A double-nicking enzyme-driven double-free-foot DNA walker biosensor and its preparation method and application

Abstract

The present invention discloses a double-nicking enzyme-driven double-free-foot DNA walker biosensor, as well as its preparation method and application. First, the DNA double foot is released through the competitive binding between the biotinylated aptamer on the functional magnetic beads and AβO and Tau. Then, the free DNA double foot walks on the electrode surface with the assistance of the double-nicking enzyme, and releases the substrates of AβO and Tau from the electrode surface, generating a one-to-many signal amplification effect. DNA walkers have been widely explored and applied as elements for preparing biosensors to detect disease-related biomarkers. The sensor of the present invention can not only simultaneously and sensitively detect AβO and Tau, but also make the diagnosis of AD more accurate in a complementary manner.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a double-nicking enzyme-driven double-free-foot DNA walker biosensor and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disorder, clinically characterized by progressive memory loss, impaired cognitive function, and difficulty with daily activities. This creates significant financial and care burdens for families and society. As a progressive neurological disease, AD's delayed diagnosis poses significant challenges to treatment. Therefore, the development of reliable and efficient early diagnostic technologies is crucial for the early diagnosis and treatment of AD.

[0003] Clinical diagnosis of AD primarily relies on pathological sections, positron emission tomography (PET), and molecular biomarker detection. Researchers have identified molecular biomarkers in the body fluids of AD patients as promising targets for diagnosis and treatment due to their stability, early onset, and non-invasive availability. Clinical studies have demonstrated that amyloid β (Aβ) and tau play a synergistic role in AD. Aβ aggregation leads to massive synaptic loss, while abnormal tau modification impedes normal neuronal communication, leading to memory loss and cognitive impairment in AD patients. Studies have shown that AβO and tau expression levels are increased in the cerebrospinal fluid and serum of nearly all AD patients. Therefore, AβO and tau have become the most promising targets for the diagnosis and treatment of AD, and their precise analysis can be used for early diagnosis. To this end, researchers have developed a variety of biosensors in recent years, including colorimetric, fluorescent, electrochemical, and electrochemiluminescent sensors. Although these sensors have been used in AD analysis, the detection of either AβO or tau alone still suffers from limitations in reliability and accuracy. Preparing a platform for the combined analysis of AβO and Tau is an effective way to solve the above problems.

[0004] Electrochemical sensors have been widely used in the detection of disease biomarkers due to their high sensitivity, flexible interface assembly, and simple operation. Furthermore, DNA walkers, which mimic biological protein motors and are molecular machines capable of autonomous mechanical motion at the nanoscale, have demonstrated robust recognition and signal amplification capabilities in fabricated biosensors. For protein-responsive biosensors, protein affinity sequences are typically incorporated into DNA walkers for target recognition and signal transduction. However, target protein residues at the interface may sterically hinder the movement of the DNA walker, and the swinging arm motion range of DNA walkers fixed to the interface is limited, preventing them from fully realizing their signal amplification capabilities.

[0005] However, traditional interface-anchored DNA walkers usually have a fixed swing arm range and preset track, which makes the sensor system complicated and limits its application in more scenarios.

[0006] Currently, there is a lack of a preparation method and application of a double-nicking enzyme-driven double-free-foot DNA walker biosensor. Summary of the Invention

[0007] In order to overcome the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a sensitive detection double-nicking enzyme-driven double-free-foot DNA walker biosensor and its preparation method and application.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows: A method for preparing a double-nicking enzyme-driven double-free-foot DNA walker biosensor of the present invention comprises the following steps:

[0009] (1) The biotinylated AβO aptamer T1 and the AβO DNA walker W1 were mixed in a molar ratio of 1:1 to form dsDNA T1W1.

[0010] The biotinylated Tau aptamer T2 was mixed with the Tau DNA walker W2 at a molar ratio of 1:1 to form dsDNA T2W2.

[0011] Strep-MB binds to T1W1 and T2W2 to form nanoparticles with recognition and capture functions. In the presence of AβO and Tau, the AβO DNA walker W1 and Tau DNA walker W2 are released under kinetic competition due to the competitive binding of the biotinylated AβO aptamer T1 and the biotinylated Tau aptamer T2 to AβO and Tau.

[0012] (2) AβO DNA walker W1 and Tau DNA walker W2 can hybridize with the AβO anchor chain A1 carrying methylene blue MB and the Tau anchor chain A2 carrying ferrocene FC on the gold electrode surface to form dsDNA containing Nt.A1WI and Nb.BbvCI cleavage sites, thereby preparing a double-nicking enzyme-driven free-foot DNA walker biosensor.

[0013] Furthermore, in step (1), the gene sequence of the biotinylated AβO aptamer T1 is the nucleotide sequence shown in SEQ ID No. 1; the gene sequence of the biotinylated Tau aptamer T2 is the nucleotide sequence shown in SEQ ID No. 2; the gene sequence of the AβO DNA walker W1 is the nucleotide sequence shown in SEQ ID No. 3; and the gene sequence of the Tau DNA walker W2 is the nucleotide sequence shown in SEQ ID No. 4.

[0014] Furthermore, in step (1), T1, W1 and T2, W2 were mixed at a molar ratio of 1:1:1:1 to a final concentration of 1 μM and incubated at 37° C. for 1 h to form dsDNA T1W1 and T2W2.

[0015] Furthermore, in step (2), A1 carrying the MB tag and A2 carrying the FC tag were added dropwise to the gold electrode surface at a final concentration of 1 μM and incubated at 4°C for 8 h. Detection instrument: Electrochemical current and electrochemical impedance spectroscopy were detected using an electrochemical workstation (CH manufacturer), instrument name: CHI760.

[0016] Furthermore, in step (2), the AβO anchor chain A1 carries the MB tag, and the Tau anchor chain A2 carries the FC tag. The MB tag and the FC tag are removed from the surface of the gold electrode by nicking enzyme cleavage, and the freed bipedal DNA walker continues to move on the electrode surface, generating a cyclic amplification effect; therefore, when AβO and Tau are added, the DNA walker W chain is released, and with the assistance of the nicking enzyme, the substrate is cyclically cleaved on the electrode surface, generating an amplified electrochemical signal.

[0017] Furthermore, in step (2), 0.5 μL Nt.A1WI and 0.5 μL Nb.BbvCI were simultaneously added dropwise to the magnetically separated W chain on a gold electrode at 37° C. for a reaction time of 1 h.

[0018] The double-nicking enzyme-driven double-free-foot DNA walker biosensor prepared by the preparation method of the present invention.

[0019] Application of the double-nicking enzyme-driven double-free-foot DNA walker biosensor of the present invention in detecting biomarkers related to Alzheimer's disease.

[0020] Beneficial effects: The sensor of the present invention can not only simultaneously and sensitively detect AβO and Tau, but also make the diagnosis of AD more accurate in a complementary manner.

[0021] Compared with the prior art, the present invention has the following advantages: (1) The electrochemical sensor of the present invention uses an electrode as a reaction interface and utilizes a double-nicking enzyme-powered bipedal DNA walker to achieve signal amplification for sensitive analysis of AβO and Tau. First, the DNA bipedal is released through the competitive binding between the biotinylated aptamer on the functional magnetic beads (MBs) and AβO and Tau. Then, the free DNA bipedal walks on the electrode surface with the assistance of the double-nicking enzyme and releases the substrates of AβO and Tau from the electrode surface, resulting in a one-to-many signal amplification effect.

[0022] (2) The sensor prepared by the present invention achieved satisfactory accuracy and reliability in detecting AβO and Tau in clinical samples. This invention provides a competitive tool for the accurate early diagnosis of AD and the diagnosis of Alzheimer's disease (differentiation between AD and normal subjects), and may have broad application prospects in the future.

[0023] (3) In this method, bifunctional magnetic nanoparticles are used to identify and capture Aβ oligomers (AβO) and Tau, and release free bipedal DNA walkers. When the DNA walkers move freely on the electrode surface, Nt.A1WI and Nt.BbvCI cyclically cut double-stranded DNA and release the two substrate chains, resulting in significant signal amplification. In addition, it has been confirmed that this method can distinguish between normal subjects and AD patients in actual samples. The excellent performance of this biosensor makes it more promising in the clinical diagnosis of AD patients and prognosis assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] For ease of explanation, the present invention is described in detail with reference to the following specific embodiments and accompanying drawings;

[0025] Figure 1 This is a diagram of a double-nicking enzyme-driven double-free-foot DNA walker for AβO and Tau detection according to the present invention;

[0026] Figure 1 a is a diagram of the dual-functional magnetic nanoparticles of the present invention identifying and capturing AβO and Tau and releasing a double-free-foot DNA walker;

[0027] Figure 1 b is a diagram showing the bipedal DNA walker of the present invention walking on the gold electrode surface, and the nicking enzyme cleaves the substrates of AβO and Tau to release MB and FC signal labels.

[0028] Figure 2 This is a diagram of PAGE verification of the binding of the aptamer to the target and the cleavage ability of the nicking enzyme of the present invention.

[0029] Figure 2A is a graph showing the binding properties of AβO to T1 and the cleavage ability of Nt.A1WI of the present invention. Lane 1: T1; Lane 2: A mixture of T1 and AβO; Lane 3: T1W1; Lane 4: A mixture of T1W1 and AβO; Lane 5: W1A1; Lane 6: A mixture of W1A1 and Nt.A1WI.

[0030] Figure 2 B shows the binding properties of Tau and T2 and the cleavage ability of Nb.BbvCI according to the present invention. Lane 1: T2; Lane 2: T2 and Tau mixture; Lane 3: T2W2; Lane 4: T2W2 and Tau mixture; Lane 5: W2A2; Lane 6: W2A2 and Nb.BbvCI mixture. PAGE analysis of DNA strands at 250 nM, AβO (50 μg / mL), and Tau (20 μg / mL).

[0031] Figure 2 C is the EIS graph of the sensor assembled on the electrode surface of the present invention:

[0032] a. Bare gold electrode (GE); b. GE+A1; c. GE+A1+MCH; d. GE+A1+MCH+W1; e. GE+A1+MCH+W1+Nt.A1WI. Figure 2 D is a feasibility diagram of the DPV of the present invention for simultaneous detection of AβO and Tau;

[0033] EIS: a.GE; b.GE+A2; c.GE+A2+MCH; d.GE+A2+MCH+W2; E.GE+A2+MCH+W2+Nb.BbvCI. (E) DPV: a.GE+A1+A2+MCH; b.GE+A1+A2+MCH+Nt.A1WI+Nb.BbvCI+AβO+Tau (A1 and A2 concentration, 1 μM; AβO concentration, 20×10 3 pg / mL; Tau concentration, 1×10 3 pg / mL).

[0034] Figure 3 This is a sensitivity experiment diagram for simultaneous detection of AβO and Tau in the present invention;

[0035] Figure 3 A is the AβO of different concentrations (0, 20, 200, 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 , 2×10 7 pg / mL) and Tau (0, 1, 10, 100, 1×10 3 , 1×104 , 1×10 5 , 1×10 6 pg / mL) incubation DPV curve;

[0036] Figure 3 B is a linear relationship diagram of the peak current of the DPV response of the present invention and the AβO concentration.

[0037] Figure 3 C is a linear relationship diagram of the peak current of the DPV response of the present invention and Tau concentration. Target value, SD, n=3.

[0038] Figure 4 This is a diagram showing the specificity and stability of the present invention's simultaneous detection of AβO and Tau;

[0039] Figure 4 A is a schematic diagram of the dual-functional magnetic beads of the present invention for capturing AβO and Tau and releasing free bipedal DNA walkers (top), and the selective response of this method to AβO (200 μg / mL) and Tau (10 μg / mL) and several other serum proteins (400 μg / mL) (bottom).

[0040] Figure 4 B is the peak current intensity of DPV obtained by detecting different concentrations of AβO in PBS, 10% HS and aCSF in the present invention.

[0041] Figure 4 C is the peak current intensity of the DPV of the present invention by detecting different concentrations of Tau in PBS, 10% HS and aCSF. Error bars, SD, n=3. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples. It should be understood that these examples are provided for illustration and exemplification of the present invention and are not intended to limit the scope of the present invention in any form.

[0043] Example 1

[0044] The preparation method of a double-nicking enzyme-driven double-free-foot DNA walker biosensor of the present invention comprises the following steps:

[0045] (1) The biotinylated AβO aptamer T1 and the AβO DNA walker W1 were mixed in a molar ratio of 1:1 to form dsDNA T1W1.

[0046] The biotinylated Tau aptamer T2 was mixed with the Tau DNA walker W2 at a molar ratio of 1:1 to form dsDNA T2W2.

[0047] Strep-MB combines with T1W1 and T2W2 to form nanoparticles with recognition and capture functions; in the presence of AβO and Tau, due to the competitive binding of aptamers T (biotinylated AβO aptamer T1 and biotinylated Tau aptamer T2) to AβO and Tau, AβO DNA walker W1 and Tau DNA walker W2 are released under kinetic competition; the gene sequence of the biotinylated AβO aptamer T1 is the nucleotide sequence shown in SEQ ID No.1; the gene sequence of the biotinylated Tau aptamer T2 is the nucleotide sequence shown in SEQ ID No.2; the gene sequence of the AβO DNA walker W1 is the nucleotide sequence shown in SEQ ID No.3; and the gene sequence of the Tau DNA walker W2 is the nucleotide sequence shown in SEQ ID No.4.

[0048] T1, W1 and T2, W2 were mixed at a molar ratio of 1:1:1:1 to a final concentration of 1 μM and incubated at 37° C. for 1 h to form dsDNA T1W1 and T2W2.

[0049] (2) AβO DNA walkers W1 and Tau DNA walkers W2 can hybridize with AβO anchor chain A1 carrying methylene blue (MB) and Tau anchor chain A2 carrying ferrocene (FC) on the gold electrode surface to form dsDNA containing Nt.A1WI and Nb.BbvCI cleavage sites. A1 (carrying MB) and A2 (carrying FC) were added to the gold electrode surface at a final concentration of 1 μM and incubated at 4°C for 8 h (CHI760, CH Instruments). A double-nicking enzyme-driven free-foot DNA walker biosensor was prepared.

[0050] The MB and FC tags are removed from the surface of the gold electrode by nicking enzyme cleavage, and the newly freed bipedal DNA walker continues to move on the electrode surface, producing a cyclic amplification effect; therefore, when AβO and Tau are added, the DNA walker W is released, and with the assistance of nicking enzyme, it cyclically cuts the substrate on the electrode surface, generating an amplified electrochemical signal.

[0051] 0.5 μL Nt.A1WI and 0.5 μL Nb.BbvCI were added dropwise simultaneously with the magnetically separated W chains onto a gold electrode at 37°C for 1 h.

[0052] The double-nicking enzyme-driven double-free-foot DNA walker biosensor prepared by the preparation method of the present invention.

[0053] Application of the double-nicking enzyme-driven double-free-foot DNA walker biosensor of the present invention in detecting biomarkers related to Alzheimer's disease.

[0054] Test Example 1

[0055] Experimental part

[0056] 1.1 Materials and Instruments

[0057] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-mercapto-1-hexanol (MCH), hexaammineruthenium(III) chloride, and ultrapure water were provided by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Phosphate buffer saline (PBS), dimethyl sulfoxide (DMSO), and Coomassie Brilliant Blue R-250 were provided by Sangon Biotech Co., Ltd. (Shanghai, China). Streptavidin-coated MBs (strep-MB) were purchased from Beaver Biopharmaceuticals (Suzhou, China). Aβ peptide and Tau were provided by Abcam Trading Co., Ltd. Nt.A1WI and Nb.BbvCI were provided by Biolabs Technology Co., Ltd. (Beijing, China). Hexafluoroisopropanol (HFIP) was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). All oligonucleotides used in this work were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0058] Transmission electron microscopy (TEM) imaging was performed using a JEOL JEM-1400 microscope. Gel imaging was performed using a ChemiScope 6200 (Shanghai, China). Zeta potential analysis was performed using a Zetasizer (Malvern, UK). Electrochemical measurements were performed using a CHI 760E electrochemical workstation (CH Instruments, China).

[0059] 1.2 Preparation of AβO

[0060] AβO was prepared according to a slightly modified method from the literature. First, Aβ peptide was dissolved to 1 mg / mL with HFIP, and the solution was incubated at room temperature for 1 hour, vortexing every 20 minutes. Subsequently, the solution was sonicated in a water bath sonicator for 10 minutes. Then, the solution was evaporated in an air drying oven overnight. Aβ monomer (AβM) was dissolved in DMSO. After incubating the AβM solution at 25°C for 24 hours and 7 days, AβO and Aβ fiber (AβF) were finally obtained. The three forms of Aβ obtained were placed at 80°C for use.

[0061] 1.3PAGE

[0062] 8% PAGE was performed using 1×TBE buffer (89 mM Tris-HCl, 2 mM EDTA, pH 8.0). The loading sample was prepared by mixing 10 μL of nucleic acid sample, 1 μL of Gel Red dye, and 2 μL of 6× loading buffer, and electrophoresis was performed at 100 V for 45 min.

[0063] 1.4 Preparation of working electrode

[0064] The gold electrode was first cleaned and then modified. Briefly, the bare electrode was soaked in piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) for 10 minutes and then polished with 1 μm and 0.3 μm aluminum powder, respectively. Subsequently, the electrode was sonicated in anhydrous ethanol and double-distilled water for 5 minutes and then immersed in 50% H₂NO₃ for 5 minutes. The electrode was activated in H₂SO₄ (0.5 M) solution. The probe was incubated in TCEP (100 mM) at 25°C for 1 hour to remove disulfide bonds. AβO-anchor and Tau-anchor were incubated on the bare electrode at a final concentration of 1 μM for 8 hours. Finally, the electrode was blocked with MCH (1 mM) solution for 30 minutes and used.

[0065] 1.5 Electrochemical measurements

[0066] Electrochemical measurements were performed at room temperature using an electrochemical workstation (CHI760, CH Instruments). A conventional three-electrode system consisting of a gold working electrode, a saturated calomel reference electrode, and a counter electrode was used. Electrochemical impedance spectroscopy (EIS) was performed in a mixture of 5 mM Fe(CN)63- / 4- and KNO3 (1 M). The EIS operating parameters were as follows: frequency range, 0.01–10 kHz; amplitude, 5 mV. Differential pulse voltammetry (DPV) was performed in PBS (pH 7.4). The DPV operating parameters were as follows: potential range, -0.6 V–0.6 V; amplitude, 50 mV.

[0067] result

[0068] 2.1 Working Mechanism

[0069] The present invention has developed an electrochemical platform for the combined detection of AβO and Tau, as shown in Scheme 1. First, a biotinylated AβO aptamer (T1) and a biotinylated Tau aptamer (T2) are pre-mixed with an AβO DNA walker (W1) and a Tau DNA walker (W2) in equal proportions to form dsDNA T1W1 and T2W2. Strep-MB binds to T1W1 and T2W2 to form nanoparticles with recognition and capture functions. In the presence of AβO and Tau, W (W1 and W2) is released under kinetic competition due to the competitive binding of T (T1 and T2) with AβO and Tau. W1 and W2 can then hybridize with the AβO anchor chain (A1) carrying methylene blue (MB) and the Tau anchor chain (A2) carrying ferrocene (FC) on the gold electrode surface, forming dsDNA containing Nt.A1WI and Nb.BbvCI cleavage sites. Finally, the MB and FC tags are removed from the gold electrode surface by nicking enzyme cleavage, and the newly freed bipedal DNA walker continues to move on the electrode surface, generating a cyclic amplification effect. Therefore, when AβO and Tau are added, W is released and, with the assistance of nicking enzyme, cyclically cleaves the substrate on the electrode surface, generating an amplified electrochemical signal.

[0070] Figure 1 This is a diagram of the double-nicking enzyme-driven double-free-foot DNA walker for AβO and Tau detection of the present invention.

[0071] 2.2 Method feasibility verification

[0072] In order to verify the feasibility of the proposed dual-nicking enzyme-powered bipedal DNA walker for AβO and Tau detection, the present invention verified several steps involved. First, PAGE was used to verify the affinity of the aptamer to the protein and the nicking enzyme's cleavage ability. Figure 2 A and Figure 2 As shown in Figure B, the bright bands in lane 1 indicate the presence of T1 and T2, respectively. The dark band at the bottom of lane 2 demonstrates the consumption of T1 and T2, while the bright band at the top demonstrates the formation of T1-AβO and T2-Tau complexes, respectively. The bright bands in lane 3 demonstrate the presence of double-stranded DNA T1W1 and T2W2, respectively. The dark band at the bottom of lane 4 demonstrates the consumption of T1W1 and T2W2, while the bright band at the top demonstrates the formation of T1W1-AβO and T2W2-Tau complexes, respectively. The T1W1-AβO complex in lane 4 remains in the gel wells and is lost during imaging. The bright bands in lane 5 demonstrate the presence of dsDNA W1A1 and W2A2, respectively. The dark band at the bottom of lane 6 demonstrates the cleavage of W1A1 and W2A2 by Nt.A1WI and Nb.BbvCI, respectively. These results indicate that the selected AβO and Tau aptamers have good affinity for AβO and Tau, and the formed specific duplexes can be cleaved by Nt.A1WI and Nb.BbvCI nicking enzymes.

[0073] Then, electrochemical impedance spectroscopy was used to verify the performance of the electrode at different experimental stages. Figure 2 C and Figure 2 As shown in Figure D, compared to the bare electrode, the electrode assembled with A1 and A2 anchor chains showed increased charge transfer resistance (Rct), indicating that A1 and A2 were successfully modified. The subsequent assembly of W1 and W2 resulted in an increase in Rct, indicating that W was successfully assembled. However, when Nt.A1WI and Nb.BbvCI were added, Rct decreased due to the cleavage of dsDNA by the nickase. These results demonstrate the successful assembly and disassembly of nucleic acid chains on the gold electrode surface.

[0074] Finally, the feasibility of the sensor for simultaneous detection of AβO and Tau was verified by differential pulse voltammetry (DPV). As shown in Figure 2E, when anchor chains A (A1 and A2) were assembled on the gold electrode, DPV detected a high current signal. When the nicking enzyme (0.5 μL Nt.A1WI and 0.5 μL Nb.BbvCI) was added to the AβO (20×10 3 pg / mL) and Tau (1×10 3 The above results confirmed the feasibility and effectiveness of this method.

[0075] Figure 2 This is a diagram of PAGE verification of the binding of the aptamer to the target and the cleavage ability of the nicking enzyme of the present invention.

[0076] 2.3 Sensitivity of electrochemical methods

[0077] The sensitivity of the method was investigated by analyzing the peak currents of AβO and Tau at different concentrations. The peak current of the DPV reaction decreased with increasing concentrations of AβO (0–20 μg / mL) and Tau (0–1 μg / mL). Figure 3 A linear relationship between peak current and the logarithm of AβO and Tau concentrations was obtained within the dynamic ranges of 20 pg / mL–20 μg / mL and 1 pg / mL–1 μg / mL ( Figure 3 B and Figure 3 C). The linear equations were: I = -0.0699logC(AβO) - 0.0114 (R² = 0.989) and I = -0.0734logC(Tau) - 0.3227 (R² = 0.959), respectively. I represents the peak current at different AβO and Tau concentrations. The peak current of the DPV showed a good linear relationship with the logarithmic values ​​of the AβO and Tau concentrations. At a signal-to-noise ratio of 3σ, the detection limits for AβO and Tau were 1.28 pg / mL and 0.04 pg / mL, respectively, meeting the sensitivity requirements for clinical diagnosis of AD.

[0078] Figure 3 This is a sensitivity experiment diagram for simultaneous detection of AβO and Tau in the present invention;

[0079] Figure 3 A is the AβO of different concentrations (0, 20, 200, 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 , 2×10 7 pg / mL) and Tau (0, 1, 10, 100, 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 pg / mL) incubation DPV curve;

[0080] Figure 3 B is a linear relationship diagram of the peak current of the DPV response of the present invention and the AβO concentration.

[0081] Figure 3 C is a linear relationship diagram of the peak current of the DPV response of the present invention and Tau concentration. Target value, SD, n=3.

[0082] 2.4 Specificity and stability of electrochemical methods

[0083] Specificity is an important factor in evaluating analytical performance. In this study, other AD markers (AβM, AβF) and serum components (BSA, IgG) were selected as interfering substances because they may be present in biological samples of AβO and Tau ( Figure 4 A). In the presence of 200 μg / mL AβO and 10 μg / mL Tau, a significant decrease in DPV current signal was observed, while in the presence of 400 μg / mL AβM, AβF, BSA, and IgG, no significant change in DPV current signal was observed ( Figure 4 A), confirming that the method has good selectivity. The stability of the method was verified by detecting different concentrations of AβO and Tau in 10% human serum (HS) and artificial cerebrospinal fluid (aCSF). In different matrices, the DPV peak current intensity decreased with the addition of AβO and Tau. There was almost no difference between different substrates, indicating that the method has high stability in detecting biological samples ( Figure 4 B and Figure 4 C).

[0084] Figure 4 This is a diagram showing the specificity and stability of the present invention's simultaneous detection of AβO and Tau;

[0085] Figure 4 A is a schematic diagram of the dual-functional magnetic beads of the present invention for capturing AβO and Tau and releasing free bipedal DNA walkers (top), and the selective response of this method to AβO (200 μg / mL) and Tau (10 μg / mL) and several other serum proteins (400 μg / mL) (bottom).

[0086] Figure 4 B is the peak current intensity of DPV obtained by detecting different concentrations of AβO in PBS, 10% HS and aCSF in the present invention.

[0087] Figure 4 C is the peak current intensity of the DPV of the present invention by detecting different concentrations of Tau in PBS, 10% HS and aCSF. Error bars, SD, n=3.

[0088] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. A method for preparing a double-nicking enzyme-driven double-free-foot DNA walker biosensor, characterized in that The steps include: (1) The biotinylated AβO aptamer T1 and the AβO DNA walker W1 were mixed in a molar ratio of 1:1 to form dsDNA T1W1. The biotinylated Tau aptamer T2 was mixed with the Tau DNA walker W2 at a molar ratio of 1:1 to form dsDNA T2W2. Streptavidin-coated magnetic beads Strep-MB bind to T1W1 and T2W2 to form nanoparticles with recognition and capture functions. In the presence of AβO and Tau, the AβO DNA walker W1 and the Tau DNA walker W2 are released under kinetic competition due to the competitive binding of the aptamer T biotinylated AβO aptamer T1 and the biotinylated Tau aptamer T2 to AβO and Tau. The gene sequence of the biotinylated AβO aptamer T1 is the nucleotide sequence shown in SEQ ID No. 1; the gene sequence of the biotinylated Tau aptamer T2 is the nucleotide sequence shown in SEQ ID No. 2; the gene sequence of the AβO DNA walker W1 is the nucleotide sequence shown in SEQ ID No. 3; and the gene sequence of the Tau DNA walker W2 is the nucleotide sequence shown in SEQ ID No.

4. (2) AβO DNA walkers W1 and Tau DNA walkers W2 can hybridize with the AβO anchor chain A1 carrying methylene blue MB and the Tau anchor chain A2 carrying ferrocene FC on the gold electrode surface to form dsDNA containing Nt.A1WI and Nb.BbvCI cleavage sites, and the corresponding sites are located on the anchor chain A1 and anchor chain A2; the methylene blue MB label and FC label are removed from the surface of the gold electrode by nicking enzyme cleavage, and the newly freed bipedal DNA walkers continue to move on the electrode surface, producing a cyclic amplification effect, thereby preparing a dual-nicking enzyme-driven free-footed DNA walker biosensor.

2. The method for preparing the double-nicking enzyme-driven double-free-foot DNA walker biosensor according to claim 1, characterized in that: In step (1), T1, W1 and T2, W2 were mixed at a molar ratio of 1:1:1:1 to a final concentration of 1 μM and incubated at 37° C. for 1 h to form dsDNA T1W1 and T2W2.

3. The method for preparing the double-nicking enzyme-driven double-free-foot DNA walker biosensor according to claim 1, characterized in that: In step (2), A1 carrying a methylene blue MB label and A2 carrying a FC label were added dropwise to the surface of the gold electrode at a final concentration of 1 μM and incubated at 4° C. for 8 h.

4. The method for preparing the double-nicking enzyme-driven double-free-foot DNA walker biosensor according to claim 1, characterized in that: In step (2), the AβO anchor chain A1 carries the MB tag, and the Tau anchor chain A2 carries the FC tag. The methylene blue MB tag and FC tag are removed from the surface of the gold electrode by nicking enzyme cleavage, and the freed bipedal DNA walker continues to move on the electrode surface, producing a cyclic amplification effect; therefore, when AβO and Tau are added, the DNA walker W chain is released, and with the assistance of the nicking enzyme, the substrate is cyclically cleaved on the electrode surface, generating an amplified electrochemical signal.

5. The method for preparing the double-nicking enzyme-driven double-free-foot DNA walker biosensor according to claim 1, characterized in that: In step (2), 0.5 μL Nt.A1WI and 0.5 μL Nb.BbvCI were added dropwise simultaneously with the magnetically separated W chains on a gold electrode at 37°C for 1 h.

6. The double-nicking enzyme-driven double-free-foot DNA walker biosensor prepared by the preparation method of claim 1.

7. Use of the double-nicking enzyme-driven double-free-foot DNA walker biosensor according to claim 1 in detecting biomarkers related to Alzheimer's disease.

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