An electrochemiluminescent biosensor based on bimetallic nanozyme and DNA walker and its application

Abstract

A kind of electrochemiluminescent biosensor based on bimetallic nanozyme and DNA walker and its application belong to the field of biosensor technology. The sensor is based on nuclear pore membrane, gold nanoparticles are loaded on the inner wall of the membrane, thiolated aptamers are designed, the aptamers are fixed to the surface of gold nanoparticles through Au-S bonds, thiolated trigger DNA and Pd@Pt nanozymes containing gold sites are mixed in PBS, coupled through Au-S bonds, and then the trigger DNA-Pd@Pt complex is drop-coated on the membrane surface modified by the aptamer, hybridized with the aptamer, fixed to the inner wall of the membrane, and then Au@Ag nanoclusters are self-assembled layer by layer on the carboxyl sites of the inner wall of the membrane by electrostatic adsorption, Track DNA is fixed to the inner wall of the membrane, then Nt.BbvCI endonuclease is added, bovine serum albumin is used to block nonspecific binding sites, CDS quantum dots are fixed on the other side of the membrane, and an electrochemiluminescent biosensor of bimetallic nanozyme and DNA walker is obtained. The sensor has the advantages of high sensitivity, low detection limit, good stability, simple preparation steps, and short detection time.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and specifically relates to a biosensor that integrates aptamer-specific recognition, bimetallic nanozyme catalytic amplification, 3D DNA walker cascade reaction, and ratiometric electrochemiluminescence (ECL) detection. Background Art

[0002] As a key technology for early disease diagnosis, efficacy assessment, and prognosis monitoring, biomarker detection holds an important position in clinical medicine and life science research. Biomolecules, such as tumor markers and proteins associated with neurodegenerative diseases, typically have extremely low concentrations in body fluids. For example, the concentration of specific proteins in the cerebrospinal fluid of Alzheimer's patients is in the femtogram (fg / mL) range, placing extremely high demands on the sensitivity of detection technology. However, existing biomarker detection technologies still have the following shortcomings: 1. Insufficient sensitivity: Traditional enzyme-linked immunosorbent assay (ELISA) technology relies on specific antigen-antibody binding. While it has a certain degree of specificity, due to limited signal amplification capabilities, the detection limit is typically at the picogram (pg / mL) level, making it difficult to meet the needs of ultra-early disease diagnosis. For example, in the early diagnosis of breast cancer, the detection limit of conventional ELISA for the circulating tumor marker HER2 is approximately 1 pg / mL. However, clinical studies have shown that HER2 concentrations below 0.1 pg / mL can indicate early disease onset, and existing technologies are unable to effectively detect levels in this concentration range. 2. Weak anti-interference ability: Biological samples (such as blood, urine, and cerebrospinal fluid) are extremely complex. In addition to the target biomarker, they also contain a large number of interfering substances, such as high-abundance proteins, metabolites, and cellular debris. For example, human serum contains over 10,000 proteins, including high-abundance proteins such as albumin and immunoglobulins, which can reach concentrations of mg / mL, while the target biomarker may be present at concentrations of only fg / mL to pg / mL. Traditional detection technologies struggle to effectively distinguish between target and interfering substances, and are prone to nonspecific adsorption or cross-reactions, leading to false positive / negative results. 3. Low signal amplification efficiency: Existing detection technologies often rely on a single signal amplification strategy, making it difficult to effectively detect low-concentration biomarkers. For example, although enzyme-catalyzed signal amplification technology can generate a large number of detectable products through enzymatic reactions, the activity of the enzyme is easily affected by factors such as temperature, pH value, and inhibitors, resulting in unstable amplification efficiency; nanomaterial-assisted signal amplification strategies, such as gold nanoparticles and quantum dots, have unique optical and electrical properties, but the amplification factor of a single nanomaterial is limited, making it difficult to meet the needs of ultra-sensitive detection. In addition, a single signal amplification strategy lacks synergistic enhancement between signals and cannot fully utilize the advantages of different amplification mechanisms, limiting further improvements in detection sensitivity. Therefore, there is an urgent need to develop a biosensor with high sensitivity, strong anti-interference ability, and an efficient signal amplification mechanism to promote the development and clinical application of biomarker detection technology. Summary of the Invention

[0003] The present invention proposes an innovative solution, which realizes ultra-sensitive detection of biomarkers through highly specific recognition of aptamers, synergistic amplification of bimetallic nanozymes and 3D DNA walkers, and ratiometric ECL detection technology, breaking through the limitations of traditional sensors in sensitivity and anti-interference.

[0004] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a nuclear pore membrane with a carboxyl functional group on its inner wall is used as a substrate, gold nanoparticles are loaded on the inner wall of the nuclear pore membrane to form a uniform anchoring point; then a thiolated aptamer is designed for the detection object, and the aptamer is directionally fixed to the surface of the gold nanoparticles pre-modified on the inner wall of the nuclear pore membrane through the Au-S bond to form a high-density recognition interface; then a core-shell structure Pd@Pt nanozyme is synthesized by a liquid phase reduction method, the thiolated trigger DNA and the Pd@Pt nanozyme containing gold sites are mixed in PBS, coupled through the Au-S bond, and then the trigger DNA-Pd@Pt complex is drop-coated on the surface of the aptamer-modified nuclear pore membrane, so that the 3' end of the trigger DNA hybridizes with the complementary sequence of the aptamer end and is fixed to the inner wall of the nuclear pore membrane; then Au@Ag nanoclusters are prepared by sodium citrate reduction method, and the negative charge on the surface causes them to self-assemble layer by layer on the carboxyl site on the inner wall of the nuclear pore membrane through electrostatic adsorption; then DNA is mixed with gold nanoparticles and modified at a high density through salt aging. After centrifugal purification, it is fixed to the inner wall of the nuclear pore membrane through Au-S bonds. Subsequently, Nt.BbvCI endonuclease is added, which can specifically recognize and cut DNA sequences, triggering a cyclic cutting reaction to form a signal amplification system. Bovine serum albumin is used to block nonspecific binding sites, and CDS quantum dots are fixed on the other side of the nuclear pore membrane to obtain an electrochemiluminescent biosensor with synergistic amplification of dual-metal nanozymes.

[0005] In addition, the present invention also provides a preparation method of an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker, and the specific preparation steps are as follows: Step (1) nuclear pore membrane pretreatment: using a nuclear pore membrane containing a carboxyl functional group as a substrate, immersing the nuclear pore membrane in a 10-20 mM EDC / NHS mixed solution, and performing an activation reaction at room temperature for 2-3 hours, and finally immersing the nuclear pore membrane in a 10-20 μM gold nanoparticle solution for 12-15 hours to uniformly form a layer of gold nanoparticles on the inner wall of the nuclear pore membrane; Step (2) aptamer immobilization: designing a thiolated aptamer (Apt) for the target biomarker, and then incubating the 10-20 μM thiolated aptamer solution with the pretreated nuclear pore membrane, and the reaction time is set to 20-24 h, so that the thiolated aptamer is firmly connected to the gold nanoparticle layer on the inner wall of the nuclear pore membrane through the Au-S bond. After the incubation, the nuclear pore membrane is washed several times with a PBS buffer solution with a pH of 7.0-8.0 to remove the unbound aptamer molecules. Subsequently, 1% BSA solution is added for blocking treatment. The reaction time is 1-2 h; Step (3) Nanozyme loading: The thiolated trigger DNA (10-15 μM) and the Pd@Pt nanozyme containing gold sites (1-2 mg / mL) are incubated in PBS with a pH of 7.0-8.0 at 4-5°C for 12-15 h, through Au-S bond coupling, the trigger DNA-Pd@Pt complex is drop-coated on the surface of the aptamer-modified nuclear pore membrane, incubated at 37-40℃ for 3-4h, so that the 3' end of the trigger DNA hybridizes with the complementary sequence of the aptamer end, and washed with PBS 3-4 times to remove the unbound nanozyme complex; step (4) nanocluster loading: Au@Ag nanoclusters (concentration of 5-8 μM, pH = 7.0-8.0) are assembled on the surface of the nuclear pore membrane using layer-by-layer self-assembly technology, with an interval of 20-30 min between each round of assembly, and a total of 5-6 rounds of assembly are performed to construct a multilayer nanostructure and enhance the signal amplification ability of the sensor; step (5) 3D DNA walker construction: AuNP (10-20 nM) and Track DNA are mixed at a molar ratio of 1:100, and NaCl is slowly added to a final concentration of 0.5-0.7 M. The mixture is stirred at room temperature for 12-15 h for salt aging, so that each AuNP is loaded with about 50-70 Track DNA. DNA, the Track DNA-AuNP complex was drop-coated on the surface of the modified trigger DNA-Pd@Pt nuclear pore membrane and incubated at 37-40℃ for 4-5 h. It was fixed to the inner wall of the membrane through Au-S bonds. Subsequently, Nt.BbvCI endonuclease (concentration of 1-2 U / μL) can specifically recognize and cut DNA sequences, triggering a cyclic cutting reaction to form a signal amplification system; Step (6) Blocking and CDS quantum dot fixation: The composite membrane is placed in a bovine serum albumin solution to block the active sites. On the other side of the nuclear pore membrane, the membrane surface is silanized using 3-aminopropyltriethoxysilane (APTES) to form an amino active layer. Cadmium sulfide (CdS) quantum dots are then fixed by covalent coupling as the sensitizing element of the electrochemiluminescence signal to achieve multi-dimensional signal synergistic amplification.

[0006] Furthermore, in step (1), the nuclear pore membrane is a track-etched membrane formed by ion irradiation, etching, and ultraviolet irradiation of polyethylene terephthalate (PET) membrane, polycarbonate (PC) membrane, or polyimide (PI) membrane, the nanopores are conical in shape, 5-20 μm in thickness, the pore diameter of the surface pores is 0.1-1 μm, and the diameter of the gold nanoparticles is 5-20 nm;

[0007] Furthermore, in steps (3) and (4), the nanozyme (Pd core diameter 5-10 nm, Pt shell thickness 2-5 nm) and the Au@Ag nanoclusters have a particle size of 2-5 nm;

[0008] Furthermore, in steps (5) and (6), the ratio of Track DNA to gold nanoparticles is 100:1, each gold nanoparticle is loaded with approximately 50 Track DNA strands, the concentration of bovine serum albumin is (8-10 mg / mL, pH 7.0-8.0), the particle size of the CDS quantum dots is 5-10 nm, and the emission peak is 500-550 nm.

[0009] In addition, the present invention also provides an application of an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker, and the specific steps are as follows: (1) detecting the response intensity of the electrochemiluminescent biosensor containing target solutions of different concentrations, wherein the concentration range of the target solution is 0.01 fg / mL to 1 pg / mL; (2) placing the prepared composite film on an ITO conductive glass as a working electrode, a platinum wire electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a three-electrode system; the three-electrode system is placed in a 0.1-0.2 mol / L PBS solution with a pH of 7.0-7.5 for measurement; (3) electrochemical detection: the scanning potential is -1.0-1.0 V, and the current values ​​I0 and I of the target standard before and after incubation are obtained, and a standard curve is drawn with the logarithm of the target concentration as the horizontal axis and the current change rate as the vertical axis; (4) ECL ratio detection: the photomultiplier is 500-600 V, the scanning potential is -1.0-1.0 V, and the scanning rate is 100-200 mV / s, 20 mmol / L TPA was added as the ECL detection solution, and the ECL signal of the reaction system was measured by cyclic voltammetry (CV). The potential-luminescence intensity curve was recorded, and a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration was established to obtain the corresponding linear regression equation;

[0010] The detection principle of the present invention is as follows: using the nuclear pore membrane as the substrate, loading gold nanoparticles on the inner wall of the membrane, designing a thiolated aptamer for the detection object, and directionally fixing the aptamer to the surface of the gold nanoparticles pre-modified on the inner wall of the membrane through the Au-S bond, mixing the thiolated trigger DNA with the Pd@Pt nanozyme containing gold sites in PBS, coupling through the Au-S bond, and then drop-coating the trigger DNA-Pd@Pt complex on the surface of the aptamer-modified nuclear pore membrane, so that the 3' end of the trigger DNA hybridizes with the complementary sequence of the aptamer end and is fixed to the inner wall of the nuclear pore membrane, and then the Au@Ag nanoclusters are self-assembled layer by layer on the carboxyl sites of the inner wall of the nuclear pore membrane through electrostatic adsorption, and then the Track DNA is mixed with gold nanoparticles and modified at a high density by salt aging method. After centrifugal purification, it is fixed to the inner wall of the nuclear pore membrane through Au-S bond. Subsequently, Nt.BbvCI endonuclease is added. This endonuclease can specifically recognize and cut the DNA sequence, triggering a cyclic cutting reaction to form a signal amplification system. Bovine serum albumin is used to block nonspecific binding sites, and CDS quantum dots are fixed on the other side of the nuclear pore membrane to obtain an electrochemiluminescent biosensor with synergistic amplification of bimetallic nanozymes. In the target-unbound state, Pd@Pt nanozymes trigger DNA to bind complementary to the aptamer and are stably anchored to the inner wall of the nuclear pore membrane. Its unique bimetallic active site can efficiently catalyze the reduction of H2O2 and generate a stable catalytic current. (1) When the target binds to the aptamer, the conformational change of the aptamer is triggered, the DNA dissociation is triggered, and the Pd@Pt nanozyme is detached. The electrochemical signal is significantly reduced, forming a high-contrast detection signal. (2) When the target binds to the aptamer, the trigger DNA is released, and the trigger DNA and Track DNA hybridization forms a double-stranded cleavage site recognizable by the Nt.BbvCI enzyme, initiating a DNA walking reaction. Each walking cycle releases multiple Pd@Pt nanozymes. The electrocatalytic action of the Pd@Pt nanozymes accelerates the oxidation of luminol. The localized surface plasmon resonance (LSPR) effect of the Au@Ag nanoclusters can increase the anodic luminescence intensity of luminol by 5 times, further enhancing the luminescence efficiency. By detecting the anodic luminescence signal of luminol and forming a ratio with the stable cathodoluminescence signal generated by the CdS quantum dots fixed on the other side of the nuclear pore membrane, more accurate detection can be achieved.

[0011] Innovations: 1. Functional modification technology of the inner wall of nuclear pore membrane: For the first time, bimetallic nanozymes (Pd@Pt), nanoclusters (Au@Ag NCs) and 3D DNA walkers are synergistically modified on the inner wall of nuclear pore membrane, and the porous structure is used to shorten the signal conduction path, thereby improving the detection efficiency several times; high specific surface area is combined with a multi-level sealing strategy to achieve ultra-low background signal; 2. Dual amplification synergistic innovation (1) Nanozyme and DNA walker synergy: The current inhibition of Pd@Pt nanozyme and the cascade cutting reaction of 3D DNA walker cooperate to form a three-level cascade amplification of "target recognition → DNA walking → nanozyme release", which improves the signal amplification efficiency; (2) Electrochemical-optical dual-mode enhancement: The current change of Pd@Pt nanozyme and the LSPR effect of Au@Ag nanoclusters synchronously amplify the signal, with a detection limit as low as 0.01 fg / mL, breaking through the bottleneck of picogram-level detection.

[0012] This invention utilizes bimetallic nanozymes integrated within the nuclear pore membrane, aptamer conformational switching triggering, and ratiometric ECL detection technology to construct a highly sensitive and specific detection platform. Its innovative multi-stage signal amplification mechanism and anti-interference strategy address bottlenecks in traditional detection technologies, providing a highly effective solution for rapid bedside detection of neurodegenerative diseases and possessing significant potential for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows a flow chart of the preparation method of an electrochemiluminescent biosensor based on bimetallic nanozymes and DNA walker.

[0014] Figure 2 Schematic diagram of the principle of detecting target objects by an electrochemiluminescent biosensor based on bimetallic nanozymes and DNA walker.

[0015] Figure 3 This is an electron micrograph of the nuclear pore membrane;

[0016] Figure 4 (A) CV diagram and (B) electrochemical impedance diagram of the electrochemical biosensor of the present invention;

[0017] Figure 5 (A) Current-time graph and (B) standard curve graph of the response to different concentrations of target substances;

[0018] Figure 6 (A) ECL light intensity-potential diagram and (B) standard curve diagram of the response to different concentrations of target;

[0019] Figure 7 Figures for exploring (A) reproducibility and (B) stability;

[0020] Figure 8 is a specific identification map; DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Any changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0022] In the following examples, the nucleic acid sequences involved are shown in Table 1

[0023] Table 1 Related nucleic acid sequences in the examples

[0024]

[0025] Example 1: The present invention also provides a method for preparing an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker, and the specific preparation steps are as follows: Step (1) Pretreatment of nuclear pore membrane: Using a nuclear pore membrane with a pore size of 100 nm and containing carboxyl functional groups as a substrate, the nuclear pore membrane is immersed in a 10 mM EDC / NHS mixed solution, and an activation reaction is carried out at room temperature for 2 hours. Finally, the nuclear pore membrane is immersed in a 10 μM gold nanoparticle solution for 12 hours to uniformly form a layer of gold nanoparticles on the inner wall of the nuclear pore membrane; Step (2) Aptamer immobilization: A thiolated aptamer (Apt) is designed for the target biomarker, and then the 10 μM thiolated aptamer solution is incubated with the pretreated nuclear pore membrane, and the reaction time is set to 20 h, so that the thiolated aptamer is firmly connected to the gold nanoparticle layer on the inner wall of the nuclear pore membrane through the Au-S bond. After the incubation, the nuclear pore membrane is washed several times with a PBS buffer solution with a pH of 7.4 to remove unbound aptamer molecules. Subsequently, 1% BSA solution is added for blocking treatment. The reaction time is 1 h; Step (3) Nanozyme loading: The thiolated trigger DNA (10 μM) and the Pd@Pt nanozyme containing gold sites (1 mg / mL) are incubated in PBS with a pH of 7.0-8.0 at 4°C for 12 h. Through Au-S bond coupling, the trigger DNA-Pd@Pt complex is drop-coated on the surface of the aptamer-modified nuclear pore membrane and incubated at 37°C for 3 h to hybridize the 3' end of the trigger DNA with the complementary sequence of the aptamer end. The membrane is washed three times with PBS to remove unbound nanozyme complexes; Step (4) Nanocluster loading: Au@Ag nanoclusters (concentration of 5 μM, pH = 7.4) were assembled on the surface of the nuclear pore membrane, with an interval of 20 min between each round of assembly, and a total of 5 rounds of assembly were performed to construct a multilayer nanostructure and enhance the signal amplification ability of the sensor; Step (5) 3D DNA walker construction: AuNP (10 nM) and Track DNA were mixed at a molar ratio of 1:100, and NaCl was slowly added to a final concentration of 0.5 M. The mixture was stirred at room temperature for 12 h for salt aging, so that each AuNP was loaded with about 50 Track DNAs. The Track DNA-AuNP complex was drop-coated on the surface of the nuclear pore membrane modified with trigger DNA-Pd@Pt, incubated at 37°C for 4 h, and fixed to the inner wall of the membrane through Au-S bonds. Subsequently, Nt.BbvCI endonuclease (concentration of 1 U / μL) can specifically recognize and cut DNA sequences, triggering a cyclic cutting reaction to form a signal amplification system; Step (6) Blocking and CDS quantum dot fixation: The composite membrane is placed in a bovine serum albumin solution to block the active sites. On the other side of the nuclear pore membrane, the membrane surface is silanized using 3-aminopropyltriethoxysilane (APTES) to form an amino active layer. Cadmium sulfide (CdS) quantum dots are then fixed by covalent coupling as the sensitizing element of the electrochemiluminescence signal to achieve multi-dimensional signal synergistic amplification.

[0026] Furthermore, in step (1), the nuclear pore membrane is a track-etched membrane formed by ion irradiation, etching, and ultraviolet irradiation of polyethylene terephthalate (PET) membrane, polycarbonate (PC) membrane, or polyimide (PI) membrane, the shape of the nanopore is conical, the thickness is 15 μm, the pore diameter of the surface pore is 0.1 μm, and the diameter of the gold nanoparticles is 10 nm;

[0027] Furthermore, in steps (3) and (4), the nanozyme (Pd core diameter 5 nm, Pt shell thickness 2 nm) and the particle size of the Au@Ag nanocluster are 5 nm; the preparation scheme of the nanozyme is as follows: chloroauric acid is prepared into a chloroauric acid solution, heated to boiling, sodium citrate solution is added, reacted for 30 minutes, and cooled to room temperature; the gold nanoparticle solution is taken, silver nitrate solution is added, stirred, and a mixed solution of sodium citrate and sodium borohydride is added dropwise. After the addition is completed, continue stirring for a period of time to allow the reaction to proceed fully. The general stirring time is 1 hour to obtain the Au@Ag core-shell nanocluster solution; Nanocluster preparation scheme: Preparation steps of bimetallic nanozymes: Dissolve chloropalladium acid to form a uniform solution, add reducing agent solution sodium borohydride solution, continue stirring for a period of time, and then centrifuge to obtain Pd nanoparticles, wash them multiple times, and finally disperse the Pd nanoparticles in an appropriate amount of solvent for use; Add the prepared Pd nanoparticle solution to a solution containing chloroplatinic acid, add a reducing agent, reduce and deposit Pt ions on the surface of Pd nanoparticles to form a Pt shell, centrifuge and wash to obtain purified Pd@Pt nanozyme;

[0028] Furthermore, in step (5), the ratio of Track DNA to gold nanoparticles is 100:1, and each gold nanoparticle is loaded with approximately 50 Track DNA strands;

[0029] Furthermore, in step (6), the concentration of bovine serum albumin is (10 mg / mL, pH 7.4); the particle size of the CDS quantum dots is 5 nm, and the emission peak is 500 nm.

[0030] Example 2: In addition, the present invention also provides an application of an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker, and the specific steps are as follows: (1) detecting the response intensity of the electrochemiluminescent biosensor containing target solutions of different concentrations, wherein the concentration range of the target solution is 0.01 fg / mL to 1 pg / mL; (2) placing the prepared composite film on an ITO conductive glass as a working electrode, a platinum wire electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a three-electrode system; the three-electrode system is placed in a 0.1 mol / L PBS solution with a pH of 7.4 for measurement; (3) electrochemical detection: the scanning potential is -1.0-1.0 V, and the current values ​​I0 and I of the target standard before and after incubation are obtained, and a standard curve is drawn with the logarithm of the target concentration as the horizontal axis and the current change rate as the vertical axis; (4) ECL ratio detection: the photomultiplier is 500 V, the scanning potential is -1.0-1.0 V, the scanning rate is 100 mV / s, and 20 mmol / L TPA was used as the ECL detection solution. Cyclic voltammetry (CV) was used to measure the ECL signal of the reaction system. The potential-luminescence intensity curve was recorded, and a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration was established to obtain the corresponding linear regression equation.

[0031] Example 3: This example uses scanning electron microscopy (SEM) as a powerful technique to help observe the morphology and structure of the prepared materials. The nuclear pore membranes were examined by electron microscopy. Figure 3 The results showed that the membrane had penetrating columnar nanopores with a porosity of 30%. Under the same conditions, the pore diameters of 100 groups of pores were statistically analyzed, and the pore diameter of the constructed nanopores was 0.1 μm. The shape of the nanopores was cylindrical, the thickness was 10 μm, and the pore density was 1×10 3 pieces / cm 2 .

[0032] Example 4: Experimental verification of the principle: In order to prove the feasibility of this technical solution, this embodiment uses a method of preparing an electrochemiluminescent biosensor using different modified electrodes. The results are as follows Figure 4 As shown in A and 4B, after the nuclear pore membrane was coated on the ITO conductive glass, the modification process was characterized by CV and electrochemical impedance spectroscopy (EIS) in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7.4. Figure 4A is the CV curve of the sensor. It can be seen that the peak current increases when gold nanoparticles are connected, and then decreases when thiolated aptamers are connected. The peak current increases rapidly when nanozymes and nanoclusters are loaded. This is because a redox reaction is formed on the electrode surface. Track DNA fixation, bovine serum albumin blocking, and CDA quantum dot modification all cause the peak current to decrease, but the rate of decrease is slow, indicating that the effect of these modification steps on the current is minimal. Then, the peak current drops sharply after the target is detected. This indicates that when target binding triggers conformational changes in the aptamer, DNA dissociation is triggered, causing the Pd@Pt nanozyme to detach, and the electrochemical signal is significantly reduced, forming a high-contrast detection signal. Figure 5 (b) is the EIS curve of the sensor, and the results are consistent with the CV curve.

[0033] Example 5: Sensitivity experiment: In order to evaluate the sensitivity of the present invention in detecting the target, the current of the target with different concentrations was measured under the optimal experimental conditions. Figure 5 As shown in the figure, the current decreases with the increase of the concentration of the target. At the same time, there is a certain functional relationship between the change of current and the concentration of the target. The linear regression equation is (I-I0) / I0 (µA) = -0.04737 lgC (fg / mL) -0.29714 (R 2 =0.998), therefore, the target can be analyzed by electrochemiluminescence biosensor.

[0034] Example 6: Sensitivity experiment: In order to evaluate the sensitivity of the present invention in detecting the target, the ECL luminescence intensity of the target at different concentrations was measured under the optimal experimental conditions. Figure 6 As shown in the figure, the ECL luminescence intensity increases with the increase of the concentration of the target. At the same time, there is a certain functional relationship between the change of ECL luminescence intensity and the concentration of the target. The linear regression equation is (ECL-ECL0) / ECL0(au)=0.44759 lgC(fg / mL)+1.94143(R 2 =0.999), so the target can be analyzed by electrochemiluminescence biosensor.

[0035] Example 7: Reproducibility experiment: Figure 7 As shown in A and B, a standard curve of ECL / EC versus concentration was obtained. Five replicates of the ECL / EC response signal were compared with the signal detected using ECL alone, demonstrating the improved reproducibility of this method. The storage stability of the sensor was investigated. After storing the prepared biosensor at 4°C for 7 days, the ECL / EC signal ratios obtained from five replicates were nearly identical to the initial value, demonstrating the excellent stability of the constructed ratiometric sensor.

[0036] Example 8: Specificity experiment: In order to study the specificity of the electrochemiluminescent biosensor, Figure 7 As shown, this embodiment uses five mismatches and one specific target as controls to perform a specificity experiment; the results are shown in Figure 8 As shown in the figure, under the same reaction conditions, the current difference of the target is much greater than that of the non-target.

[0037] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An electrochemiluminescent biosensor based on bimetallic nanozymes and DNA walker, characterized in that: The nuclear pore membrane with carboxyl functional groups on the inner wall is used as the substrate, and gold nanoparticles are loaded on the inner wall of the nuclear pore membrane. Then, thiolated aptamers are designed for the detection object, and the aptamers are directionally fixed to the surface of the gold nanoparticles pre-modified on the inner wall of the nuclear pore membrane through the Au-S bond. The thiolated trigger DNA and the Pd@Pt nanozyme containing gold sites are mixed in PBS and coupled through the Au-S bond. The trigger DNA-Pd@Pt complex is then drop-coated on the surface of the aptamer-modified nuclear pore membrane, so that the 3' end of the trigger DNA hybridizes with the complementary sequence of the aptamer end and is fixed to the inner wall of the nuclear pore membrane. Then, the Au@Ag nanoclusters are self-assembled layer by layer on the carboxyl sites on the inner wall of the nuclear pore membrane through electrostatic adsorption. DNA is mixed with gold nanoparticles and modified at a high density through salt aging method. After centrifugal purification, it is fixed to the inner wall of the nuclear pore membrane through Au-S bonds. Nt.BbvCI endonuclease is added, which can specifically recognize and cut DNA sequences, triggering a cyclic cutting reaction to form a signal amplification system. Bovine serum albumin is used to block nonspecific binding sites, and CDS quantum dots are fixed on the other side of the nuclear pore membrane to obtain an electrochemiluminescent biosensor with synergistic amplification of dual-metal nanozymes.

2. The method for preparing an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker according to claim 1, wherein: The specific preparation steps are as follows: (1) nuclear pore membrane pretreatment: using a nuclear pore membrane containing carboxyl functional groups as a substrate, immersing the nuclear pore membrane in an EDC / NHS mixed solution, and performing an activation reaction at room temperature, finally immersing the nuclear pore membrane in a gold nanoparticle solution to uniformly form a layer of gold nanoparticles on the inner wall of the nuclear pore membrane; (2) aptamer immobilization: designing a thiolated aptamer (Apt) for the target biomarker, and then incubating the thiolated aptamer solution with the pretreated nuclear pore membrane so that the thiolated aptamer is firmly connected to the gold nanoparticle layer on the inner wall of the nuclear pore membrane through the Au-S bond. After the incubation, the nuclear pore membrane is washed multiple times with PBS buffer solution to remove unbound aptamer molecules; (3) Nanozyme loading: The thiolated trigger DNA and the Pd@Pt nanozyme containing gold sites were mixed in PBS and coupled through Au-S bonds. The trigger DNA-Pd@Pt complex was then drop-coated on the surface of the aptamer-modified nuclear pore membrane, so that the 3' end of the trigger DNA hybridized with the complementary sequence of the aptamer end and fixed to the inner wall of the nuclear pore membrane; (4) Nanocluster loading: The Au@Ag nanoclusters were assembled on the surface of the nuclear pore membrane using layer-by-layer self-assembly technology to construct a multilayer nanostructure and enhance the signal amplification capability of the sensor; (5) 3D DNA walker construction: AuNPs were mixed with Track DNA, NaCl was slowly added, and salt aging was performed by stirring at room temperature so that each AuNP was loaded with 50-70 Track DNAs. The DNA-AuNP complex is drop-coated on the surface of the modified trigger DNA-Pd@Pt nuclear pore membrane and fixed to the inner wall of the membrane through the Au-S bond. Subsequently, Nt.BbvCI endonuclease is added, which can specifically recognize and cut the DNA sequence, triggering a cyclic cutting reaction to form a signal amplification system; (6) Sealing and CDS quantum dot fixation: the composite membrane is placed in a bovine serum protein solution to seal the active site. The composite membrane is placed in a bovine serum protein solution to seal the active site. On the other side of the nuclear pore membrane, the membrane surface is silanized using 3-aminopropyltriethoxysilane (APTES) to form an amino active layer. Cadmium sulfide (CdS) quantum dots are then fixed by covalent coupling as the sensitizing element of the electrochemiluminescence signal to achieve multi-dimensional signal synergistic amplification.

3. The use of an electrochemiluminescent biosensor based on a bimetallic nanozyme and a DNA walker according to claim 1, characterized in that: The specific steps are as follows: (1) detecting the response intensity of the electrochemiluminescent biosensor containing target solutions of different concentrations, wherein the concentration range of the target solution is 0.01 fg / mL to 1 pg / mL; (2) placing the prepared composite film on ITO conductive glass as a working electrode, a platinum wire electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode to form a three-electrode system; placing the three-electrode system in a 0.1-0.2 mol / L PBS solution with a pH of 7.0-7.5 for measurement; (3) electrochemical detection: scanning potential is -1.0-1.0 V, and the obtained The current values ​​I0 and I of the target standard before and after incubation were obtained, and a standard curve was drawn with the logarithm of the target concentration as the horizontal axis and the current change rate as the vertical axis; (4) ECL ratio detection: the photomultiplier was 500-600 V, the scanning potential was -1.0-1.0 V, the scanning rate was 100-200 mV / s, 20 mmol / L TPA was added as the ECL detection solution, and the ECL signal of the reaction system was measured by cyclic voltammetry (CV). The potential-luminescence intensity curve was recorded, and a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration was established to obtain the corresponding linear regression equation.

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