL, 10² fg / mL, 10³ fg / mL, and 10⁻⁶ fg / mL. 4 fg / mL, 10 5 fg / mL, 10 6 fg / mL, 10 7 fg / mL, 10 8 A standard solution of fg / mL was prepared; 6 μL of different concentrations of Tau protein standard solution were added to the sensor surface and incubated at room temperature for 1 hour. The sensor was then rinsed three times with PBS buffer and air-dried. The sensor was placed in 0.1 M PBS buffer containing 200 mM K2S2O8 at pH 7.4. An electrochemiluminescence assay system was used, with the PMT voltage set to 1200 V, the potential scan range from 0 to -2.0 V, and the scan rate at 0.1 V / s. The ECL intensity at each concentration was recorded. A standard curve was plotted with the logarithm of Tau protein concentration (lg C) on the x-axis and the ECL intensity on the y-axis.

[0114] Calculation of detection limit: Following the above method, test the ECL intensity of blank sample (PBS buffer without Tau protein), repeat the test 3 times, and calculate the standard deviation (SD) of the blank signal; calculate the detection limit of the sensor (LOD=3SD / k) based on the ratio of 3 times the standard deviation (3SD) to the slope of the standard curve.

[0115] The results showed that as the concentration of Tau protein increased, the ECL intensity of the sensor gradually decreased. Figure 12 A); at 10 fg / mL to 10 8 Within the concentration range of fg / mL, the logarithm of Tau protein concentration showed a good linear relationship with ECL intensity, with the standard curve equation being y = -561.75 lg C + 5952.07 and a correlation coefficient R² = 0.996. Figure 12 B); The calculated detection limit of the sensor is 0.80 fg / mL (S / N=3), indicating that the sensor has extremely high detection sensitivity and can achieve accurate detection of low concentrations of Tau protein.

[0116] Example 7: Sensor Stability Test

[0117] After incubating the sensor with a 10 fg / mL Tau protein solution, 10 consecutive cyclic scans were performed according to the test conditions of Example 6. The ECL intensity of each scan was recorded, and the relative standard deviation (RSD) was calculated.

[0118] The results showed that the ECL intensity did not fluctuate significantly after 10 consecutive cyclic scans, with an RSD of 1.09%. Figure 13 This indicates that the sensor has good stability.

[0119] Example 8: Selectivity Test of the Sensor

[0120] Alpha-fetoprotein (AFP), bovine serum albumin (BSA), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) were selected as interfering substances to test the selectivity of the sensor. The specific steps are as follows:

[0121] Tau protein, AFP, BSA, CEA, and PSA were diluted to 10 μL with 0.1 M, pH 7.4 PBS buffer, respectively. 8 fg / mL;

[0122] Take 6 μL of each of the above-mentioned solutions, as well as a mixed solution of Tau protein and four interfering substances (each substance concentration is 10). 8 (fg / mL) was added to the sensor surface, incubated at room temperature for 1 hour, rinsed 3 times with PBS buffer, and air-dried.

[0123] According to the test conditions of Example 6, the ECL intensity of each sample was recorded, and the influence of each interfering substance on the sensor ECL signal was calculated.

[0124] The results showed that the presence of AFP, BSA, CEA, and PSA had minimal impact on the ECL signal; when 10 8 When fg / mL of Tau protein was mixed with interfering substances, the ECL signal decreased rapidly; compared with detection alone, 10 8 The observed signal deviation for fg / mL Tau protein is negligible. Figure 14 This indicates that the sensor has high selectivity for the Tau protein.

[0125] Example 9 Detection of Tau protein in mouse cerebrospinal fluid (spiked recovery experiment)

[0126] Mouse cerebrospinal fluid diluted 1000 times (1‰ MCSF) was selected as the detection sample, and a spiked recovery experiment was performed to verify the sensor's detection performance in real samples. The specific steps are as follows:

[0127] (1) Plotting the standard curve:

[0128] Tau protein standard was added to 1‰ MCSF samples to achieve spiked concentrations of 10 fg / mL, 10² fg / mL, 10³ fg / mL, and 10, respectively. 4 fg / mL, 10 5 fg / mL, 10 6 fg / mL, 10 7 fg / mL, 10 8 A standard solution of fg / mL was prepared; 6 μL of different concentrations of Tau protein standard solution were added to the sensor surface and incubated at room temperature for 1 hour. The sensor was then rinsed three times with PBS buffer and air-dried. The sensor was placed in 0.1 M PBS buffer containing 200 mM K2S2O8 at pH 7.4. An electrochemiluminescence assay system was used, with the PMT voltage set to 1200 V, the potential scan range from 0 to -2.0 V, and the scan rate at 0.1 V / s. The ECL intensity at each concentration was recorded. A standard curve was plotted with the logarithm of Tau protein concentration (lg C) on the x-axis and the ECL intensity on the y-axis.

[0129] (2) After the spiked concentration test

[0130] Tau protein standard was added to 1‰ MCSF samples to achieve spiked concentrations of 1 pg / mL, 10 pg / mL, and 100 pg / mL, with three replicates for each concentration. 6 μL of each spiked sample was added to the sensor surface and incubated at room temperature for 1 hour. The sensor was then rinsed three times with PBS buffer and air-dried. The sensor was placed in 0.1 M PBS buffer (pH 7.4) containing 200 mM K₂S₂O₈. An electrochemiluminescence assay system was used, with a PMT voltage of 1200 V, a potential scan range of 0 to -2.0 V, and a scan rate of 0.1 V / s. The ECL intensity at each concentration was recorded. The measured concentration of Tau protein was calculated based on the standard curve. The spiked recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 1 below.

[0131] Table 1

[0132] Sample number Dosage (pg / mL) Measured value (pg / mL) Recovery rate (%) Relative standard deviation (%, n=3) 1 1 1.04 104.02 2.89 2 10 9.72 97.20 3.20 3 100 108.60 108.60 6.50

[0133] The results showed that the spiked recoveries ranged from 97.20% to 108.60%, with RSD < 6.50% (n=3); and in 1‰ MCSF samples, a good linear relationship was still observed between Tau protein concentration and ECL intensity, with the standard curve equation being y = -560.16lg C + 5943.77, R² = 0.993. Figure 15This indicates that the sensor still exhibits excellent detection performance in complex biological samples, has strong anti-interference capabilities, and can be used for the detection of Tau protein in real samples.

[0134] Example 10: Comparative Experiment of the Method of the Present Invention with Methods Reported in Existing Literature

[0135] Tau protein detection systems using different materials and detection methods reported in recent literature were selected and systematically compared with the method of this invention. The comparison indicators included detection materials, detection methods, linear range, and limit of detection (LOD). The specific comparison results are shown in Table 2 below.

[0136] Table 2

[0137] Testing materials Detection methods Linear range Detection limit source <![CDATA[F-TiO2]]> Electrochemical method 1.0 ng / mL ~ 200 ng / mL 1.774 pg / mL Reference 1 AuNs@g-CN Electrochemiluminescence 0.1 ng / mL ~ 100 ng / mL 0.034 ng / mL Reference 2 FeMOF Electrochemiluminescence <![CDATA[0.01 pg / mL~10 3 pg / mL]]> 3.38 fg / mL Reference 3 G4 hydrogel Electrochemical method 0.01 ng / mL ~ 100 ng / mL 1.31 pg / mL Reference 4 DTSSP Electrochemical method 2 ng / mL ~ 2000 ng / mL 1 ng / mL Reference 5 PBAS Colorimetric method 200 pg / ml~2 µg / mL 153 pg / mL Reference 6 <![CDATA[CsPbBr3@PVP / Au]]> Electrochemiluminescence <![CDATA[10 fg / mL~10 8 fg / mL]]> 0.80 fg / mL This invention

[0138] The results show that the Tau protein electrochemiluminescence aptamer sensor constructed in this invention exhibits superior sensitivity and a wider linear range in Tau protein detection, with overall performance exceeding that of existing detection methods, providing a more efficient and accurate detection platform for early AD diagnosis. Specific performance is as follows:

[0139] (1) Wider linear range: The linear range of the method of the present invention is 10 fg / mL~10 8 With a concentration of fg / mL, covering eight orders of magnitude, compared to all existing detection systems (which cover up to six orders of magnitude), the linear range is significantly broadened, enabling one-time quantitative detection of Tau protein in different concentration ranges without the need for multiple sample dilutions, thus improving detection efficiency.

[0140] (2) Lower detection limit: The detection limit of the method of the present invention is 0.80 fg / mL, which is much lower than that of detection systems such as F-TiO2 (1.774 pg / mL), AuNs@g-CN (0.034 ng / mL), and G4 hydrogel (1.31 pg / mL). The detection sensitivity is significantly improved, making it more suitable for the accurate detection of low concentrations of Tau protein in the early diagnosis of AD.

[0141] Among them, Literature 1: Zhang, Z.-h., Hu, J., Zhu, H., Chen, Q., Koh, K., Chen, H., Xu, X.-h., 2022. A facile and effective immunoassay for sensitive detection of phosphorylated Tau: The role of flower-shaped TiO2 in specificity and signal amplification. Sensors and Actuators B: Chemical 366, 132015。

[0142] Literature 2: Jalili, R., Chenaghlou, S., Khataee, A., Khalilzadeh, B., Rashidi, M.-R., 2022. An electrochemiluminescence biosensor for the detection of Alzheimer’s Tau protein based on gold nanostar decorated carbon nitride nanosheets. Molecules 27(2), 431。

[0143] Literature 3: Yuan, W., Tao, Q., Chen, X., Liu, T., Wang, J., Wang, X., 2025. Using machine learning to design a FeMOF bidirectional regulator for electrochemiluminescence sensing of Tau protein. ACS Applied Materials & Interfaces 17(6), 8924-8936

[0144] Document 4: Chen, Q., Hu, J., Mao, Z., Koh, K., Chen, H., 2022. Loachmucus-like guanosine-based hydrogel as an antifouling coating for electrochemical detection of Tau protein. Sensors and Actuators B: Chemical370, 132419.

[0145] Document 5: Yang, M., Chen, Y., Sun, H., Li, D., Li, Y., 2024. A simplesandwich electrochemical immunosensor for rapid detection of the Alzheimer'sdisease biomarker Tau protein. Biosensors 14(6), 279.

[0146] Document 6: Duan, C., Jiao, J., Zheng, J., Li, D., Ning, L., Xiang, Y., Li, G., 2020. Polyvalent biotinylated aptamer scaffold for rapid and sensitive detection of Tau proteins. Analytical Chemistry 92(22), 15162-15168.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Tau protein electrochemiluminescent aptamer sensor, characterized in that, The invention includes a working electrode, a perovskite nanocomposite material modified on the surface of the working electrode, and a Tau protein-specific aptamer immobilized on the surface of the perovskite nanocomposite material. The perovskite nanocomposite material is a CsPbBr3@PVP / Au composite material, composed of polyvinylpyrrolidone-encapsulated cesium bromide nanocrystals and gold nanoparticles. The polyvinylpyrrolidone achieves the encapsulation and passivation of the cesium bromide nanocrystals through coordination between its carbonyl groups and lead ions on the surface of the cesium bromide nanocrystals. The gold nanoparticles, which are zero-valent gold, are modified on the surface of the polyvinylpyrrolidone-encapsulated cesium bromide nanocrystals. The Tau protein-specific aptamer is a thiol-modified aptamer with a base sequence of 5'-3': CGG ACA CCA ACA ACC CCG CCC ACG C-C6-SH, which is immobilized on the surface of the gold nanoparticles through gold-sulfur bonds for the specific recognition of Tau protein.

2. The Tau protein electrochemiluminescence aptamer sensor according to claim 1, characterized in that, The working electrode is a glassy carbon electrode; the CsPbBr3 nanocrystals have a cubic structure with an average particle size of 7.87±1.73nm; the CsPbBr3@PVP / Au lattice fringe spacing is 0.41nm, corresponding to the (110) crystal plane; In the X-ray diffraction pattern of the water-dispersed CsPbBr3@PVP / Au composite material, characteristic diffraction peaks of both cesium lead bromide and gold are observed.

3. The sensor according to claim 2, characterized in that, The polyvinylpyrrolidone-encapsulated cesium lead bromide nanocrystals contain 6% polyvinylpyrrolidone by mass.

4. The sensor according to claim 1, characterized in that, It also includes a 6-mercapto-1-hexanol blocking layer, which is modified on the surface of the aptamer-immobilized CsPbBr3@PVP / Au composite material to suppress non-specific adsorption.

5. A method for preparing the sensor according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of CsPbBr3@PVP composite material: Lead bromide and cesium bromide were dissolved in N,N-dimethylacetamide and heated and stirred to obtain a mixed solution; oleic acid, oleylamine and polyvinylpyrrolidone solution were heated and added to the above mixed solution, and the reaction was carried out by stirring and temperature control to obtain a precursor solution; the precursor solution was added to ethyl acetate, stirred and mixed, precipitated under controlled temperature, and dried by centrifugation to obtain CsPbBr3@PVP powder; S2. Preparation of CsPbBr3@PVP / Au composite material: CsPbBr3@PVP was dispersed in deionized water, chloroauric acid solution was added and stirred, sodium borohydride solution was added dropwise under ice bath conditions to carry out reduction reaction, centrifugation purification and freeze drying were performed to obtain CsPbBr3@PVP / Au powder; S3. Sensor Assembly: Sensor assembly includes the following steps: S301. Pretreatment of working electrode: The glassy carbon electrode is successively polished, cleaned and activated; S302. Composite material modified working electrode: CsPbBr3@PVP / Au solution is drop-coated onto the surface of the pretreated glassy carbon electrode and dried to form a uniform sensing film; S303. Immobilization of aptamers: The Tau protein-specific aptamer activated by tris(2-carboxyethyl)phosphine was drop-coated onto the surface of the working electrode modified with the composite material, and incubated at a controlled temperature to immobilize the aptamer on the surface of the working electrode modified with the composite material. After incubation, the aptamer was rinsed off to remove the non-specifically adsorbed aptamer. S304. Sealing treatment: 6-mercapto-1-hexanol solution was dropped onto the sensor and incubated to seal non-specific adsorption sites. After incubation, the sensor was rinsed to obtain the sensor.

6. The preparation method according to claim 5, characterized in that, In S1, the feed ratio of cesium bromide, lead bromide, and N,N-dimethylacetamide was 0.0851 g: 0.1468 g: 10 ml, the mixing temperature was 60 °C, and the mixing time was 60 minutes. The concentration of polyvinylpyrrolidone solution was 6%, and the feed ratio of cesium bromide, oleic acid, oleylamine, and polyvinylpyrrolidone was 0.0851 g: 200 µL: 500 µL: 200 µL, the reaction temperature was 60 °C, and the reaction time was 30 minutes. The feed ratio of precursor solution to ethyl acetate was 1 ml: 10 ml, the precipitation temperature was 60 °C, and the precipitation time was 60 minutes.

7. The preparation method according to claim 5, characterized in that, In S2, the concentration of chloroauric acid solution is 10 mM, the concentration of sodium borohydride solution is 10 mM, and the feed ratio of CsPbBr3@PVP, deionized water, chloroauric acid solution, and sodium borohydride solution is 10 mg: 10 ml: 20 µL: 100 µL.

8. The preparation method according to claim 5, characterized in that, In S301, the glassy carbon electrode was polished using 0.3µm and 0.05µm aluminum oxide powders; the cleaning method was ultrasonic cleaning with deionized water and ethanol; the activation method was activation in 0.5M sulfuric acid solution, characterized by a redox potential difference of less than 120mV using 5mM potassium ferricyanide / potassium ferrocyanide solution. In S302, the concentration of CsPbBr3@PVP / Au solution was 1 mg / mL, the drop volume was 6µL, the drying temperature was 37℃, and the drying time was 4 hours. In S303, the Tau protein-specific aptamer solution was activated with 3 mg / mL tris(2-carboxyethyl)phosphine at a concentration of 10µM, the drop volume was 6µL, the incubation temperature was 4℃, and the incubation time was 12 hours; after incubation, 0.1... Rinse with PBS buffer at pH 7.4 (M); In S304, the concentration of 6-mercapto-1-hexanol solution is 10 mM, the drop volume is 6 µL, the incubation temperature is room temperature, the incubation time is 1 hour, and after incubation, rinse with PBS buffer at pH 7.4 (0.1 M).

9. A method for detecting Tau protein, characterized in that, Includes the following steps: The sample to be tested is drop-coated onto the surface of the working electrode of the sensor according to any one of claims 1-4, incubated at 37°C for 1 h, and rinsed with PBS buffer. The rinsed sensor is placed in PBS buffer containing K2S2O8, and electrochemiluminescence detection is performed using a three-electrode system. The quantitative detection of Tau protein is achieved based on the change in the electrochemiluminescence signal intensity.

10. The method according to claim 9, characterized in that, The concentration of K2S2O8 was 200mM. During the test, the PMT voltage was set to 1200 V, the potential range was 0 to -2.0 V, and the cyclic voltammetry scan rate was 0.1 V / s.

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