Tau protein immunosensor and method of making the same

Abstract

The application provides a Tau protein immunosensor and a preparation method thereof, and the preparation method of the Tau protein immunosensor comprises the following steps: mixing manganese-doped molybdenum disulfide and a gold source to obtain a gold nanoparticle-manganese-doped molybdenum disulfide complex; covering a suspension containing the gold nanoparticle-manganese-doped molybdenum disulfide complex on a screen-printed electrode to form a complex layer on the surface of the screen-printed electrode; S3: covering an anti-Tau protein antibody solution on the complex layer and incubating for at least 1 hour, then washing away the excess anti-Tau protein antibody solution to obtain an anti-Tau protein antibody layer; and S4: blocking non-specific binding sites on the anti-Tau protein antibody layer to obtain the Tau protein detection sensor. According to tests, the detection limit of the Tau protein immunosensor constructed by the application can be as low as 20 fM.

Description

Technical Field

[0001] This invention relates to the field of electrochemical immunosensors, and more particularly to a Tau protein immunosensor and its preparation method. Background Technology

[0002] Alzheimer's disease (AD), a chronic neurodegenerative disease, is difficult to slow once its neurodegenerative mechanisms are established, and effective treatments are lacking. The formation of neurofibrillary tangles (NFTs) is one of the key pathological features of AD in the preclinical stage and typical pathological characteristics. Tau protein aggregates undergo soluble hyperphosphorylation on microtubule assemblies, thereby disrupting intracellular transport, impairing dendritic integrity, and producing neurotoxicity to neuronal function, subsequently accumulating into neurofibrillary tangles within neurons. NFTs in AD patients begin 20 to 30 years before the onset of symptoms, and the number of NFTs is positively correlated with the severity of dementia in AD. Therefore, early detection of functional and structural changes in Tau protein before significant accumulation can predict the onset of AD. However, as a major blood biomarker of AD, Tau protein levels in the blood are relatively low; therefore, effective detection requires immunosensors with excellent specificity and sensitivity.

[0003] Numerous immunosensors for Tau protein detection have been reported in existing technologies, such as: In 2019, Haoyu Wang et al. prepared an Au-TH-CG / GCE electrochemical aptamer sensor with a Tau 381 detection range of 1.0 pM to 100 pM and a detection limit of 700 fM (7 × 10⁻⁶). -13 M).

[0004] Shui, B, et al. prepared the Aptamer-Au-CS electrochemical biosensor in 2018, with a Tau 381 protein detection range of 500 fM–100.0 pM and a detection limit of 420 fM (4.2 × 10⁻⁶ pM). -13 M).

[0005] In 2025, Haoyu Wang et al. fabricated a PDMS-NiFe-FeMn actively driven biosensor with a Tau protein detection range of 2.22 pM to 13.33 pM and a detection limit of 25.7 pM (2.57 × 10⁻⁶ pM). -11 M).

[0006] In 2025, Claudia A. Razzino et al. prepared a 3D-Au-PAMAM-p-ABA-SPCE electrochemical immunosensor with a Tau protein detection range of 110 fM to 91 pM and a detection limit of 31 pM (3.1 × 10⁻⁶). -11 M).

[0007] It can be seen that among the many known Tau protein immunosensors, the lowest detection limit is generally around 10. -11 ~10 -13 Above M. Therefore, given the instability and low concentration of Tau protein in blood, it is necessary to further improve the detection sensitivity of immunosensors for Tau protein to increase its clinical applicability. Summary of the Invention

[0008] This invention provides a novel immunosensor capable of detecting trace amounts of Tau protein, its preparation method, and its applications.

[0009] The first aspect of this invention provides a method for preparing a Tau protein immune sensor, comprising: S1: React manganese-doped molybdenum disulfide with a gold source to obtain a gold nanoparticle-manganese-doped molybdenum disulfide composite. S2: Cover the screen-printed electrode with a suspension containing the gold nanoparticle-manganese-doped molybdenum disulfide composite to form a composite layer on the surface of the screen-printed electrode. S3: Cover the complex layer with anti-Tau protein antibody solution and incubate for at least 1 hour, then wash away excess anti-Tau protein antibody solution to obtain an anti-Tau protein antibody layer; S4: Block the non-specific binding sites on the anti-Tau protein antibody layer to obtain the Tau protein detection sensor.

[0010] In this invention, the term "nanoparticle" refers to a particle with at least one dimension being nanometer-sized, such as 1-1000 nm.

[0011] The "manganese-doped molybdenum disulfide" used in this invention can be prepared using methods known in the prior art, and this invention does not limit its preparation method.

[0012] Furthermore, the anti-Tau protein antibody used in this invention can be implemented using antibodies known or commonly used in the prior art, and this invention does not limit it.

[0013] In some embodiments, in step S1, the mass ratio of the gold source to manganese-doped molybdenum disulfide is 1:0.5 to 1:2.

[0014] In some embodiments, the manganese-doped molybdenum disulfide used in step S1 is prepared by reacting a manganese source, a molybdenum source, and a sulfur source in a molar ratio of 1:(1.5-2.5):(7-15).

[0015] In some embodiments, in step S2, the coating amount of the gold nanoparticle-manganese-doped molybdenum disulfide composite is 2-4 mg / cm² based on the working electrode surface area of ​​the screen-printed electrode. 2 .

[0016] In some embodiments, in step S3, the concentration of the anti-Tau protein antibody solution is 3-5 μg / mL.

[0017] In some embodiments, the coverage of the anti-Tau protein antibody solution is 5-10 μL / mm, based on the surface area of ​​the screen-printed electrode. 2 .

[0018] In some embodiments, the incubation time for the anti-Tau protein antibody in step S3 is 1.5-2.5 hours.

[0019] A second aspect of the present invention provides a Tau protein immunosensor prepared by the preparation method of the first aspect.

[0020] In some embodiments, when using the Tau protein immunosensor to detect Tau protein, the Tau protein detection sensor is incubated with the sample to be detected for at least 45 minutes.

[0021] Beneficial effects This invention utilizes a screen-printed carbon electrode (SPCE) as a substrate, forming an Au / Mn-MoS2 nanocomposite with abundant functional sites on it. The introduction of gold nanoparticles significantly enhances electron transfer kinetics. Furthermore, anti-Tau antibodies are immobilized on the Au / Mn-MoS2 nanocomposite via Au-S covalent bonds, ultimately developing an electrochemical immunosensor for Tau antigen capture. This sensor exhibits excellent surface active sites, promoting increased antibody loading and achieving ultra-trace detection of Tau protein.

[0022] Based on experiments, the linear range of the immunosensor constructed in this invention is 20 fM to 2 nM, and the detection limit can be as low as 20 fM (2 × 10⁻⁶). -14 M). Attached Figure Description

[0023] Figure 1 A TEM image of Mn-MoS2 is shown; Figure 2 SEM images of Au / Mn-MoS2 are shown; Figure 3 The EDS spectrum of Au / Mn-MoS2 is shown; Figure 4 The elemental distribution of Au / Mn-MoS2 is shown; Figure 5 The XRD patterns of MoS2, Mn-MoS2 and Au / Mn-MoS2 are shown. Figure 6 This demonstrates the effect of the Au to Mn-MoS2 ratio in the Au / Mn-MoS2 complex on the performance of the immunosensor; Figure 7 The effect of antibody concentration on the performance of the immunosensor is shown; Figure 8 The effect of antibody incubation time on the performance of the immunosensor is shown; Figure 9 The effect of incubation time of Tau protein on sensor detection is shown; Figure 10 Cyclic voltammetry (CV) curves of modified electrodes at different stages of the fabrication of the immune sensor are shown. Figure 11 Electrochemical impedance spectroscopy (EIS) spectra of modified electrodes at different stages of the preparation of the immune sensor are shown. Figure 12A The DPV response signals of the immune sensor at different concentrations of Tau protein are shown. Figure 12B The current difference-concentration calibration curve of Tau antigen is shown; Figure 13 The results are from the selectivity test of the sensor; Figure 14 The results are from the sensor's reproducibility test. Figure 15 This is the result of the sensor's stability test. Detailed Implementation

[0024] To provide a clearer understanding of the technical solution, objectives, and effects of the present invention, specific embodiments of the present invention will now be described in conjunction with the accompanying drawings.

[0025] It should be noted that, unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] First, the preparation process of some of the raw materials required for the preparation and testing of the immune sensor will be explained.

[0027] Preparation of manganese-doped molybdenum disulfide (Mn-MoS2) 0.5 mmol manganese nitrate hexahydrate, 0.95 mmol sodium molybdate dihydrate, and 5 mmol thiourea were added to pure water and mixed thoroughly. The mixture was then hydrothermally reacted at 200 °C for 24 hours. The resulting powder was washed with pure water and ethanol, and then dried overnight at 60 °C to obtain a black powder, Mn-MoS2. 2 mg of the obtained powder was weighed and added to 10 mL of pure water, and sonicated to obtain a Mn-MoS2 suspension for later use.

[0028] Preparation of gold nanoparticle-manganese-doped molybdenum disulfide complex (Au / Mn-MoS2) 4 mL of previously prepared Mn-MoS2 (5 mg•mL) -1 ), 4 mL HAuCl·3H2O (5 mg·mL -1 The Au / Mn-MoS2 was mixed with 4 mL of NaOH solution (0.1 M) and diluted to 40 mL. The mixture was then sonicated at room temperature for 12 hours. The 40 mL mixture was then transferred to a Teflon-lined autoclave and subjected to a hydrothermal reaction at 180 °C for 12 hours to obtain Au / Mn-MoS2. The prepared Au / Mn-MoS2 was allowed to cool naturally to room temperature, rinsed with pure water, and dried for later use.

[0029] Characterization of Mn-MoS2 and Au / Mn-MoS2 First, the structure and morphology of the previously prepared Mn-MoS2 were characterized using transmission electron microscopy (TEM). Figure 1 The structure and morphology of the previously prepared Au / Mn-MoS2 were characterized using scanning electron microscopy (SEM). Figure 2 ).from Figure 1 As can be seen, MoS2 exhibits uniformly sized aggregated flower-like microspheres, assembled from interconnected petal-like nanosheets. When Mn... 2+ When doped with MoS2, granular Mn grows on the petal-like surface. 2+ The presence of ions shortens the spacing between nanosheets. This also increases the number of highly active sites and active centers exposed at the edges of MoS2.

[0030] from Figure 2 Au particles are clearly visible, indicating that Au has been successfully incorporated into the MoS2 nanosheets. This proves that Au... 3+ Au can be reduced to AuNPs, which are uniformly distributed on the MoS2 surface, thus hindering the aggregation of AuNPs. Au promotes a high electron transfer rate through covalent coupling with the MoS2 interface, which helps to enhance its intrinsic activity and reaction kinetics.

[0031] Further EDS spectral characterization of Au / Mn-MoS2 was performed, and the results are as follows: Figure 3 As shown. In Figure 3In the diffraction angle, the (100) / (110) crystal plane can be observed, indicating that the crystal structure of MoS2 is not destroyed and the spacing between Mn-doped crystal planes changes very little. Among them, when AuNPs nucleate and grow on the Mn-MoS2 surface, the (111) crystal plane preferentially forms the (111) plane with the lowest energy, which also indicates that AuNPs crystallize well and are relatively uniform.

[0032] Further characterization of Au / Mn-MoS2 was performed using transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS). Figure 4 The results further indicated the elemental concentrations as 1.0% (Mn), 6.5% (Au), 15.0% (O), 29% (Mo), and 48.4% (S). The presence of Au and its uniform distribution within Mn-MoS2 were also confirmed.

[0033] Further XRD analysis confirmed the successful synthesis of MoS2 and Mn-MoS2, as well as the complexation of AuNPs, revealing a clear compositional pattern. Figure 5 The XRD spectrum shown indicates that the peaks corresponding to the hexagonal crystal structure of MoS2 are located at 9.36°, 32.56°, and 57.61°, indicating the presence of the (002), (102), and (110) planes. The (002) plane shows that the nanosheets preferentially stack along the c-axis, forming a nanoflower-like structure. Furthermore, the (002) plane exhibits a significant blue shift to 13.38°, presumably due to the increased interlayer spacing caused by Mn doping. Materials with high specific surface area and porous structure can provide more active sites. The more exposed active sites, the larger the contact area between the electrode surface and the electrolyte, resulting in higher charge transfer and electrode reaction rates. The peaks at 33.72° and 57.61° on the remaining crystal planes indicate the formation of Mn-MoS2. Due to the incorporation of AuNPs, 38.16°, 44.25°, 64.62°, and 77.54° were observed in Au / Mn-MoS2, corresponding to the (111), (200), (220), and (311) planes, respectively, indicating a significant interaction between Au and Mn-MoS2 materials.

[0034] General process for preparing electrochemical immunosensors Pretreatment of the screen-printed electrode (SPCE). Before electrode modification, the SPCE is first activated to improve its conductivity: 100 μL of 0.5 M H2SO4 solution is dropped onto the SPCE surface, and then cyclic voltammetry (CV) is used to scan 10 times from -0.2 V to +0.6 V at a scan rate of 0.1 V / s. After completion, the SPCE is rinsed 3 times with deionized water. In this invention, the parameters of the screen-printed electrode used are as follows: working electrode: 3 mm / 0.0071 cm 2 Counter electrode (carbon electrode) (0.050cm) 2Reference electrode (silver electrode) (0.010cm) 2 ).

[0035] Then, droplets of Au / Mn-MoS2 suspension were placed on the working electrode of the newly activated SPCE electrode to cover its surface, and dried at 37°C.

[0036] Antibody (recombinant Anti-Tau (phospho T231) antibody, Abcam, UK) PBS solution was dropped onto the Au / Mn-MoS2 / SPCE surface and incubated at 4°C. The surface was then washed three times with PBS (0.01 M, pH 7.4) to remove excess antibody and dried.

[0037] Then, non-specific binding sites were blocked with bovine serum albumin (BSA, 1%) and incubated for 45 minutes. The cells were then washed three times with PBS (0.01 M, pH=7.4) to remove excess BSA, and dried to obtain the immunosensor.

[0038] General preparation process of immune sensors incorporating Tau protein The subsequent characterization and optimization process requires the use of an immunosensor containing Tau protein, and its preparation process is described here.

[0039] Tau protein (human TAU microtubule-associated protein antigen, Shanghai Sangon Biotech Co., Ltd.) was added to the surface of the immunosensor prepared according to the aforementioned immunosensor preparation method. The immunosensor was incubated at 37°C and then washed three times with PBS (0.01 M, pH=7.4) to remove excess Tau, thus obtaining an immunosensor bound to Tau protein.

[0040] Condition optimization experiment To construct a sensitive and efficient immunosensor, the formation process and related parameters of the immunosensor were monitored using differential pulse voltammetry (DPV) to screen for the optimal process parameters.

[0041] DPV measurements were performed in a solution containing 5.0 mM [Fe(CN)6]. 3- / 4- The experiment was conducted in PBS solution (pH 7.4) containing 0.1 M KCl. The scan range was from -0.2 V to +0.6 V, the amplitude was 0.05 V, the pulse period was 0.2 seconds, and the change in peak current was recorded.

[0042] It should be noted that all DPV measurements in this article are performed under the conditions described above.

[0043] Optimization of the ratio of Au to Mn-MoS2 in Experiment Example 1 First, following the aforementioned preparation method for Au / Mn-MoS2, only the amount of HAuCl·3H2O was varied (the mass ratio of HAuCl·3H2O to Mn-MoS2 was 1:0.5, 1:1, 1:1.5, and 1:2, respectively), to prepare a series of Au / Mn-MoS2 complexes. Then, following the general process for preparing the aforementioned immunosensor, the Au / Mn-MoS2 complexes with different ratios of Au and Mn-MoS2 (the concentration of Au / Mn-MoS2 complex was 2 mg / mL and the volume was 10 μL) were loaded onto an SPCE to obtain an SPCE electrode with an Au / Mn-MoS2 complex layer. DPV detection was then performed on these electrodes. The results are as follows: Figure 6 As shown.

[0044] from Figure 6 As can be seen, the mass ratio of Au to Mn-MoS2 has a significant impact on the overall stability of the electrode. When Au:Mn-MoS2 = 1:0.5, the sensor sensitivity is low, possibly due to insufficient Mn-MoS2 content and weak electron transport capability. As the Mn-MoS2 ratio increases, the sensitivity reaches its maximum when Au:Mn-MoS2 = 1:1. At this point, the two-dimensional conductive plane constructed by Mn-MoS2 fully integrates with the active sites of AuNPs, and the electron transport efficiency and the number of active sites reach saturation.

[0045] Optimization of Anti-Tau Antibody Concentration in Experiment Example 2 Optimal antigen-antibody ratio is crucial in biosensing assays. Insufficient antibody concentration may lead to underestimated antigen levels due to reduced binding efficiency, while excessive antibody can induce antigen aggregation and hinder accurate quantification. In this experimental example, the concentration of anti-Tau antibody was systematically optimized to achieve maximum binding efficiency and signal-to-noise ratio. Therefore, based on the results of the aforementioned optimized experimental example 1, after forming a complex layer using 10 μL of 1:1 Au / Mn-MoS2 suspension (concentration 2 mg / mL), the antibody layer was prepared following the general procedure for immunosensor preparation described above. Different concentrations of 5 μL antibody PBS solution (1, 2, 3, 4, 5 μg / mL) were dropped onto the Au / Mn-MoS2 / SPCE surface and incubated at 4°C for 2 hours. The antibody layer was then subjected to DPV detection. The detection results are as follows: Figure 7 As shown.

[0046] from Figure 7 As can be seen, when the anti-Tau concentration increases to 5 μg / mL, the differential current (ΔI) reaches a plateau, indicating that the antigen binding site is saturated and the recognition efficiency is the highest at this concentration.

[0047] Optimization of Anti-Tau Antibody Incubation Time (Experiment Example 3) Antibody incubation time is a key factor affecting specific recognition. Therefore, based on the results of the aforementioned optimized experiments 1 and 2, after forming a complex layer using 10 μL of Au:Mn-MoS2=1:1 Au / Mn-MoS2 suspension (2 mg / mL), the antibody layer was prepared by dropping 5 μL of antibody (antibody concentration 5 μg / mL) onto the Au / Mn-MoS2 / SPCE surface and incubating at 4°C for different times (0.5, 1, 1.5, 5, 2.5 hours). DPV detection was then performed on the antibody layer. The detection results are as follows: Figure 8 As shown.

[0048] from Figure 8 As can be seen, as the incubation time increases from 0.5 hours to 2 hours, (ΔI) continuously decreases and tends to stabilize, indicating that the binding of anti-Tau antibody to Au / Mn-MoS2 reaches saturation. Therefore, the preferred antibody incubation time is 2 hours.

[0049] Optimization of Antigen Incubation Time in Experiment Example 4 This optimized experimental example aims to investigate the effect of Tau antigen incubation time on the detection performance of the immunosensor. Following the general procedure for immunosensor preparation described above and using the optimal preparation conditions determined in the previous optimization experiment (10 μL (2 mg / mL) Au:Mn-MoS2 suspension = 1:1, 5 μL antibody (5 μg / mL concentration) incubated for 2 hours), the immunosensor was prepared. Then, following the general preparation procedure for Tau-bound immunosensors described above, 5 μL (10 pg / mL) of Tau in PBS solution was incubated with the sensor for different times (15, 30, 45, 60, 75 minutes), and the Tau-bound sensors were detected. The detection results are as follows: Figure 9 As shown.

[0050] from Figure 9 As can be seen, the current difference (ΔI) significantly increased with the incubation time increasing from 15 minutes to 45 minutes, indicating that the specific recognition and binding between the antigen and the antibody modified on the Au / Mn-MoS2 composite material gradually stabilized. When the incubation time exceeded 45 minutes, the ΔI value remained essentially constant, indicating that the antigen-antibody specific binding had reached saturation, and further extending the incubation time did not significantly enhance the response signal. To maintain experimental efficiency, 60 minutes was selected as the optimal antigen incubation time.

[0051] Preferred embodiment 1 of the immune sensor Pretreatment of screen-printed electrodes (SPCE). Before electrode modification, the SPCE was first activated to improve its conductivity: 10 μL of 0.5 M H2SO4 solution was dropped onto the SPCE surface, and then cyclic voltammetry (CV) was used to scan 10 times from -0.2 V to +0.6 V at a scan rate of 0.1 V / s. After completion, the SPCE was rinsed 3 times with deionized water.

[0052] Then, 10 μL of Au / Mn-MoS2 suspension (Au:Mn-MoS2=1:1, 2 mg / mL) was dropped onto the freshly activated SPCE electrode, so that the Au / Mn-MoS2 suspension covered the surface of the SPCE electrode, and dried at 37°C.

[0053] 5 μL of antibody (5 μg / mL) was dropped onto the Au / Mn-MoS2 / SPCE surface and incubated at 4 °C for 2 hours. Then, the surface was washed three times with PBS (0.01 M, pH=7.4) to remove excess antibody and dried.

[0054] Then, non-specific binding sites were blocked with bovine serum albumin (BSA, 1%) and incubated for 45 minutes. The cells were then washed three times with PBS (0.01 M, pH=7.4) to remove excess BSA, and dried to obtain the immunosensor.

[0055] Preferred Example 2: An immune sensor incorporating Tau protein On the surface of the immunosensor prepared, for example, in preferred embodiment 1, 5 μL (10 pg / mL) of Tau in PBS solution was added, and the mixture was incubated at 37°C for 60 minutes. Then, it was washed three times with PBS (0.01 M, pH=7.4) to remove excess Tau.

[0056] Electrochemical monitoring of immunosensor fabrication Electrochemical monitoring of the sensor mainly utilizes cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to verify each layer of material modified on the electrode surface. Changes in current and impedance values ​​can indicate whether the added material has been successfully modified on the electrode surface.

[0057] CV measurements were performed in a solution containing 5.0 mM [Fe(CN)6]. 3- / 4- The experiment was conducted in PBS buffer solution of 0.1 M KCl (pH 7.4) with a potential range of –0.2 V to +0.6 V and an amplitude of 0.05 V.

[0058] EIS measurements were performed in a solution containing 5.0 mM [Fe(CN)6]. 3- / 4- The experiments were conducted in PBS buffer (pH 7.4) with 0.1 M KCl, at frequencies ranging from 0.1 Hz to 10 Hz.6 Hz, amplitude is 5 mV.

[0059] It should be noted that the above conditions were followed when discussing CV and EIS measurements in this article.

[0060] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed on the various immunosensor intermediates (also referred to herein as modified electrodes at different preparation stages) from different preparation stages in Preferred Example 1, the final immunosensor, and the immunosensor bound to Tau protein in Preferred Example 2. The results are as follows: Figure 10 and 11 As shown.

[0061] exist Figure 10 as well as Figure 11 In this context, SPCE represents a bare screen-printed electrode; Mn-MoS2 / SPCE represents a screen-printed electrode further having a Mn-MoS2 layer formed; Au / Mn-MoS2 / SPCE represents a screen-printed electrode further loaded with gold nanoparticles; Ab / Au / Mn-MoS2 / SPCE represents a screen-printed electrode loaded with antibodies; BSA / Ab / Au / Mn-MoS2 / SPCE represents a screen-printed electrode further blocked by BSA (preferred embodiment 1); and Tau / BSA / Ab / Au / Mn-MoS2 / SPCE represents an immunosensor bound to Tau protein (preferred embodiment 2).

[0062] like Figure 10 As shown, loading Mn-MoS2 onto the SPCE significantly enhanced the electrochemical response and increased the electrochemical signal. Furthermore, Mn-MoS2 provided numerous active sites for antibody immobilization. The addition of AuNPs further improved the electron transfer rate on the electrode surface, resulting in the highest reduction and oxidation current peaks. These results confirm the successful immobilization of the material on the electrode surface and its excellent conductivity. The peak current was somewhat reduced due to the bioconductive properties of the Tau protein antibody (Ab) and BSA. When the prepared sensor was incubated with Tau antigen, a further decrease in the peak current at target binding was observed. This indicates that the specific recognition of Tau antigen by the immunosensor further hindered electron transfer.

[0063] Electrochemical impedance spectroscopy (EIS) uses the Nyquist plot, which consists of semicircular and linear segments, to represent electrode detection and can be used to evaluate the interfacial properties of modified electrodes. Figure 11 The EIS plots of the modified electrodes at different fabrication stages are shown. Figure 11As can be seen, the electron transfer resistance (retrofit) of Mn-MoS2 is lower than that of the bare electrode, indicating that Mn-MoS2 enhances the electrode's conductivity. After loading AuNPs onto Mn-MoS2, a significant decrease in retrofit was observed, suggesting that Au / Mn-MoS2 improves the interfacial charge transfer rate. The sequential immobilization of Ab (antibody), BSA, and antigen Tau protein on the electrode surface leads to a gradual increase in electron transfer resistance (retrofit). This indicates that the antibody Tau protein, BSA, and Tau protein act as an insulating protein layer, hindering interfacial charge transfer due to their non-conductive properties. These findings are consistent with the CV results, confirming the successful construction of the Tau immunosensor.

[0064] Determination of concentration linearity The immunosensor prepared in Preferred Example 1 was used to detect DPV of different concentrations of Tau protein (1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL) in PBS buffer at pH 7.4. As the concentration of Tau protein increased, the peak current of the immunosensor gradually decreased. Figure 12A This is mainly attributed to the specific recognition and binding of the antibody to the Tau protein, forming a biological complex on the electrode surface, which in turn hinders electron transfer between the electrode interface and the potassium ferricyanide probe. Figure 12B The calibration curve for this Tau protein immunosensor shows a good linear relationship between the peak current change rate and the logarithm of the Tau protein concentration. The linear regression equation is ΔI = 15.468x + 191.184, and the linear correlation coefficient R0 is [value missing]. 2 =0.996. The limit of detection (LOD) for Tau protein by this immunosensor is 1 pg (20 fM).

[0065] Therefore, the detection limit of the immune sensor provided by the present invention is significantly lower than that of various known Tau immune sensors described in the background art, and it has higher sensitivity, enabling the detection of trace amounts (ultra-low concentrations) of Tau. This indicates that the sensor has great potential for the early diagnosis of Alzheimer's disease and is more clinically applicable.

[0066] Determination of reproducibility, stability, and selectivity In practical applications, electrochemical immunosensors must possess strong anti-interference capabilities to ensure accurate and timely measurements even in the presence of non-target substances. To evaluate the selectivity of Tau protein, a common biological interfering agent, β-amyloid 42 (Aβ), was selected. 42The Tau protein and interfering agents were dissolved in PBS at a concentration ratio of 1:100, and the specificity of the Tau electrochemical immunosensor was verified using DPV. Results are as follows: Figure 13 As shown in the figure, the current differences generated by each individual interfering agent are significantly lower than the specific current difference of the Tau protein, indicating that the interfering agents do not undergo a specific recognition reaction with the anti-Tau antibody. This further confirms the high selectivity of the Tau protein against the anti-Tau antibody and the excellent interference performance of the electrochemical immunosensor.

[0067] To evaluate the reproducibility of the sensor, nine identical Tau detection sensors were manufactured and tested according to the preparation method of preferred embodiment 1. Detection results ( Figure 14 The results showed that the relative standard deviation (RSD) of the peak current of the Tau antigen and the immune sensor was 1.72%, reflecting good reproducibility.

[0068] The prepared immunosensor was stored at 4°C, and its stability was tested at 1, 3, 5, 7, and 14 days to observe the changes in the DPV peak current. Figure 15 ).from Figure 15 It can be seen that although the signal decreased slightly on the 14th day compared with the initial current, the sensor's response rate still maintained good detection performance.

[0069] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a Tau protein immunosensor, characterized in that, include: S1: Manganese-doped molybdenum disulfide and gold source are combined to obtain gold nanoparticle-manganese-doped molybdenum disulfide composite. S2: Cover the screen-printed electrode with a suspension containing the gold nanoparticle-manganese-doped molybdenum disulfide composite to form a composite layer on the surface of the screen-printed electrode. S3: Cover the complex layer with anti-Tau protein antibody solution and incubate for at least 1 hour, then wash away excess anti-Tau protein antibody solution to obtain an anti-Tau protein antibody layer; S4: Block the non-specific binding sites on the anti-Tau protein antibody layer to obtain the Tau protein immunosensor.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of gold source to manganese-doped molybdenum disulfide is 1:0.5 to 1:

2.

3. The preparation method according to claim 1, characterized in that, The manganese-doped molybdenum disulfide used in step S1 is prepared by reacting manganese source, molybdenum source and sulfur source in a molar ratio of 1:(1.5-2.5):(7-15).

4. The preparation method according to claim 1, characterized in that, In step S2, based on the working electrode surface area of ​​the screen-printed electrode, the coating amount of the gold nanoparticle-manganese-doped molybdenum disulfide composite is 2-4 mg / cm². 2 .

5. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the anti-Tau protein antibody solution is 3-5 μg / mL.

6. The preparation method according to claim 6, characterized in that, Based on the surface area of ​​the screen-printed electrode, the coverage of the anti-Tau protein antibody solution is 5-10 μL / mm. 2 .

7. The preparation method according to claim 1, characterized in that, In step S3, the incubation time for the anti-Tau protein antibody is 1.5-2.5 hours.

8. The Tau protein immunosensor prepared by the preparation method according to any one of claims 1-7.

9. The Tau protein immunosensor according to claim 8, characterized in that, when using the Tau protein immunosensor for Tau protein detection, the Tau protein immunosensor is incubated with the sample to be detected for at least 45 minutes.

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