Biosensor for detecting Alzheimer's disease marker as well as construction method and application of biosensor

Abstract

The invention discloses a biosensor for detecting Alzheimer's disease markers as well as a construction method and application thereof, and relates to the technical field of biosensors. The construction method comprises the following steps: after a glassy carbon electrode is cleaned, dropwise adding a RuSi NPs (at) CS compound on the surface of the glassy carbon electrode to obtain a RuSi NPs (at) CS / GCE electrode; and immersing the RuSi NPs coated CS / GCE electrode into a PHL: S double-chain solution, and carrying out an incubation reaction to obtain the biosensor. The RuSi NPs (at) CS compound is obtained by carrying out mixed reaction on ruthenium-silicon nanoparticles and chitosan; pHL: S double chains contained in the PHL: S double-chain solution are obtained by carrying out incubation reaction on carboxyl-activated PHL chains and S chains. The biosensor has the advantages of high accuracy, strong sensitivity, good selectivity and the like, and can be applied to accurate detection of the Alzheimer's disease marker A beta oligomer.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, in particular to a biosensor for detecting Alzheimer's disease markers, a construction method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease that affects the central nervous system. It is usually caused by abnormally deposited β-amyloid (Aβ) in the nervous system and abnormally accumulated tau protein in neurofibrillary tangles, resulting in the loss of brain neurons and ultimately causing brain atrophy and memory loss. In particular, the intermediate Aβ oligomers formed during this process are considered to be the most cytotoxic entities. Compared with healthy people, the level of Aβ oligomers in the cerebrospinal fluid of Alzheimer's disease patients is significantly increased. Therefore, Aβ oligomers are widely used as biomarkers for the early diagnosis of Alzheimer's disease. Given the association between the content of Aβ oligomers and the progression of Alzheimer's disease, it is crucial to design an accurate and sensitive method to evaluate the content of Aβ oligomers.

[0003] Researchers have designed various signal amplification strategy-based schemes for quantitative analysis of the content of Aβ oligomers, such as porous materials as sensing platforms, silver nanoparticles as dual-regulating co-reaction accelerators, electrocatalysis of covalent organic framework nanocomposites, hybridization chain reaction-driven DNA walkers, and DNA enzyme-driven DNA strand dissociation. For DNA assembly technology, cascaded DNA cycles have great application potential in constructing highly sensitive, low-background-signal, and programmable biosensors in a short time. The present invention aims to develop a biosensor for detecting Alzheimer's disease markers to achieve efficient detection of Aβ oligomers. Summary of the Invention

[0004] The purpose of the present invention is to provide a biosensor for detecting Alzheimer's disease markers, a construction method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. This biosensor has the advantages of high accuracy, strong sensitivity, good selectivity, etc., and can be applied to the accurate detection of the Alzheimer's disease marker Aβ oligomers, and has important application value in the fields of clinical diagnosis and drug research of Alzheimer's disease.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a construction method of a biosensor for detecting Alzheimer's disease markers, comprising the following steps:

[0007] After the glassy carbon electrode is cleaned, a RuSi NPs@CS composite is dropped onto its surface to obtain a RuSi NPs@CS / GCE electrode;

[0008] Immerse the RuSi NPs@CS / GCE electrode into the PHL:S double-stranded solution for incubation reaction to obtain the biosensor;

[0009] The RuSi NPs@CS composite is obtained by mixing and reacting ruthenium-silicon nanoparticles and chitosan;

[0010] The PHL:S double-stranded solution contains PHL:S double strands obtained by incubating carboxyl-activated PHL strands and S strands;

[0011] The nucleotide sequence of the PHL strand is shown in SEQ ID NO.3; the nucleotide sequence of the S strand is shown in SEQ ID NO.4.

[0012] Further, the cleaning treatment includes the steps of polishing with aluminum powder and then ultrasonic cleaning with ethanol and deionized water.

[0013] Further, the carboxyl-activated PHL strand is obtained by reacting the PHL strand with a mixture containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

[0014] Further, the preparation method of the RuSi NPs@CS composite includes the following steps:

[0015] Add n-hexanol, Triton-X-100 and Ru(bpy)3 2+ to cyclohexane, stir and react to form an oil-in-water system, then add tetraethyl orthosilicate and NH3·H2O, after reaction, add a demulsifier to demulsify, centrifuge and collect to obtain ruthenium-silicon nanoparticles, and mix and react the ruthenium-silicon nanoparticles and chitosan to obtain the RuSi NPs@CS composite.

[0016] Further, the demulsifier is acetone.

[0017] The present invention also provides a biosensor constructed according to the above construction method.

[0018] The present invention also provides the application of the above biosensor in the preparation of a kit for detecting Alzheimer's disease biomarker Aβ oligomers.

[0019] The present invention also provides a kit for detecting Alzheimer's disease biomarker Aβ oligomers, including the above biosensor.

[0020] Further, the kit also includes M chain, F chain, H1-Fc chain, H2-Fc chain, H3-Fc chain, H4-Fc chain and 3'-end amino-functionalized Aβ oligomer aptamer;

[0021] The nucleotide sequence of the Aβ oligomer aptamer is shown in SEQ ID NO.1;

[0022] The nucleotide sequence of the M chain is shown in SEQ ID NO.2;

[0023] The nucleotide sequence of the F chain is shown in SEQ ID NO.5;

[0024] The nucleotide sequence of the H1-Fc chain is shown in SEQ ID NO.6;

[0025] The nucleotide sequence of the H2-Fc chain is shown in SEQ ID NO.7;

[0026] The nucleotide sequence of the H3-Fc chain is shown in SEQ ID NO.8;

[0027] The nucleotide sequence of the H4-Fc chain is shown in SEQ ID NO.9.

[0028] Furthermore, the kit further includes carboxylated magnetic beads.

[0029] The present invention discloses the following technical effects:

[0030] The present invention constructs a dual-mode ratiometric biosensor for detecting the content of Alzheimer's disease biomarker Aβ oligomers. This biosensor is based on magnetic bead separation technology and specific binding of aptamers, converting the detection of the content of Alzheimer's disease biomarker Aβ oligomers into nucleic acid analysis; using ruthenium-silicon nanoparticles (RuSi NPs) as the electrochemiluminescence active substrate and ferrocene as the quencher and electrochemical beacon for in-situ quenching of electrochemiluminescence signals and electrochemical response; relying on entropy-driven DNA cycling to activate hybridization chain reaction to achieve cyclic amplification of signal molecules; based on the ratiometric response of electrochemical and electrochemiluminescence dual-mode signals, thereby realizing highly sensitive and accurate detection of Alzheimer's disease biomarker Aβ oligomers.

[0031] The present invention uses magnetic bead separation technology and aptamer binding for signal transduction of Aβ oligomers, improving the selectivity of the sensing system; using RuSi NPs as the electrochemiluminescence active substrate and combining with in-situ quenching of ferrocene to reduce the interference of background signals and improve signal output and response; based on the signal amplification strategy of entropy-driven DNA cycling to activate hybridization chain reaction, improving the diversity of substrate selection and detection sensitivity; due to the reverse change trend of electrochemical and electrochemiluminescence dual-modes, improving the accuracy of the biosensor. Therefore, the biosensor prepared by the present invention has the advantages of high accuracy, strong sensitivity, good selectivity, etc., and can be applied to the accurate detection of Alzheimer's disease biomarker Aβ oligomers, having important application value in the fields of clinical diagnosis and drug research of Alzheimer's disease. Brief Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 It is a schematic diagram of the preparation process and sensing mechanism of the Aβ oligomer dual-mode ratio-type biosensor for Alzheimer's disease markers of the present invention;

[0034] Figure 2 It is a transmission electron microscope (SEM) image of RuSi NPs;

[0035] Figure 3 It is an X-ray photoelectron spectroscopy (XPS) image of RuSi NPs;

[0036] Figure 4 It is an XPS image of the Ru 3d orbital electrons of RuSi NPs;

[0037] Figure 5 It is the ultraviolet-visible (UV-vis) absorption spectrum of RuSi NPs and Ru(bpy)3 2+ ;

[0038] Figure 6 It is a feasibility gel electrophoresis image of Aβ oligomer signal transduction and entropy-driven DNA cycle-activated hybridization chain reaction; among them, lane 1 represents the aptamer of Aβ oligomer, lane 2 represents the mixture of aptamer and M, lane 3 represents the supernatant after the aptamer:M double strand is treated with Aβ oligomer, lane 4 represents the PHL:S double strand, lane 5 represents the PHL:S double strand treated with the released M strand, lane 6 represents the F strand, lane 7 represents lane 5 further treated with the F strand, lane 8 represents the H1-Fc strand, lane 9 represents the reaction of lane 7 with the H1-Fc strand, and lane 10 represents the reaction of lane 7 with the mixture of H1-Fc strand, H2-Fc strand, H3-Fc strand and H4-Fc strand;

[0039] Figure 7 It is an electrochemical impedance (EIS) characterization image of the sensing substrate interface construction process; among them, a represents the bare GCE, b represents RuSi NPs@CS / GCE, c represents PHL:S / RuSi NPs@CS / GCE, d represents the sensing substrate after the reaction of c with the M strand transduced by Aβ oligomer, e represents the sensing substrate after the reaction of d with the F strand, and f represents the sensing substrate after the further reaction of e with the mixture containing H1-Fc strand, H2-Fc strand, H3-Fc strand and H4-Fc strand;

[0040] Figure 8 Electrochemiluminescence-time response diagram for the construction process of the sensing substrate interface; wherein, a represents RuSi NPs@CS / GCE, b represents PHL:S / RuSi NPs@CS / GCE, c represents the sensing substrate after the reaction of b with the M chain transduced by Aβ oligomers, d represents the sensing substrate after the reaction of c with the F chain, and e represents the sensing substrate after d further reacts with the mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain and H4-Fc chain;

[0041] Figure 9 Electrochemical response diagram before and after the reaction of the constructed sensing system with the mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain and H4-Fc chain; a represents before the reaction, and b represents after the reaction;

[0042] Figure 10 Electrochemical and electrochemiluminescence response curve diagrams for different concentrations of Aβ oligomers; wherein, the concentrations corresponding to a~i are 0, 50 fM, 100 fM, 500 fM, 1 pM, 10 pM, 100 pM, 1 nM and 10 nM respectively;

[0043] Figure 11 Linear relationship diagram between the logarithm of the ratio of the electrochemical peak current to the electrochemiluminescence signal and the logarithm of the Aβ oligomer concentration;

[0044] Figure 12 Bar chart of the selective response of the biosensor;

[0045] Figure 13 Comparison diagram of the detection results of the Aβ oligomer content in the blood samples of Alzheimer's patients (numbered 1~3) and healthy volunteers by using the biosensor and ELISA method; wherein, the constructed method refers to the method of detecting by using the biosensor of the present invention, and the standard method refers to the ELISA method. Detailed implementation manners

[0046] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0047] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0050] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0051] The sequence information related to this invention is as follows:

[0052] The nucleotide sequence of the Aβ oligomer aptamer (SEQ ID NO.1) is: 5′-GCCTGTGGTGTTGGGGCGGGTGCG-NH2-3′;

[0053] The nucleotide sequence of the M chain (SEQ ID NO.2) is: 5′-CGTCCGCCCCAACACCACATCGAGAAG-3′;

[0054] The nucleotide sequence of the PHL chain (SEQ ID NO.3) is: 5′-COOH-GCTTCATCTTCATCTCCGACACTCGAGATGAAGATGAAGCGGGTAATACTGTGGTGTTGGGGCGGGCG-3′;

[0055] The nucleotide sequence of the S chain (SEQ ID NO.4) is: 5′-CCAACACCACAGTATTACCCATGT-3′;

[0056] The nucleotide sequence of the F chain (SEQ ID NO.5) is: 5′-ATGACTATTGAGTATTACCCGCTTCATCTTCATCTC-3′;

[0057] The nucleotide sequence of the H1-Fc chain (SEQ ID NO.6) is: 5′-Fc-GAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCATCTTCATCTCCG-3′;

[0058] The nucleotide sequence of the H2-Fc chain (SEQ ID NO.7) is: 5′-Fc-GCTTCATCTTCATCTCCGGTTTTGCGGAGATGAAGATGAAGCACGATG-3′;

[0059] The nucleotide sequence of the H3-Fc chain (SEQ ID NO.8) is: 5′-Fc-CAAAACCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCG-3′;

[0060] The nucleotide sequence of the H4-Fc chain (SEQ ID NO.9) is: 5′-Fc-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-3′.

[0061] Based on the magnetic bead separation technology and the specific binding of aptamers, the present invention converts the detection of the content of Alzheimer's disease biomarker Aβ oligomers into nucleic acid analysis; uses ruthenium-silicon nanoparticles (RuSi NPs) as the electrochemiluminescence active substrate, ferrocene as the quencher and electrochemical beacon to perform in-situ quenching of electrochemiluminescence signals and electrochemical responses, significantly reducing the interference of background signals; realizes the cyclic amplification of signal molecules by means of entropy-driven DNA cycle-activated hybridization chain reaction; and based on the dual-mode signal ratio response of electrochemistry and electrochemiluminescence, thereby realizing highly sensitive and accurate detection of Alzheimer's disease biomarker Aβ oligomers (for the schematic diagram of the sensing mechanism of this biosensor, see Figure 1 ) and the specific details are as follows:

[0062] Example 1

[0063] The construction process of a biosensor for detecting Alzheimer's disease biomarkers is as follows:

[0064] First, the glassy carbon electrode (GCE) was polished successively with 0.3 and 0.05 μm aluminum powder, then ultrasonically cleaned with ethanol and deionized water for 5 min, and dried with nitrogen. 10 μL of RuSi NPs@CS complex was dropped onto its surface to obtain the RuSi NPs@CS / GCE electrode.

[0065] Next, 100 μL of PHL strand (5.0 μM) was reacted with a mixture containing 20 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL N-hydroxysuccinimide to obtain the carboxyl-activated PHL strand. Then, it was incubated with 5.0 μM S strand in an equal volume at 37 °C for 1 h to obtain the PHL:S double-stranded solution. The RuSi NPs@CS / GCE electrode was immersed in the PHL:S double-stranded solution and incubated at 37 °C for 60 min to obtain PHL:S / RuSi NPs@CS / GCE, which is the biosensor.

[0066] Among them, the preparation method of the RuSi NPs@CS complex: 1.8 mL of n-hexanol, 1.7 mL of Triton X-100, and 340 μL of Ru(bpy)3 2+ (40 mM) were successively added to 7.5 mL of cyclohexane, and the reaction was carried out under magnetic stirring for 30 min to form an oil-in-water system. Then, 100 μL of tetraethyl orthosilicate and 60 μL of NH3·H2O were successively added, and the reaction was carried out at room temperature for 24 h. Then, 50 mL of acetone was slowly added to break the emulsion, and the generated RuSi NPs were collected by centrifugation (8000 rpm, 5 min) and washed with ethanol and water. Then, 0.1 mg / mL RuSi NPs and 0.5 wt% chitosan (CS) were mixed in an equal volume and sonicated for 5 min to obtain the RuSi NPs@CS complex.

[0067] The usage method of this biosensor is as follows: Alzheimer's disease biomarker Aβ oligomers are added to the aptamer:M / magnetic bead complex for signal transduction. After magnetic separation, the released M strand is collected. Then, PHL:S / RuSi NPs@CS / GCE is reacted with the Aβ oligomer-dependent intermediate M strand and the fuel F strand solution. Subsequently, the sensing substrate is further placed into a mixed solution containing H1-Fc strand, H2-Fc strand, H3-Fc strand, and H4-Fc strand. After incubation, the sensing substrate is collected. Finally, with the incubated sensing substrate as the working electrode, Ag / AgCl electrode as the reference electrode, and platinum wire as the auxiliary electrode, a three-electrode system is formed. The electrochemiluminescence signal is detected in tripropylamine solution, and the electrochemical signal is detected in phosphate buffer under a nitrogen atmosphere. A multifunctional electrochemical and chemiluminescence instrument is used to detect the electrochemiluminescence signal, with a photomultiplier tube at 700 V and a cyclic voltage range of 0 - 1.4 V. An electrochemical workstation records the differential pulse voltammetry (DPV) signal, with a voltage range of 0 - 0.4 V, a pulse amplitude of 25 mV, and a pulse period of 0.5 s. The specific details are as follows:

[0068] (1) Signal transduction of Alzheimer's disease biomarker Aβ oligomers.

[0069] The specific operation is as follows: 0.5 mg / mL carboxylated magnetic beads are added to a mixture containing N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL) and N-hydroxysuccinimide (10 mg / mL), and the reaction is carried out at room temperature for 1 h to obtain surface carboxyl-activated magnetic beads. Then, these magnetic beads are added to 100 μL of 3'-terminally amino-functionalized Aβ oligomer aptamer (2.5 μM), and the reaction is carried out at room temperature for 2 h. Through an amide bond, the aptamer is assembled onto the surface of the magnetic beads. The formed aptamer / magnetic beads are separated by a magnet and washed with a buffer solution, and then placed into 100 μL of M strand (2.5 μM), and the reaction is carried out at 37 °C for 1 h to form an aptamer:M / magnetic bead complex. After magnetic separation and buffer solution washing of this complex, it is added to 100 μL of a buffer solution containing different concentrations of Aβ oligomers, and the reaction is carried out at 37 °C for 2 h. After magnetic separation, the Aβ oligomer signal transduction intermediate M strand is obtained.

[0070] (2) Construct an entropy-driven DNA cycle-activated hybridization chain reaction at the PHL:S / RuSi NPs@CS / GCE electrode interface to obtain a sensing substrate assembled with a DNA long-chain polymer.

[0071] The PHL:S / RuSi NPs@CS / GCE electrode was immersed in 100 μL of a mixed solution containing the Aβ oligomer signal transduction intermediate M strand and 2.5 μM F strand, and incubated at 37 °C for 60 min to activate the entropy-driven DNA cycling reaction, obtaining PHL:F / RuSi NPs@CS / GCE. The PHL:F / RuSi NPs@CS / GCE was placed into 100 μL of a mixed solution containing H1-Fc strand, H2-Fc strand, H3-Fc strand, and H4-Fc strand, and reacted at 37 °C for 1 h to initiate a hybridization chain reaction, forming a DNA long-chain polymer at the electrode interface and introducing a large amount of Fc into the electrode interface. The final concentrations of H1-Fc strand, H2-Fc strand, H3-Fc strand, and H4-Fc strand were all 5 μM.

[0072] (3)Using the sensing substrate obtained in step (2) as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode to form a three-electrode system, detecting the electrochemiluminescence signal in a tripropylamine solution and detecting the electrochemical signal in a nitrogen atmosphere phosphate buffer solution. According to the standard curve between the logarithm of the electrochemistry-electrochemiluminescence signal ratio and the logarithm of the Aβ oligomer concentration, calculate the Aβ oligomer content in the blood sample of Alzheimer's patients. A multifunctional electrochemistry and chemiluminescence instrument was used to detect the electrochemiluminescence response, with the photomultiplier tube at 700 V and the cyclic voltage range of 0 - 1.4 V. An electrochemical workstation was used to record the differential pulse voltammetry (DPV) signal, with the voltage range of 0 - 0.4 V, the pulse amplitude of 25 mV, and the pulse period of 0.5 s.

[0073] Example 2

[0074] The performance of the biosensor prepared in Example 1 was detected in this example, and the specific process is as follows:

[0075] (1)The biosensor of the present invention uses RuSi NPs to improve the electrochemiluminescence signal response, and ferrocene realizes signal in-situ quenching. To verify the successful synthesis of RuSi NPs, the RuSi NPs prepared in Example 1 were characterized. From Figure 2 it can be seen that RuSi NPs have a spherical structure with a particle size of about 90 nm. Figure 3 And Figure 4 shows that the material contains Si, C, Ru, N, and O elements. In addition, Figure 5 shows that RuSi NPs have the same characteristic absorption peaks (295.0 nm and 453.0 nm) as the Ru(bpy)3 2+ molecule. The above results indicate the successful embedding of the Ru(bpy)3 2+ molecule in the silica nanospheres.

[0076] (2) The biosensor of the present invention uses RuSi NPs as the sensing substrate, specifically recognizes Aβ oligomers through aptamers, and activates the hybridization chain reaction by entropy-driven DNA cycling to amplify the signal and improve the detection sensitivity. Therefore, the construction process of the sensing interface and the feasibility of the entropy-driven DNA cycling-activated hybridization chain reaction are monitored and verified. As Figure 6 shown, lane 1 represents the aptamer strand of Aβ oligomers, lane 2 represents the mixture of aptamer and M, and the lagging new band represents the formation of the aptamer:M double strand. Lane 3 represents the supernatant after the aptamer:M double strand is treated with Aβ oligomers, and a new fast-migrating band appears, indicating that the release of the M strand induced by the recognition of Aβ oligomers by the aptamer is feasible. Lane 4 represents the PHL:S double strand, lane 5 represents the PHL:S double strand treated with the released M strand, and two new bands are obtained. The slower band is attributed to the formation of the PHL:M double strand, and the other band indicates the release of the S strand. Lane 6 represents the F strand. When the PHL:M double strand is incubated with the F strand, two bands can be observed in lane 7. The band with a lower mobility represents the formation of the PHL:F double strand, and the faster-migrating band belongs to the release of the M strand. It shows that the M strand can be replaced by the F strand, and the released M strand participates in the reaction again, activating the upstream entropy-driven DNA cycle. Lane 8 represents the H1-Fc strand. When lane 7 reacts with the H1-Fc strand, a new lagging band can be observed in lane 9, indicating that the H1-Fc strand can hybridize with the PHL strand to form the H1:PHL:F complex strand. After adding a mixture of the H1-Fc strand, H2-Fc strand, H3-Fc strand, and H4-Fc strand to lane 7, a clear bright band appears at the top of lane 10, proving the initiation of the downstream hybridization chain reaction and the formation of the DNA long-chain polymer. These observations confirm that the strategy of specific recognition of Aβ oligomers by aptamers and activation of the hybridization chain reaction by entropy-driven DNA cycling is feasible.

[0077] Then, electrochemical impedance, electrochemiluminescence, and electrochemistry are used to characterize the construction of the sensing interface, and the results are as Figures 7-9 shown. As Figure 7 shown by curve a in et , on the surface of the bare GCE, the electron transfer impedance (R et ) is about 351.8 Ω. When RuSiNPs@CS is assembled onto the GCE surface, the R et of the obtained RuSi NPs@CS / GCE electrode increases to 1942.0 Ω ( Figure 7 , curve b), and at the same time, an obvious electrochemiluminescence signal of 14460 a.u. can be observed ( Figure 8 , curve a). When the modified electrode is successively combined with the PHL:S double strand (2427.0 Ω, Figure 7 , curve c in Figure 7Curve d) in, fuel F chain (3522.0 Ω, Figure 7 Curve e) in and a mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain, H4-Fc chain (6139.0 Ω, Figure 7 After incubation with curve f) in, R et gradually increases, attributed to the electrostatic repulsion between the negative charge of the DNA phosphate backbone and [Fe(CN)6] 3- / 4- The corresponding electrochemiluminescence intensity gradually decreases to 13210 a.u. (curve b) in Figure 8 12490 a.u. (curve c) in Figure 8 11482 a.u. (curve d) in Figure 8 and 5810 a.u. (curve e) in Figure 8 The electron and energy transfer between RuSi NPs and ferrocene results in the quenching of the electrochemiluminescence signal. At the same time, the introduction of ferrocene to the electrode interface brings about the appearance of an oxidation peak, and the current intensity reaches 278 nA at 0.17 V (curve b) in Figure 9 Therefore, a reduced electrochemiluminescence signal and an enhanced electrochemical response are obtained, demonstrating the feasibility of the dual-mode ratiometric biosensor. The above-mentioned electrochemical impedance, electrochemiluminescence, and electrochemical results effectively prove the construction process of the electrode sensing interface.

[0078] (3) Different concentrations of Aβ oligomers (0, 50 fM, 100 fM, 500 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM) were used to verify the detection performance of the biosensor according to the usage method described in Example 1.

[0079] The electrochemical peak current and electrochemiluminescence response curves of different concentrations of the Alzheimer's disease biomarker Aβ oligomers are as Figure 10 shown. It can be seen that as the concentration of Aβ oligomers increases, the peak current intensity gradually increases and the electrochemiluminescence intensity decreases accordingly. The linear relationship between the logarithm of the ratio of the electrochemical and electrochemiluminescence signals and the logarithm of the Aβ oligomer concentration is as Figure 11 shown, and the linear regression equation is log EC / ECL = 0.189 logc (M) + 0.416 (R 2 = 0.996). From Figure 12It can be found that after replacing the target Aβ oligomers with amyloid-β fibrils, amyloid-β monomers, immunoglobulin G, and L-cysteine, the electrochemical and electrochemiluminescence responses showed negligible signal changes compared to the blank control. However, at a concentration of 1 nM, after mixing the interfering substances amyloid-β fibrils, amyloid-β monomers, immunoglobulin G, L-cysteine, and the target Aβ oligomers, the electrochemical and electrochemiluminescence signals were not significantly different from those of the individual target Aβ oligomers. These results indicate that the interfering substrates have no effect on the detection of Aβ oligomers. Due to the strong recognition ability of the aptamer for Aβ oligomers, satisfactory specificity for the detection of Aβ oligomers was obtained.

[0080] Example 3

[0081] In this example, blood samples from Alzheimer's disease patients (numbered AD patients 1 to 3) and healthy volunteers were used to verify the detection performance of the biosensor in Example 1. The specific process is as follows: The blood obtained from three Alzheimer's disease patients (numbered AD1 to 3) and one healthy volunteer was centrifuged after natural coagulation, and the supernatant was collected for further use. Then, the detection of Aβ oligomers was performed according to the usage method described in Example 1. Using the linear regression equation in Example 2 for calculation, the concentration of Aβ oligomers in the blood samples was obtained, and the results are as Figure 13 shown. Compared with the results detected by the standard method, the commercial kit ELISA, the concentration error was less than 5%. This indicates that even for complex blood samples, the biosensor designed in the present invention has good detection ability and application.

[0082] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a biosensor for detecting Alzheimer's disease markers, characterized in that, It includes the following steps: After the glassy carbon electrode is cleaned, a RuSi NPs@CS composite is dropped onto its surface to obtain a RuSi NPs@CS / GCE electrode; The RuSi NPs@CS / GCE electrode is immersed in a PHL:S double-stranded solution for an incubation reaction to obtain the biosensor; The RuSi NPs@CS composite is obtained by mixing ruthenium-silicon nanoparticles and chitosan for a reaction; The PHL:S double-stranded solution contains PHL:S double strands obtained by incubating a carboxyl-activated PHL strand and an S strand; The nucleotide sequence of the PHL strand is as shown in SEQ ID NO.3; the nucleotide sequence of the S strand is as shown in SEQ ID NO.

4.

2. The construction method according to claim 1, characterized in that, The cleaning treatment includes the steps of polishing with aluminum powder and then ultrasonically cleaning with ethanol and deionized water.

3. The construction method according to claim 1, characterized in that, The carboxyl-activated PHL strand is obtained by reacting the PHL strand with a mixture containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

4. The construction method according to claim 1, characterized in that The preparation method of the RuSi NPs@CS composite includes the following steps: Add n-hexanol, Triton-X-100 and Ru(bpy)3 2+ to cyclohexane, stir and react to form an oil-in-water system. Then add tetraethyl orthosilicate and NH3·H2O. After the reaction, add a demulsifier to demulsify, and collect ruthenium-silicon nanoparticles by centrifugation. Mix and react the ruthenium-silicon nanoparticles with chitosan to obtain the RuSi NPs@CS composite.

5. The construction method according to claim 4, wherein The demulsifier is acetone.

6. A biosensor constructed by the construction method according to any one of claims 1-5.

7. Use of the biosensor according to claim 6 in the preparation of a kit for detecting the Alzheimer's disease biomarker Aβ oligomer.

8. A kit for detecting the Alzheimer's disease biomarker Aβ oligomer, characterized in that, It includes the biosensor according to claim 6.

9. The kit according to claim 8, wherein The kit further includes an M strand, an F strand, an H1-Fc strand, an H2-Fc strand, an H3-Fc strand, an H4-Fc strand, and a 3'-terminal amino-functionalized Aβ oligomer aptamer; The nucleotide sequence of the Aβ oligomer aptamer is as shown in SEQ ID NO.1; The nucleotide sequence of the M strand is as shown in SEQ ID NO.2; The nucleotide sequence of the F strand is as shown in SEQ ID NO.5; The nucleotide sequence of the H1-Fc strand is as shown in SEQ ID NO.6; The nucleotide sequence of the H2-Fc strand is as shown in SEQ ID NO.7; The nucleotide sequence of the H3-Fc strand is as shown in SEQ ID NO.8; The nucleotide sequence of the H4-Fc strand is as shown in SEQ ID NO.

9.

10. The kit according to claim 9, characterized in that, The kit further includes carboxylated magnetic beads.

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