Electrochemical luminescence biosensor based on bimetallic nano-enzyme and DNA walker and application of electrochemical luminescence biosensor

Abstract

The invention discloses an electrochemical luminescence biosensor based on bimetallic nano-enzyme and DNA walker and application thereof, and belongs to the technical field of biosensing. The sensor takes a nuclear track membrane as a substrate, gold nanoparticles are loaded on the inner wall of the membrane, thiolated aptamers are designed, the aptamers are fixed on the surfaces of the gold nanoparticles through Au-S bonds, thiolated trigger DNA and Pd (at) Pt nano-enzyme containing gold sites are mixed in PBS and coupled through the Au-S bonds, then the trigger DNA-Pd (at) Pt compound is dispensed on the surface of the aptamer modified membrane, and the sensor is obtained. The preparation method comprises the following steps: preparing Au (at) Ag nano-clusters, hybridizing the Au (at) Ag nano-clusters with aptamers, fixing the Au (at) Ag nano-clusters on the inner wall of a membrane, self-assembling the Au (at) Ag nano-clusters on carboxyl sites on the inner wall of the membrane layer by layer through electrostatic adsorption, fixing Track DNA on the inner wall of the membrane, adding Nt.BbvCI endonuclease, closing non-specific binding sites by using bovine serum albumin, and fixing CDS quantum dots on the other side of the membrane. The electrochemical luminescence biosensor of the bimetallic nano enzyme and the DNA walker is obtained. The sensor has the advantages of high sensitivity, low detection limit, good stability, simple preparation steps, short detection time and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and particularly relates to a biosensor integrating aptamer specific recognition, bimetallic nanozyme catalytic amplification, 3D DNA walker cascade reaction and ratiometric electrochemiluminescence (ECL) detection Background Technique

[0002] As a key technology for early disease diagnosis, treatment efficacy evaluation, and prognosis monitoring, biomarker detection plays an important role in the fields of clinical medicine and life science research. Biomolecules represented by tumor markers, proteins related to neurodegenerative diseases, etc., usually have extremely low concentrations in body fluids. For example, the concentration of specific proteins in the cerebrospinal fluid of Alzheimer's disease patients is at the fg / mL level, which poses extremely high requirements for the sensitivity of detection techniques. However, existing biomarker detection techniques still have the following deficiencies: 1. Insufficient sensitivity: Traditional enzyme-linked immunosorbent assay (ELISA) technology relies on the specific binding of antigens and antibodies. Although it has certain specificity, due to limited signal amplification ability, the detection limit is usually 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 traditional ELISA for the circulating tumor marker HER2 is about 1 pg / mL, while clinical studies have shown that when the HER2 concentration is below 0.1 pg / mL, it can indicate the early occurrence of the disease, and existing technologies cannot effectively detect this concentration range; 2. Weak anti-interference ability: The components of biological samples (such as blood, urine, 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, cell debris, etc. Taking blood samples as an example, human serum contains more than 10,000 proteins, and the concentrations of high-abundance proteins such as albumin and immunoglobulins can reach the mg / mL level, while the concentration of the target biomarker may only be at the fg / mL - pg / mL level. Traditional detection techniques are difficult to effectively distinguish the target from the interfering substances, and are extremely prone to non-specific adsorption or cross-reaction, resulting in false positive / negative results; 3. Low signal amplification efficiency: Existing detection techniques mostly rely on a single signal amplification strategy, making it difficult to achieve effective detection of low-concentration biomarkers. For example, although signal amplification techniques based on enzyme catalysis can generate a large number of detectable products through enzymatic reactions, the activity of enzymes is easily affected by factors such as temperature, pH value, and inhibitors, resulting in unstable amplification efficiency; Signal amplification strategies assisted by nanomaterials, such as gold nanoparticles, quantum dots, etc., although having unique optical and electrical properties, the amplification factor of a single nanomaterial is limited, making it difficult to meet the needs of ultrasensitive detection. In addition, a single signal amplification strategy lacks the synergistic enhancement effect between signals and cannot fully utilize the advantages of different amplification mechanisms, limiting the further improvement of detection sensitivity. Therefore, there is an urgent need to develop a biosensor with high sensitivity, strong anti-interference ability, and efficient signal amplification mechanism to promote the development and clinical application of biomarker detection techniques. Summary of the Invention

[0003] The present invention proposes an innovative solution to achieve ultrasensitive detection of biomarkers through aptamer high-specific recognition, the synergistic amplification of dual-metal nanozymes and 3D DNA walker, and ratiometric ECL detection technology, breaking through the limitations of traditional sensors in sensitivity and anti-interference.

[0004] To achieve the above object, the technical solution of the present invention is as follows: using a nuclear pore membrane with carboxyl functional groups on the inner wall as a substrate, loading gold nanoparticles on the inner wall of the nuclear pore membrane to form uniform anchoring points; then designing a thiolated aptamer for the analyte, and orienting and fixing the aptamer on the surface of the pre-modified gold nanoparticles on the inner wall of the nuclear pore membrane through Au-S bonds to form a high-density recognition interface; then synthesizing a core-shell structure Pd@Pt nanozyme by a liquid-phase reduction method, mixing the thiolated trigger DNA with the Pd@Pt nanozyme containing gold sites in PBS, coupling through Au-S bonds, and then dropping 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 at the end of the aptamer and is fixed on the inner wall of the nuclear pore membrane; then preparing Au@Ag nanoclusters by a sodium citrate reduction method, and enabling them to self-assemble layer by layer on the carboxyl sites on the inner wall of the nuclear pore membrane through electrostatic adsorption due to their surface negative charges; then mixing Track DNA with gold nanoparticles, achieving high-density modification through a salt aging method, and fixing it on the inner wall of the nuclear pore membrane through Au-S bonds after centrifugal purification. Subsequently, adding Nt.BbvCI endonuclease, which can specifically recognize and cleave the DNA sequence, triggering a cyclic cleavage reaction to form a signal amplification system, using bovine serum albumin to block non-specific binding sites, and fixing CDS quantum dots on the other side of the nuclear pore membrane to obtain an electrochemiluminescence biosensor with synergistic amplification of bimetallic nanozymes.

[0005] In addition, the present invention also provides a preparation method of an electrochemiluminescence biosensor based on bimetallic nanozymes and DNA walker. The specific preparation steps are as follows: Step (1) Nucleopore membrane pretreatment: Using a nucleopore membrane containing carboxyl functional groups as the substrate, immerse the nucleopore membrane in a 10-20 mM EDC / NHS mixed solution and carry out an activation reaction at room temperature for 2-3 h. Finally, immerse the nucleopore membrane in a 10-20 μM gold nanoparticle solution for 12-15 h to uniformly form a layer of gold nanoparticles on the inner wall of the nucleopore membrane; Step (2) Aptamer immobilization: Design a thiolated aptamer (Apt) for the target biomarker, and then incubate a 10-20 μM thiolated aptamer solution with the pretreated nucleopore membrane for 20-24 h to firmly connect the thiolated aptamer to the gold nanoparticle layer on the inner wall of the nucleopore membrane through Au-S bonds. After the incubation, wash the nucleopore membrane multiple times with a PBS buffer solution with a pH of 7.0-8.0 to remove unbound aptamer molecules. Subsequently, add a 1% BSA solution for blocking treatment, and the reaction time is 1-2 h; Step (3) Nanozyme loading: Incubate thiolated trigger DNA (10-15 μM) with Pd@Pt nanozymes containing gold sites (1-2 mg / mL) in PBS at pH 7.0-8.0 at 4-5 °C for 12-15 h, and couple through Au-S bonds. Drop the trigger DNA-Pd@Pt complex onto the surface of the aptamer-modified nucleopore membrane and incubate at 37-40 °C for 3-4 h to hybridize the 3' end of the trigger DNA with the complementary sequence at the end of the aptamer. Wash with PBS 3-4 times to remove unbound nanozyme complexes; Step (4) Nanocluster loading: Adopt the layer-by-layer self-assembly technique to assemble Au@Ag nanoclusters (concentration 5-8 μM, pH = 7.0-8.0) on the surface of the nucleopore membrane, with an interval of 20-30 min for each round of assembly, and a total of 5-6 rounds of assembly to construct a multi-layer nanostructure to enhance the signal amplification ability of the sensor; Step (5) 3D DNA walker construction: Mix AuNP (10-20 nM) with Track DNA at a molar ratio of 1:100, slowly add NaCl to a final concentration of 0.5-0.7 M, and stir at room temperature for 12-15 h for salt aging, so that each AuNP is loaded with about 50-70 Track DNAs. Drop the Track DNA-AuNP complex onto the surface of the nucleopore membrane modified with trigger DNA-Pd@Pt and incubate at 37-40 °C for 4-5 h to fix it on the inner wall of the membrane through Au-S bonds. Subsequently, add Nt.BbvCI endonuclease (concentration: 1-2 U / μL), which can specifically recognize and cleave DNA sequences, triggering a cyclic cleavage reaction to form a signal amplification system; Step (6) Blocking and CDS quantum dot immobilization: Immerse the composite membrane in bovine serum albumin solution for active site blocking. On the other side of the nuclear pore membrane, use 3-aminopropyltriethoxysilane (APTES) to perform silanization treatment on the membrane surface to form an amino-active layer, and then immobilize cadmium sulfide (CdS) quantum dots through covalent coupling as a sensitizing element for electrochemiluminescence signals, achieving multi-dimensional signal synergistic amplification.

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

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

[0008] Further, in the steps (5) and (6), the ratio of Track DNA to gold nanoparticles is 100:1, each gold nanoparticle is loaded with about 50 strands of Track DNA, and 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 electrochemiluminescence biosensor based on bimetallic nanozymes and DNA walker, and the specific steps are as follows: (1) Detect the response intensity of the electrochemiluminescence biosensor to target solutions with different concentrations, and the concentration range of the target solutions is 0.01 fg / mL to 1 pg / mL; (2) Place the prepared composite film on the ITO conductive glass as the working electrode, a platinum wire electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode to form a three-electrode system; place the three-electrode system in a PBS solution with a concentration of 0.1-0.2 mol / L and 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. Plot a standard curve with the logarithm of the target concentration as the abscissa and the current change rate as the ordinate; (4) ECL ratio detection: The photomultiplier is 500-600 V, the scanning potential is -1.0-1.0 V, the scanning rate is 100-200 mV / s, add 20 mmol / L of TPA as the ECL detection solution, and use cyclic voltammetry (CV) to measure the ECL signal of the reaction system, record the potential-luminescence intensity curve, establish a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration, and obtain the corresponding linear regression equation;

[0010] The detection principle of the present invention is as follows: Using a nuclear pore membrane as the substrate, gold nanoparticles are loaded on the inner wall of the membrane. A thiolated aptamer is designed for the analyte, and the aptamer is directionally immobilized on the surface of the pre-modified gold nanoparticles on the inner wall of the membrane through Au-S bonds. The thiolated trigger DNA and Pd@Pt nanozyme containing gold sites are mixed in PBS and coupled through Au-S bonds. Then, the trigger DNA-Pd@Pt complex is drop-coated on the surface of the aptamer-modified nuclear pore membrane, enabling the 3'-end of the trigger DNA to hybridize with the complementary sequence at the end of the aptamer and be fixed on the inner wall of the nuclear pore membrane. Then, Au@Ag nanoclusters are layer-by-layer self-assembled on the carboxyl sites on the inner wall of the nuclear pore membrane through electrostatic adsorption. Then, the Track DNA and gold nanoparticles are mixed, and high-density modification is achieved through the salt aging method. After centrifugal purification, it is fixed on the inner wall of the nuclear pore membrane through Au-S bonds. Subsequently, Nt.BbvCI endonuclease is added. This endonuclease can specifically recognize and cleave DNA sequences, triggering a cyclic cleavage reaction to form a signal amplification system. Bovine serum albumin is used to block non-specific binding sites, and Cds quantum dots are fixed on the other side of the nuclear pore membrane, obtaining an electrochemiluminescence biosensor with synergistic amplification by bimetallic nanozymes; in the state where the target is not bound, the Pd@Pt nanozyme is complementarily bound to the aptamer through the trigger DNA and stably anchored on the inner wall of the nuclear pore membrane. Its unique bimetallic active sites can efficiently catalyze the reduction of H2O2, generating a stable catalytic current; (1) When the binding of the target causes a conformational change in the aptamer, the dissociation of the trigger DNA leads to the detachment of the Pd@Pt nanozyme, and the electrochemical signal is significantly reduced, forming a high-contrast detection signal; (2) When the binding of the target to the aptamer causes the release of the trigger DNA, the trigger DNA hybridizes with the Track DNA to form a double-stranded cleavage site recognizable by the Nt.BbvCI enzyme, initiating a DNA walking reaction. For each walking cycle, multiple Pd@Pt nanozymes can be released. The electrocatalytic action of the Pd@Pt nanozyme can accelerate the oxidation of luminol, and the local 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 cathodic luminescence signal generated by the Cds quantum dots fixed on the other side of the nuclear pore membrane, more accurate detection is achieved.

[0011] Innovative points: 1. Inner wall functionalization modification technology of nuclear pore membrane: For the first time, bimetallic nanozymes (Pd@Pt), nanoclusters (Au@Ag NCs) and 3D DNA walker are co-modified on the inner wall of the nuclear pore membrane. The porous structure is used to shorten the signal transduction path, and the detection efficiency is increased several times. The combination of high specific surface area and multi-level closed strategy realizes an ultra-low background signal. 2. Dual amplification collaborative innovation: (1) Collaboration between nanozyme and DNA walker: The current inhibition of Pd@Pt nanozyme and the cascade cleavage reaction of 3D DNA walker are coordinated to form a three-level cascade amplification of "target recognition → DNA walking → nanozyme release", and the signal amplification efficiency is improved. (2) Electrochemical-optical dual-mode enhancement: The current change of Pd@Pt nanozyme and the LSPR effect of Au@Ag nanoclusters amplify the signal synchronously, and the detection limit is as low as 0.01 fg / mL, breaking through the picogram-level detection bottleneck.

[0012] The present invention constructs a highly sensitive and highly specific detection platform through the integration of bimetallic nanozymes on the inner wall of the nuclear pore membrane, the triggering of aptamer conformational switching, and the ratio-type ECL detection technology. Its innovative multi-level signal amplification mechanism and anti-interference strategy solve the bottleneck problems of traditional detection technologies, provide an efficient solution for the bedside rapid detection of neurodegenerative diseases, and have significant clinical transformation potential. Brief description of the drawings

[0013] Figure 1 It is a flow chart of the preparation method of an electrochemiluminescence biosensor based on bimetallic nanozymes and DNA walker.

[0014] Figure 2 It is a schematic diagram of the detection of the target by an electrochemiluminescence biosensor based on bimetallic nanozymes and DNA walker.

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

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

[0017] Figure 5 It is the (A) current-time diagram and (B) standard curve diagram of the response of different concentrations of the target;

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

[0019] Figure 7 It is the (A) reproducibility and (B) stability exploration diagram;

[0020] Figure 8 It is the specific recognition diagram; Detailed implementation manners

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

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

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

[0024] Example 1: The present invention also provides a preparation method of an electrochemiluminescence biosensor based on bimetallic nanozyme and DNA walker. 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 carboxyl functional groups as the substrate, immerse the nuclear pore membrane in a 10 mM EDC / NHS mixed solution and carry out an activation reaction at room temperature for 2 h. Finally, immerse the nuclear pore membrane in a 10 μM gold nanoparticle solution for 12 h to uniformly form a layer of gold nanoparticles on the inner wall of the nuclear pore membrane; Step (2) Aptamer immobilization: Design a thiolated aptamer (Apt) for the target biomarker, and then incubate the 10 μM thiolated aptamer solution with the pretreated nuclear pore membrane. Set the reaction time to 20 h to make the thiolated aptamer firmly connect to the gold nanoparticle layer on the inner wall of the nuclear pore membrane through Au-S bonds. After the incubation, wash the nuclear pore membrane multiple times with PBS buffer solution with a pH of 7.4 to remove unbound aptamer molecules. Subsequently, add 1% BSA solution for blocking treatment, and the reaction time is 1 h; Step (3) Nanozyme loading: Incubate thiolated trigger DNA (10 μM) with Pd@Pt nanozyme containing gold sites (1 mg / mL) in PBS at pH 7.0 - 8.0 at 4 °C for 12 h. Couple through Au-S bonds and drop-coat the trigger DNA-Pd@Pt complex on the surface of the aptamer-modified nuclear pore membrane. Incubate at 37 °C for 3 h to hybridize the 3' end of the trigger DNA with the complementary sequence at the end of the aptamer. Wash 3 times with PBS to remove unbound nanozyme complexes; Step (4) Nanocluster loading: Adopt the layer-by-layer self-assembly technique to assemble Au@Ag nanoclusters (concentration of 5 μM, pH = 7.4) on the surface of the nuclear pore membrane. The interval between each round of assembly is 20 min, and a total of 5 rounds of assembly are carried out to construct a multi-layer nanostructure and enhance the signal amplification ability of the sensor; Step (5) 3D DNA walker construction: Mix AuNP (10 nM) and Track DNA at a molar ratio of 1:100, slowly add NaCl to a final concentration of 0.5 M, and stir at room temperature for 12 h for salt aging to make each AuNP load about 50 Track DNAs. Drop-coat the Track DNA-AuNP complex on the surface of the nuclear pore membrane modified with trigger DNA-Pd@Pt and incubate at 37 °C for 4 h to fix it on the inner wall of the membrane through Au-S bonds. Subsequently, add Nt.BbvCI endonuclease (at a concentration of 1 U / μL), which can specifically recognize and cleave DNA sequences, triggering a cyclic cleavage reaction to form a signal amplification system; Step (6) Blocking and CDS quantum dot immobilization: Immerse the composite membrane in a bovine serum albumin solution for active site blocking. On the other side of the nuclear pore membrane, use 3-aminopropyltriethoxysilane (APTES) to perform silanization treatment on the membrane surface to form an amino active layer, and then fix cadmium sulfide (CdS) quantum dots through covalent coupling as a sensitizing element for electrochemiluminescence signals, realizing multi-dimensional signal synergistic amplification.

[0025] Further, in the said step (1), the nuclear pore membrane is a track-etched membrane formed by ion irradiation, etching, and ultraviolet light irradiation of a polyethylene terephthalate (PET) membrane, a polycarbonate (PC) membrane, or a polyimide (PI) membrane. The shape of the nanopores is conical, the thickness is 15 µm, the pore diameter of the surface pores is 0.1 µm, and the diameter of the gold nanoparticles is 10 nm;

[0026] Further, in the said steps (3) and (4), the nanozyme (Pd core diameter 5 nm, Pt shell thickness 2 nm), the particle size of the Au@Ag nanoclusters is 5 nm; Preparation scheme of the nanozyme: Prepare a chloroauric acid solution by dissolving chloroauric acid, heat it to boiling, add a sodium citrate solution, react for 30 minutes, and cool to room temperature; Take the gold nanoparticle solution, add a silver nitrate solution, stir, and dropwise add a mixed solution of sodium citrate and sodium borohydride. After dropping, continue to stir for a period of time to allow the reaction to proceed fully, generally the stirring time is 1 hour, and then the Au@Ag core-shell nanocluster solution can be obtained; Preparation scheme of the nanoclusters: Preparation steps of the bimetallic nanozyme Dissolve chloropalladic acid to form a homogeneous solution, add a reducing agent solution of sodium borohydride solution, continue to stir for a period of time, then centrifuge to separate Pd nanoparticles, wash them multiple times, and finally disperse the Pd nanoparticles in an appropriate amount of solvent for standby; Add the prepared Pd nanoparticle solution to a solution containing chloroplatinic acid, add a reducing agent to reduce and deposit Pt ions on the surface of the Pd nanoparticles to form a Pt shell layer, and centrifuge and wash to obtain purified Pd@Pt nanozymes;

[0027] Further, in the said step (5), the ratio of Track DNA to gold nanoparticles is 100:1, and each gold nanoparticle is loaded with about 50 Track DNAs;

[0028] Further, in the said 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.

[0029] Example 2: In addition, the present invention also provides an application of an electrochemiluminescence biosensor based on bimetallic nanozymes and DNA walker, and the specific steps are as follows: (1) Detect the response intensity of the electrochemiluminescence biosensor for solutions containing different concentrations of the target, and the concentration range of the target solution is 0.01 fg / mL to 1 pg / mL; (2) Place the prepared composite film on the ITO conductive glass as the working electrode, a platinum wire electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode to form a three-electrode system; Place the three-electrode system 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. A standard curve is plotted with the logarithm of the target concentration as the abscissa and the current change rate as the ordinate; (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 of TPA is added as the ECL detection solution. The ECL signal of the reaction system is measured using cyclic voltammetry (CV), and the potential-luminescence intensity curve is recorded to establish a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration, and the corresponding linear regression equation is obtained;

[0030] Example 3: In this example, scanning electron microscopy (SEM) is used as a powerful technique to help observe the morphology and structure of the prepared material. The above nuclear pore membrane is subjected to electron microscopy detection, Figure 3 The results show that the membrane has through-columnar nanopores with a porosity of 30%. Under the same conditions, the pore diameters of 100 groups of pores are statistically analyzed, and the constructed nanopore diameter is 0.1 µm. The shape of the nanopores is cylindrical, the thickness is 10 µm, and the pore density is 1×10 3 pcs / cm 2 .

[0031] Example 4: Experimental verification of the principle: To prove the feasibility of the present technical solution, this example uses a method of preparing an electrochemiluminescence biosensor with different modified electrodes. The results are as Figure 4 shown in FIGS. 4A and 4B. After coating the nuclear pore membrane on the ITO conductive glass, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are used to characterize the modification process in a 0.1 mol / L PBS buffer solution with a pH of 7.4, Figure 4A is the CV curve of the sensor. It can be seen that connecting gold nanoparticles increases the peak current, then connecting thiolated aptamer decreases the peak current, and loading nanozyme and nanoclusters rapidly increases the peak current. This is because a redox reaction forms on the electrode surface. Fixing Track DNA, bovine serum albumin blocking, and CDA quantum dot modification all decrease the peak current, but this decrease is slow, indicating that these modification steps have a minor impact on the current. Then, after detecting the target, the peak current drops sharply, which shows that when the target binds, it triggers a conformational change in the aptamer, triggering DNA dissociation and causing the Pd@Pt nanozyme to detach, resulting in a significant decrease in the electrochemical signal and 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.

[0032] Example 5: Sensitivity experiment: To evaluate the sensitivity of the detection target of this technical solution, under the optimal experimental conditions, the currents of different concentrations of the target were measured. As Figure 5 shown, the current decreases as the concentration of the target increases. At the same time, there is a certain functional relationship between the change in 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 the electrochemiluminescence biosensor.

[0033] Example 6: Sensitivity experiment: To evaluate the sensitivity of the detection target of this technical solution, under the optimal experimental conditions, the ECL luminescence intensities of different concentrations of the target were measured. As Figure 6 shown, the ECL luminescence intensity increases as the concentration of the target increases. At the same time, there is a certain functional relationship between the change in ECL luminescence intensity and the concentration of the target. The linear regression equation is (ECL - ECL0) / ECL0 (a.u.) = 0.44759 lgC (fg / mL) + 1.94143 (R 2 = 0.999). Therefore, the target can be analyzed by the electrochemiluminescence biosensor.

[0034] Example 7: Reproducibility experiment: As Figure 7 shown in A and B, from the standard curve of ECL / EC and concentration obtained, the ECL / EC response signals were repeatedly detected 5 times and compared with the signals detected by the ECL method alone, indicating that this method has better reproducibility. The storage stability of this sensor was investigated. After the prepared biosensor was stored at 4°C for 7 days, the ratio of the ECL / EC signal measured repeatedly 5 times was almost the same as the initial value, indicating that the constructed ratio sensor has good stability.

[0035] Example 8: Specificity experiment: To study the specificity of the electrochemiluminescence biosensor, as Figure Seven shown, in this example, a specificity experiment was carried out with five mismatches and one specific target as a control; the results are as Figure 8 shown. Under the same reaction conditions for different types of analytes, the current difference of the target is much larger than that of the non-targets.

[0036] It should be noted that the above examples are only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention according to needs, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An electrochemiluminescence biosensor based on bimetallic nanozyme and DNA walker, characterized in that, Using a nuclear pore membrane with a carboxyl functional group on the inner wall as the substrate, gold nanoparticles are loaded on the inner wall of the nuclear pore membrane. Then, a thiolated aptamer is designed for the analyte, and the aptamer is directionally immobilized on the surface of the pre-modified gold nanoparticles on the inner wall of the nuclear pore membrane through Au-S bonds. The thiolated trigger DNA and Pd@Pt nanozyme containing gold sites are mixed in PBS and coupled through Au-S bonds. Then, the trigger DNA-Pd@Pt complex is drop-coated on the surface of the aptamer-modified nuclear pore membrane to hybridize the 3'-end of the trigger DNA with the complementary sequence at the end of the aptamer and immobilize it on the inner wall of the nuclear pore membrane. Then, Au@Ag nanoclusters are assembled layer by layer on the carboxyl sites on the inner wall of the nuclear pore membrane through electrostatic adsorption. The Track DNA and gold nanoparticles are mixed, and high-density modification is achieved through the salt aging method. After centrifugal purification, it is fixed on the inner wall of the nuclear pore membrane through Au-S bonds. Nt.BbvCI endonuclease is added, which can specifically recognize and cleave the DNA sequence to trigger a cyclic cleavage reaction to form a signal amplification system. Bovine serum albumin is used to block non-specific binding sites, and CDS quantum dots are fixed on the other side of the nuclear pore membrane to obtain an electrochemiluminescence biosensor with synergistic amplification of bimetallic nanozymes.

2. The preparation method of an electrochemiluminescence biosensor based on bimetallic nanozyme and DNA walker according to claim 1, wherein The specific preparation steps are as follows: (1) Pretreatment of the nuclear pore membrane: Using a nuclear pore membrane containing carboxyl functional groups as the substrate, immerse the nuclear pore membrane in an EDC / NHS mixed solution for an activation reaction at room temperature. Finally, soak 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: Design a thiolated aptamer (Apt) for the target biomarker, and then incubate 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 Au-S bonds. After the incubation, wash the nuclear pore membrane with PBS buffer solution multiple times to remove unbound aptamer molecules; (3) Nanzyme loading: Mix the thiolated trigger DNA with Pd@Pt nanzymes containing gold sites in PBS and couple them through Au-S bonds. Then, drop-coat the trigger DNA-Pd@Pt complex on the surface of the aptamer-modified nuclear pore membrane to hybridize the 3'-end of the trigger DNA with the complementary sequence at the end of the aptamer and immobilize it on the inner wall of the nuclear pore membrane; (4) Nanocluster loading: Use the layer-by-layer self-assembly technique to assemble Au@Ag nanoclusters on the surface of the nuclear pore membrane to construct a multi-layer nanostructure and enhance the signal amplification ability of the sensor; (5) 3D DNA walker construction: Mix AuNP with Track DNA, slowly add NaCl, and stir at room temperature for salt aging so that each AuNP loads about 50 Track DNAs. Drop-coat the Track DNA-AuNP complex on the surface of the nuclear pore membrane modified with trigger DNA-Pd@Pt and fix it on the inner wall of the membrane through Au-S bonds. Subsequently, add Nt.BbvCI endonuclease, which can specifically recognize and cleave the DNA sequence to trigger a cyclic cleavage reaction to form a signal amplification system; (6) Blocking and CDS quantum dot immobilization: Immerse the composite membrane in a bovine serum albumin solution for active site blocking. On the other side of the nuclear pore membrane, use 3-aminopropyltriethoxysilane (APTES) to perform silanization treatment on the membrane surface to form an amino active layer, and then fix cadmium sulfide (CdS) quantum dots through covalent coupling as a sensitizing element for electrochemiluminescence signals to achieve multi-dimensional signal synergistic amplification.

3. Application of an electrochemiluminescence biosensor based on bimetallic nanozyme and DNA walker according to claim 1, characterized in that, The specific steps are as follows: (1) Detect the response intensity of an electrochemiluminescence biosensor to target solutions with different concentrations, where the concentration range of the target solutions is from 0.01 fg / mL to 1 pg / mL; (2) Place the prepared composite film on the ITO conductive glass as the working electrode, a platinum wire electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode to form a three-electrode system; Place the three-electrode system in a PBS solution with a concentration of 0.1 - 0.2 mol / L and 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. Plot a standard curve with the logarithm of the target concentration as the abscissa and the current change rate as the ordinate; (4) ECL ratio detection: The photomultiplier is 500 - 600 V, the scanning potential is -1.0 - 1.0 V, the scanning rate is 100 - 200 mV / s. Add 20 mmol / L of TPA as the ECL detection solution, and use cyclic voltammetry (CV) to measure the ECL signal of the reaction system. Record the potential-luminescence intensity curve, establish a linear relationship between the luminescence intensity ratio and the logarithm of the target concentration, and obtain the corresponding linear regression equation.

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