A ptau231 aptamer, ptau231 probe, electrochemical biosensor and construction method

Abstract

This invention discloses a pTau231 aptamer, a pTau231 probe, an electrochemical biosensor, and its construction method, belonging to the field of biodetection technology. The electrochemical biosensor uses the pTau231 aptamer and pTau231 probe as core biorecognition elements, combined with an immunomagnetic bead enrichment module, a TDT enzyme-catalyzed signal amplification module, and a target probe-induced ZIF-8 in situ growth signal amplification module for ultrasensitive detection of pTau231 protein. This electrochemical biosensor integrates three major modules: immunomagnetic bead enrichment, TDT enzyme-catalyzed signal amplification, and ZIF-8 in situ growth signal amplification, achieving multiple signal amplification and efficient signal conversion. It converts protein signals into nucleic acid signals, and then into measurable strong electrochemical signals, achieving highly sensitive detection of low-abundance pTau231 with a wide detection linear range.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, specifically to a pTau231 aptamer, a pTau231 probe, an electrochemical biosensor, and a method for constructing them. Background Technology

[0002] Alzheimer's disease (AD) is an insidious, irreversible, progressive neurodegenerative disease with a continuously rising global incidence and mortality rate. It has become one of the leading causes of death for urban and rural residents, imposing a heavy economic and social burden on society. Currently, there are no specific drugs to cure AD or reverse its progression. Most patients are diagnosed at an advanced stage. Therefore, early screening and accurate diagnosis of AD are crucial for disease intervention, slowing progression, and reducing medical costs. pTau231 is highly correlated with the pathological process of AD and is an important biomarker for early diagnosis. Developing efficient detection technologies for pTau231 is key to addressing the clinical need for early AD screening.

[0003] Existing methods for detecting Alzheimer's disease (AD) biomarkers have many limitations: neuroimaging techniques are expensive and poorly accessible; cerebrospinal fluid analysis is invasive, easily causing discomfort and making it difficult to guarantee consistent results; while blood tests are non-invasive and low-cost, the blood-brain barrier results in extremely low concentrations of pTau231 in the blood, making trace detection difficult. Current technologies for detecting pTau231 in blood are either insufficiently sensitive and susceptible to interference, or involve complex equipment and are extremely expensive, making large-scale population screening difficult.

[0004] Electrochemical biosensors are an important means of detecting trace amounts of biomarkers, but traditional electrochemical immunosensors suffer from high cost and poor stability. While nucleic acid aptamers offer advantages as novel biorecognition elements, they alone cannot achieve trace detection. Signal amplification strategies are the core of electrochemical biosensor research. Techniques such as magnetic bead enrichment, TDT enzyme-catalyzed signal amplification, and ZIF-8 nanomaterial self-assembly signal amplification each have their advantages, but these techniques have not yet been effectively combined for the construction of electrochemical biosensors. Summary of the Invention

[0005] Based on the aforementioned problems in the existing technology, this invention provides a highly specific and adaptable pTau231 aptamer and pTau231 probe, and also provides an electrochemical biosensor and its construction method based on an immune enrichment and target probe-induced ZIF-8 growth signal amplification strategy, thereby achieving ultrasensitive and highly specific detection of the Alzheimer's disease biomarker pTau231.

[0006] The technical solution provided by this invention is as follows: The first aspect of the present invention provides a pTau231 aptamer, the nucleotide sequence of which is 5'-TGACTGATTTACGATGCATGTAGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCTTC-3', as shown in SEQ ID NO.1.

[0007] Optionally, the 5' end region of the pTau231 aptamer contains an identification sequence 1, and the 3' end region contains an identification sequence 2 that is associated with pTau231 identification.

[0008] Optionally, the length of the identification sequence 1 is 22 bp, and the length of the identification sequence 2 is 43 bp.

[0009] A second aspect of the present invention provides a pTau231 probe, which can hybridize with the recognition sequence 1 to achieve signal conversion and amplification of pTau231.

[0010] Optionally, the pTau231 probe is 22 bp in length and has a nucleotide sequence of 5'-T ACA TGC ATC GTAAAT CAG TCA-3', as shown in SEQ ID NO.2.

[0011] The third aspect of the present invention provides an electrochemical biosensor, wherein the electrochemical biosensor is constructed by using the above-mentioned pTau231 aptamer and the above-mentioned pTau231 probe as core biorecognition elements, combined with an immunomagnetic bead enrichment module, a TDT enzyme-catalyzed signal amplification module and a target probe-induced ZIF-8 in situ growth signal amplification module, for ultrasensitive detection of pTau231.

[0012] Optionally, the immunomagnetic bead enrichment module is composed of carboxylated magnetic beads modified with Anti-Tau antibody, which can specifically recognize and enrich pTau231; the detection signal of the electrochemical biosensor is an "attenuated" electrochemical signal, which is detected by differential pulse voltammetry, cyclic voltammetry or electrochemical impedance spectroscopy.

[0013] A fourth aspect of the present invention provides a method for constructing the above-mentioned electrochemical biosensor, characterized by comprising the following steps: S1: Activation of carboxylated magnetic beads: Take carboxylated magnetic beads, perform magnetic separation and washing with MEST solution, add EDC solution and NHS solution, and activate by rotating and mixing at room temperature; S2: Antibody-modified carboxylated magnetic beads: The supernatant was removed by magnetic separation, Anti-Tau antibody was added, and after coupling at room temperature, the mixture was rotated and incubated overnight. S3: Blocking nonspecific sites: Remove the supernatant by magnetic separation, add blocking solution to resuspend the magnetic beads, rotate and mix at room temperature, remove the supernatant by magnetic separation again, and resuspend the magnetic beads in PBS solution to obtain immunomagnetic beads; S4: Preparation of pTau231 probe product: The pTau231 probe was reacted with TDT enzyme reaction buffer, dATP, TDT enzyme and deionized water were mixed and subjected to enzymatic extension reaction and inactivation to obtain pTau231 probe product; S5: Preparation of pTau231 probe working solution: The immunomagnetic beads obtained in step S3 were mixed with pTau231 at room temperature, and then pTau231 aptamer was added and reacted at room temperature to obtain pTau231 probe working solution. S6: Preparation of target pTau231 probe product: The pTau231 probe working solution obtained in step S5 is mixed with the pTau231 probe product obtained in step S4 at room temperature. After thermal denaturation, it is naturally cooled to room temperature. The supernatant is magnetically separated to obtain the target pTau231 probe product. S7: Electrochemical signal activation: The target pTau231 probe product was dropped onto a 0.5M... The activated gold electrode was incubated at room temperature and then dried. Zinc nitrate solution and 2-methylimidazole solution were added dropwise in sequence, and the electrode was incubated at room temperature and dried to complete the construction of the electrochemical biosensor.

[0014] Optionally, in step S1, the MEST solution is washed 3 times and the activation time is 30 min; in step S2, the coupling time at room temperature is 1 h; in step S3, the blocking reaction time is 3 h, and the immunomagnetic beads are stored at 4°C.

[0015] Optionally, in step S4, the enzymatic extension reaction time at 37℃ is 2 h, and the inactivation time at 80℃ is 5 min; in step S5, the mixing time of immunomagnetic beads and pTau231 is 90 min, and the reaction time with pTau231 aptamer is 45 min; in step S7, the incubation time of the target pTau231 probe product on the gold electrode is 15 min, the concentration of zinc nitrate solution is 40 mM, the concentration of 2-methylimidazole solution is 160 mM, and the incubation time of ZIF-8 in situ growth is 15 min.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The pTau231 probe and pTau231 aptamer provided by this invention have high specificity. The pTau231 aptamer can accurately recognize both pTau231 and the pTau231 probe at the same time. The pTau231 probe can realize the signal conversion of the target protein, laying the foundation for subsequent signal amplification. The two work together to achieve accurate recognition of pTau231 and initial signal conversion. 2. The electrochemical biosensor of this invention combines three major modules: immunomagnetic bead enrichment, TDT enzyme-catalyzed signal amplification, and ZIF-8 in situ growth signal amplification. This achieves multiple signal amplification and efficient signal conversion, transforming protein signals into nucleic acid signals, and then into measurable strong electrochemical signals. This enables highly sensitive detection of low-abundance pTau231 with a wide linear detection range, reaching [value missing]. ; 3. The electrochemical biosensor construction method of the present invention is simple to operate, low in cost, and the prepared sensor has good stability, high specificity for pTau231, and minimal interference from non-target proteins on the detection results; 4. The electrochemical biosensor of the present invention exhibits good anti-interference ability and detection accuracy in complex biological samples. The recovery rate of serum spike recovery experiment is between 99.14% and 100.77%, and it can successfully detect pTau231 in the plasma of AD mice. It has important application value in early large-scale population screening, biomedical research and pharmacological applications of Alzheimer's disease. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the electrochemical biosensor provided in the embodiments of the present invention; Figure 2A This is an SDS-polyacrylamide gel electrophoresis characterization image of the antibody modified on the surface of the magnetic beads in this embodiment of the invention; Figure 2B This is a polyacrylamide gel electrophoresis characterization image of the Poly(A) extension reaction in an embodiment of the present invention; Figure 2C D represents the DPV response signal diagrams of different modified electrodes in the embodiments of the present invention; Figure 3A The above are CV curves of different modified electrodes in the embodiments of the present invention. Figure 3B These are EIS curves of different modified electrodes in embodiments of the present invention; Figure 4A This is a graph showing the effect of the reaction time of the TDT enzyme on the sensor analysis performance in an embodiment of the present invention; Figure 4B This is a graph showing the effect of the reaction time of immunomagnetic bead enrichment of pTau231 on the sensor analytical performance in an embodiment of the present invention; Figure 4C This is a graph showing the effect of the reaction time of pTau231 and pTau231 aptamer on the sensor analysis performance in an embodiment of the present invention; Figure 4D This is a graph showing the effect of the assembly time of the target pTau231 probe-Poly(A) on the AuE surface on the sensor analysis performance in an embodiment of the present invention; Figure 4E This is a graph showing the effect of the assembly time of ZIF-8 on the AuE surface on the sensor's analytical performance in an embodiment of the present invention. Figure 5A This is a fitting graph of the logarithm of pTau231 concentration versus the rate of change of DPV in an embodiment of the present invention; Figure 5B This is a linear relationship graph of the sensor's response to pTau231 in an embodiment of the present invention; Figure 6A This is a DPV response signal diagram of the sensor in the detection of non-target protein reaction system and target pTau231 reaction system in an embodiment of the present invention; Figure 6B This is a graph showing the rate of change of the DPV response signal of the sensor in the non-target protein reaction system and the target pTau231 reaction system in an embodiment of the present invention. Figure 7 This is a graph showing the recovery rate of the sensor in serum spiked with pTau231 in an embodiment of the present invention. Figure 8A This is a DPV response signal diagram of pTau231 detected by the sensor in plasma samples of control group C57BL / 6 mice and AD mice in an embodiment of the present invention; Figure 8B This is a graph showing the rate of change of the DPV response signal of pTau231 in plasma samples of control group C57BL / 6 mice and AD mice detected by the sensor in an embodiment of the present invention. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments. All equivalent transformations made based on the technical solutions of the present invention fall within the scope of protection of the present invention.

[0019] The reagent formulations involved in the embodiments of this invention are as follows: MEST buffer: 0.1M MES, pH 6.0, 0.05% Tween 20; EDC solution: 1 mL MEST, 10 mg EDC powder; NHS solution: 1 mL MEST, 20 mg NHS powder; 5×SSC Buffer: 0.75M NaCl (1.3163g), sodium citrate 0.661725g, dissolved in 30mL deionized water, pH=7; 160mM 2-Methylimidazole solution: Dissolve 0.0065g of 2-methylimidazole powder in 500μL of deionized water; 40mM zinc nitrate solution: Dissolve 0.0059g of zinc nitrate hexahydrate crystals in 500μL of deionized water; 10mM electrolyte: 0.025g potassium ferricyanide, 0.032g potassium ferrocyanide, 0.22g potassium chloride, dissolved in 30mL deionized water, pH=7.0; Resistance solution: Dissolve 0.016g potassium ferricyanide, 0.021g potassium ferrocyanide, and 0.373g potassium chloride in 50mL of deionized water; The solution is prepared as follows: Take 48.6 mL of deionized water into a 50 mL beaker, slowly add 1.4 mL of concentrated sulfuric acid along the beaker wall while stirring continuously with a glass rod. After mixing, transfer to a brown wide-mouth reagent bottle and store in a cool place.

[0020] The pTau231 probe used in this embodiment of the invention is 22 bp in length and has the sequence 5'-T ACA TGC ATC GTAAAT CAG TCA-3', as shown in SEQ ID NO.2. The pTau231 aptamer used has a 5' end region and a 3' end region. The 5' end region contains the pTau231 probe recognition sequence 1, and the 3' end region contains the pTau231 recognition sequence 2. The recognition sequence 2 is 43 bp in length, and the recognition sequence 1 is 22 bp in length.

[0021] The complete sequence of the pTau231 aptamer is shown in SEQ ID NO.1, specifically: 5'-TGACTGATTTACGATGCATGTAGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCTTC-3'.

[0022] Example 1: Specificity verification of pTau231 probe and pTau231 aptamer The pTau231 probe (1 μM) and pTau231 aptamer (1 μM) of this embodiment were mixed in 5×SSC Buffer at room temperature for 30 min to perform nucleic acid hybridization. A blank control group (containing only the pTau231 probe or only the pTau231 aptamer) was also set up. The reaction products were characterized by polyacrylamide gel electrophoresis. The results showed that a clear hybridization band appeared after the pTau231 probe and pTau231 aptamer were mixed, while the blank control group only showed a single probe or aptamer band. This demonstrates that the pTau231 probe of this invention can specifically hybridize and bind to the pTau231 aptamer.

[0023] The pTau231 aptamer and pTau231 were incubated in PBS solution at room temperature for 45 min. The binding affinity between the two was detected using biomembrane interferometry. A control group containing non-target proteins (Tau441, BSA) was also included. The results showed that the binding signal between the pTau231 aptamer and pTau231 was significantly higher than that in the non-target protein control group, demonstrating that the pTau231 aptamer of this invention can specifically recognize and bind to pTau231.

[0024] Example 2: Construction of an electrochemical biosensor This embodiment constructs a novel aptamer electrochemical biosensor that combines immune enrichment with a target probe-induced ZIF-8 growth signal amplification strategy for ultrasensitive detection of pTau231 expression levels in Alzheimer's disease.

[0025] To enrich pTau231, immunocapture magnetic beads were designed, which are modified with anti-Tau antibody to specifically recognize and enrich pTau231. To further accurately identify the target pTau231, a pTau231 probe was designed that specifically recognizes pTau231. The 5' end region recognition sequence of the pTau231 aptamer can accurately recognize the pTau231 probe and hybridize with it; the 3' end region recognition sequence can specifically recognize and bind to pTau231. To convert and amplify the detection signal, a long-chain detection probe product was obtained by extending the 3' end of the pTau231 probe with a polymeric (A) sequence using TDT enzyme. This pTau231 probe product was then mixed with a pTau231 aptamer and released via thermal denaturation to obtain the target pTau231 probe product. Subsequently, the target pTau231 probe product was efficiently assembled onto the gold electrode (AuE) surface using the strong binding force between the A base and the electrode. The presence of this product hinders electron collisions on the electrode surface, thereby generating a "decreased" electrochemical detection signal, achieving the conversion and amplification of the electrochemical signal. Based on this, a ZIF-8 precursor solution was added dropwise. This solution uses the target long-chain detection probe product on the AuE surface as a nucleation site, inducing in-situ growth of ZIF-8, which significantly hinders electron transfer on the electrode surface, generating a significantly "decreased" electrochemical detection signal, further amplifying the electrochemical signal, and thus achieving ultrasensitive detection of pTau231.

[0026] Specifically, the construction steps of the electrochemical biosensor provided in this embodiment are as follows: S1: Activation of carboxylated magnetic beads: Take 20 μL of 10 mg / mL magnetic beads, wash them three times with 20 μL of MEST solution, add 20 μL of EDC solution and 20 μL of NHS solution, and activate them for 30 min on a rotary mixer at room temperature.

[0027] S2: Antibody-modified carboxylated magnetic beads: Remove the supernatant by magnetic separation, add 10 μL of 0.2 mg / mL Anti-Tau antibody, couple at room temperature for 1 h, and then mix by rotation at 4 °C overnight.

[0028] S3: Block nonspecific sites: The supernatant was removed by magnetic separation, and the magnetic beads were resuspended in 100 μL of blocking buffer. The mixture was reacted on a rotary mixer at room temperature for 3 h. The supernatant was removed by magnetic separation, and the immunomagnetic beads were resuspended in PBS solution and stored at 4 °C.

[0029] S4: Preparation of pTau231 probe product: Add 2 μL of 1 μM Tau probe, 2 μL of 10X Terminal Transferase Reaction Buffer, 2.5 μL of 10 mM dATP, 2 μL of CoCl2, 0.5 μL of 10000 u / mL TDT enzyme, and 3 μL of ddH2O to a PCR tube. Incubate the PCR tube at 37°C for 2 h. After the reaction is complete, inactivate the enzyme at 80°C for 5 minutes to generate a long-chain detection probe product that amplifies the signal.

[0030] S5: Preparation of pTau231 probe working solution: The immunomagnetic beads described in S3 were mixed with pTau231 at room temperature for 90 min, and then the pTau231 aptamer was added and reacted at room temperature for 45 min to obtain the corresponding pTau231 probe working solution. S6: Preparation of target pTau231 probe product: The corresponding pTau231 probe working solution described in S5 was mixed with the pTau231 probe product described in S4 at room temperature, denatured at 95°C for 2 min, and then naturally cooled to room temperature. The supernatant was then magnetically separated to obtain the target pTau231 probe product.

[0031] S7: Activation of electrochemical signals: 5 µL of the target pTau231 probe product was transferred and added to a gold electrode activated with 0.5 M H2SO4. After incubation at room temperature for 15 min and drying, 3 µL of 40 mM zinc nitrate solution was added to the electrode, followed immediately by 3 µL of 160 mM 2-methylimidazole solution. After incubation at room temperature for 15 min and drying at room temperature, an electrochemical biosensor was obtained. The electrochemical signal intensity can be detected by differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy.

[0032] Example 3: Feasibility Study of Electrochemical Biosensors To verify the successful modification of the carboxylated magnetic bead surface with Anti-Tau antibody, SDS-polyacrylamide electrophoresis was used to characterize the relevant experiments. Figure 2A As shown, lane 1 represents the band corresponding to the anti-Tau antibody-modified magnetic beads, and lane 2 represents the band corresponding to the anti-Tau antibody. The bands in lanes 1 and 2 are in the same position. The results indicate that immunomagnetic beads can be successfully constructed by modifying the surface of carboxylated magnetic beads with antibodies.

[0033] To verify whether Poly(A) could be successfully extended at the ends of long-chain detection probe products, polyacrylamide electrophoresis was used to characterize the relevant experiments. Figure 2BAs shown, lane 1 represents the band corresponding to the Poly(A) extension reaction system with 2 μM pTau231 probe, while lanes 2 and 3 represent the bands corresponding to the pTau231 probe and the absence of the pTau231 probe, respectively. No band was observed in lane 3 because the TDT enzyme cannot induce a Poly(A) extension reaction in the blank solution without the pTau231 probe. The band observed below the sample well in lane 2 represents the pTau231 probe-Poly(A) product formed by the Poly(A) extension reaction at the pTau231 probe tip under the action of the TDT enzyme. Due to its increased molecular weight, it failed to migrate rapidly downwards. The results indicate that a Poly(A) extension reaction at the pTau231 probe tip can produce a large molecular weight pTau231 probe-Poly(A) product.

[0034] To verify the successful construction of the electrochemical sensor based on the target-induced ZIF-8 growth strategy and its successful application in the ultrasensitive detection of pTau231, the feasibility of the sensor strategy was evaluated, and the steps are as follows: S1: Activation of carboxylated magnetic beads: Take 30 μL of 10 mg / mL magnetic beads, wash them three times with 30 μL of MEST solution, add 30 μL of 1-EDC solution and 30 μL of NHS solution, and activate them for 30 min on a rotary mixer at room temperature.

[0035] S2: Antibody-modified carboxylated magnetic beads: Remove the supernatant by magnetic separation, add 15 μL of 0.2 mg / mL Anti-Tau antibody, couple at room temperature for 1 h, and then let stand overnight at 4 °C.

[0036] S3: Block nonspecific sites: The supernatant was removed by magnetic separation, and the magnetic beads were resuspended in 100 μL of blocking buffer. The mixture was reacted on a rotary mixer at room temperature for 3 h. The supernatant was then removed by magnetic separation, and 120 μL of PBS was added to prepare 25 μg / mL immunomagnetic beads. 35 μL of the immunomagnetic bead solution was taken to obtain immunomagnetic bead 1; 35 μL of the immunomagnetic bead solution was taken to obtain control immunomagnetic bead 1-1; and 35 μL of the immunomagnetic bead solution was taken to obtain control immunomagnetic bead 1-2.

[0037] S4: Preparation of long-chain detection probe products: Add 2 μL Tau probe (1 μM), 2 μL 10' Terminal Transferase Reaction Buffer, 2.5 μL dATP (10 mM), 2 μL CoCl2, 0.5 μL TDT enzyme (10000 U / mL), and 3 μL ddH2O to a PCR tube. Incubate at 37°C for 2 h in a PCR instrument. After the reaction is complete, inactivate the enzyme at 80°C for 5 min to produce long-chain detection probe product 1.

[0038] Add 2 μL of 10×Terminal Transferase Reaction Buffer, 2.5 μL of 10 mMdATP, 2 μL of CoCl2, 0.5 μL of TDT enzyme (10000 U / mL), and 5 μL of ddH2O to a PCR tube. Incubate the PCR tube at 37°C for 2 h. After the reaction is complete, inactivate the enzyme at 80°C for 5 min to produce the long-chain control product 1-1.

[0039] Add 2 μL Tau probe (1 μM), 2 μL 10' Terminal Transferase Reaction Buffer, 2.5 μL dATP (10 mM), 2 μL CoCl2, and 5.5 μL ddH2O to the PCR tube, and react at 37°C for 2 h in a PCR instrument. After the reaction is complete, inactivate at 80°C for 5 min to produce long chain control products 1-2.

[0040] S5: Preparation of pTau231 probe working solution: Immunomagnetic beads 1 were mixed with pTau231 at room temperature for 90 min, and then the pTau231 aptamer was added and reacted at room temperature for 45 min to obtain the corresponding pTau231 probe working solution 1 (pTau231 probe-Poly(A)). Immunomagnetic beads 1-1 were mixed with pTau231 at room temperature for 90 min, and then the pTau231 aptamer was added and reacted at room temperature for 45 min to obtain the corresponding control probe working solution 1-1. Immunomagnetic beads 1-2 were mixed with pTau231 at room temperature for 90 min, and then the pTau231 aptamer was added and reacted at room temperature for 45 min to obtain the corresponding control probe working solution 1-2. S6: Preparation of target pTau231 probe product: The pTau231 probe working solution 1 was mixed with the long-chain Tau probe product 1 at room temperature, denatured at 95°C for 2 min, and then naturally cooled to room temperature. The supernatant was magnetically separated to obtain the target pTau231 probe product 1 (target pTau231 probe-Poly(A)).

[0041] The control probe working solution 1-1 was mixed with the long chain control product 1-1 at room temperature, denatured at 95°C for 2 min, and then naturally cooled to room temperature. The supernatant was obtained by magnetic separation to obtain the non-target long chain control product 1-1.

[0042] The control probe working solution 1-2 was mixed with the long chain control product 1-2 at room temperature, denatured at 95℃ for 2 min, and then naturally cooled to room temperature. The supernatant was obtained by magnetic separation to obtain the non-target long chain control product 1-2.

[0043] S7: Activation of electrochemical signals: 5 µL of target pTau231 probe-Poly(A) was transferred and dropped onto the gold electrode. After incubation at room temperature for 15 min and drying, the electrochemical signal intensity was detected by differential pulse voltammetry. The target long chain detection probe product 1 used included the target long chain detection probe product 1 from step S6 or its series of controls (non-target long chain control products 1-1 to 1-2).

[0044] After rinsing thoroughly with deionized water and air-drying, 3 µL of 40 mM zinc nitrate solution was added to the electrode, followed immediately by 3 µL of 160 mM 2-methylimidazole solution. The electrode was incubated at room temperature for 15 min and then air-dried at room temperature. The electrochemical signal intensity was then detected using differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy, respectively.

[0045] To verify whether the target pTau231 probe-Poly(A) product could be assembled onto AuE, differential pulse voltammetry (DPV) was used for characterization. Figure 2CAs shown, without Poly(A) extension, the DPV response current values ​​with and without the target detection probe are similar to those of AuE, approximately 43 μA. However, with Poly(A) extension, the DPV response current of the target pTau231 probe-Poly(A) / AuE (32 μA) is attenuated by 13.51% compared to the DPV response current of AuE and ddH2O-Poly(A) / AuE (approximately 37 μA). The assembly of the target pTau231 probe-Poly(A) product onto AuE hinders electron transfer at the electrode surface, resulting in a "attenuated" DPV response signal. These results indicate that modifying the pTau231 probe end with the Poly(A) sequence can promote rapid and efficient adsorption of the pTau231 probe on the AuE surface. In the absence of ZIF-8 nanostructures, the DPV response current of the target pTau231 probe-Poly(A) / AuE changed by only 34.69% compared to AuE. However, after the ZIF-8 nanostructures self-assembled on the AuE surface, the DPV signal of the target product significantly decreased (current value approximately 0.9 μA), with a signal change rate of 98.16% compared to AuE. These results indicate that the presence of the target pTau231 probe-Poly(A) product can induce the self-assembly of ZIF-8 nanostructures, thereby exciting a significant DPV "attenuated" response signal.

[0046] Subsequently, the feasibility of the sensor was further verified using cyclic voltammetry (CV) in an electrolyte. Figure 3A As shown, a pair of distinct redox peaks can be observed on AuE. After ddH2O-Poly(A) modification of AuE, the redox peak current is slightly reduced due to non-specific adsorption. However, after modification of AuE with the target pTau231 probe-Poly(A) product, the peak current decreases further. This is because after Poly(A) extension at the end of the pTau231 probe, the target pTau231 probe-Poly(A) product efficiently assembles on the AuE surface by utilizing the strong binding ability of the A base to AuE, thus hindering the transfer of electrons on the electrode surface. When the target pTau231 probe-Poly(A) product is present on the AuE surface, the redox peak current drops sharply after further modification of ZIF-8. This is because the presence of the target pTau231 probe-Poly(A) product can provide nucleation sites for ZIF-8. The growth of ZIF-8 encapsulates the pTau231 probe, shielding the electron transfer at the AuE interface. By monitoring this significant "attenuation" response signal, pTau231 can be detected.

[0047] Furthermore, to understand the construction of the sensor, the EIS of different modified electrodes was tested in resistive liquid. Figure 3B The semicircular portion of the Nyquist plot represents the high-frequency region controlled by electron transport processes, and its diameter approximates the electron transport resistance (Ret). The linear portion shows Warburg diffusion under low-frequency conditions, representing the diffusion-controlled process. Figure 3B As shown, the AuE semicircle diameter is small, or even shows almost no resistance. This is because after electrode pretreatment, the AuE surface is a smooth, impurity-free mirror, allowing electrons in the resistive liquid to transfer to the AuE surface. After ddH2O-Poly(A) modification, the AuE surface resistance slightly increases due to the adsorption of non-specific substances. After modification with the target pTau231 probe-Poly(A) product, the resistance further increases. This is because the target pTau231 probe-Poly(A) product enhances the adsorption of AuE through the Poly(A) tail, hindering electron transfer between AuE surfaces. After ZIF-8 assembles on the AuE surface, the semicircle diameter increases significantly. This is because the target pTau231 probe-Poly(A) product induces the growth of the ZIF-8 nanostructure, the presence of which greatly hinders the growth of [Fe(CN)6]. 3− / 4− Transfer to the AuE surface. The above results demonstrate the successful construction of the electrochemical biosensor.

[0048] Example 4: Optimization of Conditions To achieve optimal sensor detection performance, relevant experimental conditions were optimized, including the optimal reaction time for TDT enzyme, the optimal reaction time for pTau231 enrichment with immunomagnetic beads, the optimal reaction time for pTau231 and its aptamer, the optimal assembly time of the target pTau231 probe-Poly(A) on the AuE surface, and the assembly time of ZIF-8 on the AuE surface. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) In the electrolyte, the change in response signal is monitored using differential pulse voltammetry (DPV). The rate of change of DPV is represented by ΔI%, and is calculated as: ΔI% = ×100%. Where: I0 and I pTau231 These represent the DPV signals obtained by the sensor when the target P-Tau231 is absent and present, respectively.

[0049] In the optimization experiment of this embodiment, the single variable principle is adopted, that is, based on the basic system and steps described in Example 2, only the parameter of the factor to be investigated is changed each time, while all other conditions remain consistent with Example 2, so as to accurately evaluate the impact of the factor on the system performance.

[0050] To determine the optimal reaction time of the TDT enzyme, optimization was performed using 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min at 37℃. Figure 4A As shown, when the TDT enzyme amplification time was 30 min, ΔI% was approximately 22.11%; when the incubation time was increased to 120 min, ΔI% remained stable at around 85.43%; further extending the reaction time did not result in a significant change in ΔI%. The results indicate that the optimal reaction time for the TDT enzyme is 120 min.

[0051] To determine the optimal reaction time for immunomagnetic bead enrichment of pTau231, optimization was performed using incubation times of 30 min, 60 min, 90 min, 120 min, and 150 min at room temperature. Figure 4B As shown, when the incubation time was 30 min, ΔI% was approximately 77.46%; when the incubation time was increased to 90 min, ΔI% was approximately 92%; further extension of the incubation time decreased ΔI%. The results indicate that the optimal reaction time for immunomagnetic beads to enrich pTau231 is 90 min.

[0052] To determine the optimal reaction time between pTau231 and its aptamer, optimization was performed by incubating pTau231 and its aptamer at room temperature for 15 min, 30 min, 45 min, 60 min, and 90 min. Figure 4C As shown, when the incubation time was 15 min, ΔI% was approximately 35%; when the incubation time was increased to 45 min, ΔI% was approximately 93.58%; further extension of the incubation time resulted in a decrease in ΔI%. The results indicate that the optimal reaction time between pTau231 and the pTau231 aptamer is 45 min.

[0053] To determine the optimal assembly time of the pTau231 probe-Poly(A) on the AuE surface, assembly time points of 5 min, 10 min, 15 min, 25 min, and 35 min were set for investigation. Figure 4D As shown, when the target pTau231 probe-Poly(A) is assembled on the AuE surface for 5 min, ΔI% is approximately 64%. ΔI% gradually increases with time, stabilizing at approximately 92.35% when the assembly time is 15 min. Therefore, the optimal time for the pTau231 probe-Poly(A) product to assemble on the electrode is 15 min.

[0054] To determine the optimal assembly time for ZIF-8 on the AuE surface, assembly time points of 5 min, 10 min, 15 min, 30 min, and 60 min were set and examined. Figure 4E As shown, when the assembly time of ZIF-8 on the AuE surface is 5 min, ΔI% is about 60%. ΔI% gradually increases with time, stabilizing at around 91% when the assembly time is 15 min. Therefore, the optimal assembly time for ZIF-8 on the electrode is 15 min.

[0055] Example 5: Sensitivity Analysis of Electrochemical Biosensors To examine the sensitivity of the sensor, different concentrations were used. Electrochemical experiments were conducted using pTau231. For example... Figure 5A As shown in the inset, the DPV response signal gradually decreases with increasing pTau231 concentration, and in... Within the concentration range, the rate of change of the current signal is proportional to the logarithm of the pTau231 concentration ( ) exhibits a good linear relationship (see Figure 5B The linear regression equation is: The detection performance is significantly superior to many existing sensing methods. This indicates that the constructed sensor has high sensitivity, mainly due to: firstly, the strong binding ability of the Poly(A) tail at the end of the pTau231 probe to AuE allows the pTau231 probe to be directly assembled on the electrode, eliminating the need to fix and modify functional probes on the sensing electrode, thus resulting in extremely low background signal; secondly, this method, combined with ZIF-8 nanomaterials, has high signal amplification capability.

[0056] Example 6: Specificity Analysis of Electrochemical Biosensing To investigate the specificity of the sensor, electrochemical experiments were conducted using the target pTau231 and non-target proteins (Tau441, BSA, Hb, and Aβ42). In the experiments, the concentration of the non-target proteins was set at 1 pM, while the concentration of pTau231 was set at 100 fM. Figure 6A As shown, compared with the blank control, only the target pTau231 produced a significant decay current response (7.5 μA). The current values ​​of other non-target proteins (approximately 16 μA-18.5 μA) were similar to those of the blank control Blank (19.5 μA). Figure 6B The results showed that the ΔI% for Tau441, BSA, Hb, and Aβ42 were 11.7%, 10.4%, 6.2%, and 7.2%, respectively, while the ΔI% for pTau231 was 63.6%, which is 5-10 times higher than that for non-target proteins. This demonstrates that the sensor has good recognition ability for the target pTau231.

[0057] Example 7: Practical Applicability of Electrochemical Biosensors To evaluate the applicability and reliability of the proposed electrochemical biosensor in practical bioanalysis, a spiked recovery experiment was performed in 10% serum. Three representative concentrations (20 fM, 200 fM, and 1000 fM) of pTau231 were selected for spiking and determination. Figure 7 As shown, the quantitative recovery analysis results indicated a recovery rate ranging from 99.14% to 100.77%, with all relative standard deviations (RSDs) between 3.13% and 7.55%, and an average RSD of 5.68%, all within acceptable limits. These results confirm that the electrochemical biosensor system possesses excellent analytical accuracy, good reproducibility, and strong resistance to matrix interference in serum samples, providing a practical basis for its efficient detection of pTau231 in complex biological environments.

[0058] Furthermore, an electrochemical biosensor was used to detect the expression level of pTau231 in plasma samples from AD mice. An AD mouse model was established by injecting okadaic acid into the brains of C57BL / 6 mice using stereotactic brain injection (control group C57BL / 6 mice were injected with the same volume of saline using the same technique). Plasma samples were then extracted from both AD and control (WT) mice, and the expression level of pTau231 in the plasma samples was detected using the sensor. Figure 8A Compared to WT mouse plasma samples (14.5 μA), AD mouse plasma samples produced a significantly decaying current response (4.5 μA). Figure 8B The ΔI% of WT mouse plasma samples was approximately 8.8%, while that of AD mouse plasma samples was approximately 72%, which is 8 times higher than that of WT mouse plasma samples. In summary, the invented sensor has the ability to detect the expression level of pTau231 in AD mouse plasma samples.

[0059] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A pTau231 aptamer, characterized in that, The nucleotide sequence of the pTau231 aptamer is 5'-TGACTGATTTACGATGCATGTAGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCTTC-3', as shown in SEQ ID NO.

1.

2. The pTau231 aptamer of claim 1, wherein, The 5' region of the pTau231 aptamer contains a recognition sequence 1 that is complementary to the pTau231 probe, and the 3' region contains a recognition sequence 2 that specifically binds to pTau231.

3. The pTau231 aptamer of claim 2, wherein, The length of the identification sequence 1 is 22 bp, and the length of the identification sequence 2 is 43 bp.

4. A pTau231 probe, characterized in that, The pTau231 probe can hybridize with the recognition sequence 1 to achieve signal conversion and amplification of pTau231.

5. The pTau231 probe according to claim 4, characterized in that, The pTau231 probe is 22 bp in length and has a nucleotide sequence of 5'-T ACA TGC ATC GTA AAT CAG TCA-3', as shown in SEQ ID NO.

2.

6. An electrochemical biosensor, characterized in that, The electrochemical biosensor is constructed using the pTau231 aptamer as described in claim 1 or 2 and the pTau231 probe as described in any one of claims 3-5 as core biorecognition elements, combined with an immunomagnetic bead enrichment module, a TDT enzyme-catalyzed signal amplification module and a target probe-induced ZIF-8 in situ growth signal amplification module, for ultrasensitive detection of pTau231.

7. The electrochemical biosensor according to claim 6, characterized in that, The immunomagnetic bead enrichment module is composed of carboxylated magnetic beads modified with Anti-Tau antibody, which can specifically recognize and enrich pTau231; the detection signal of the electrochemical biosensor is an "attenuated" electrochemical signal, which can be detected by differential pulse voltammetry, cyclic voltammetry or electrochemical impedance spectroscopy.

8. A method for constructing an electrochemical biosensor as described in any one of claims 6 or 7, characterized in that, Includes the following steps: S1: Activation of carboxylated magnetic beads: Take carboxylated magnetic beads, perform magnetic separation and washing with MEST solution, add EDC solution and NHS solution, and activate by rotating and mixing at room temperature; S2: Antibody-modified carboxylated magnetic beads: The supernatant was removed by magnetic separation, Anti-Tau antibody was added, and after coupling at room temperature, the mixture was rotated and incubated overnight. S3: Blocking nonspecific sites: Remove the supernatant by magnetic separation, add blocking solution to resuspend the magnetic beads, rotate and mix at room temperature, remove the supernatant by magnetic separation again, and resuspend the magnetic beads in PBS solution to obtain immunomagnetic beads; S4: Preparation of pTau231 probe product: The pTau231 probe was mixed with TDT enzyme reaction buffer, dATP, CoCl2, TDT enzyme and deionized water, and enzymatic extension reaction and inactivation were performed respectively to obtain pTau231 probe product. S5: Preparation of pTau231 probe working solution: The immunomagnetic beads obtained in step S3 were mixed with pTau231 at room temperature, and then pTau231 aptamer was added and reacted at room temperature to obtain pTau231 probe working solution. S6: Preparation of target pTau231 probe product: The pTau231 probe working solution obtained in step S5 is mixed with the pTau231 probe product obtained in step S4 at room temperature. After thermal denaturation, it is naturally cooled to room temperature. The supernatant is magnetically separated to obtain the target pTau231 probe product. S7: Electrochemical signal activation: The target pTau231 probe product was dropped onto a gold electrode activated with 0.5M H2SO4 and incubated at room temperature. After drying, zinc nitrate solution and 2-methylimidazole solution were added dropwise, and the electrode was incubated at room temperature and dried to complete the construction of the electrochemical biosensor.

9. The construction method according to claim 8, characterized in that, In step S1, the MEST solution was washed 3 times and the activation time was 30 min; in step S2, the coupling time at room temperature was 1 h; in step S3, the blocking reaction time was 3 h, and the immunomagnetic beads were stored at 4℃.

10. The construction method according to claim 8 or 9, characterized in that, In step S4, the enzymatic extension reaction time at 37℃ is 2 h, and the inactivation time at 80℃ is 5 min; in step S5, the mixing time of immunomagnetic beads and pTau231 is 90 min, and the reaction time with pTau231 aptamer is 45 min; in step S7, the incubation time of the target pTau231 probe product on the gold electrode is 15 min, the concentration of zinc nitrate solution is 40 mM, the concentration of 2-methylimidazole solution is 160 mM, and the incubation time of ZIF-8 in situ growth is 15 min.

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