Biosensor for detecting Alzheimer's disease markers and its construction method and application

Abstract

The present invention discloses a biosensor for detecting Alzheimer's disease markers, as well as a construction method and application thereof, relating to the field of biosensor technology. The construction method comprises the following steps: after cleaning a glassy carbon electrode, dripping a RuSi NPs@CS complex onto the surface of the cleaned glassy carbon electrode to obtain a RuSi NPs@CS / GCE electrode; immersing the RuSi NPs@CS / GCE electrode in a PHL:S double-strand solution for incubation to obtain the biosensor; the RuSi NPs@CS complex is obtained by mixing ruthenium silicon nanoparticles and chitosan; the PHL:S double-strand solution contains PHL chains activated by carboxyl groups and S chains through an incubation reaction. The biosensor has the advantages of high accuracy, strong sensitivity, and good selectivity, and can be used to accurately detect Aβ oligomers, a marker of Alzheimer's disease.

Description

Technical Field

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

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder affecting the central nervous system. It is typically caused by abnormal deposition of β-amyloid protein (Aβ) and abnormal accumulation of tau protein in neurofibrillary tangles in the nervous system, leading to brain neuron loss, ultimately causing brain atrophy and memory loss. In particular, the intermediate Aβ oligomers formed during this process are considered the most cytotoxic entities. Compared with healthy controls, Aβ oligomer levels are significantly increased in the cerebrospinal fluid of AD patients. Therefore, Aβ oligomers are widely used as a biomarker for the early diagnosis of AD. Given the association between Aβ oligomer content and AD progression, the design of an accurate and sensitive method to assess Aβ oligomer content is crucial.

[0003] Researchers have designed various signal amplification strategies to quantitatively analyze Aβ oligomer content, including porous materials as sensing platforms, silver nanoparticles as dual-modulation co-reaction accelerators, electrocatalysis using covalent organic framework nanocomposites, hybridization chain reaction-driven DNA walkers, and DNA enzyme-driven DNA chain dissociation. Cascade DNA looping, a DNA assembly technology, holds great potential for constructing biosensors with high sensitivity, low background signals, and short programmability. This paper proposes to develop a biosensor for detecting Alzheimer's disease markers, enabling efficient detection of Aβ oligomers. Summary of the Invention

[0004] The present invention aims to provide a biosensor for detecting Alzheimer's disease markers, as well as its construction method and application, to address the aforementioned issues with the existing technology. This biosensor boasts high accuracy, strong sensitivity, and excellent selectivity. It can be used to accurately detect Aβ oligomers, a marker of Alzheimer's disease, and has significant application value in the clinical diagnosis and drug research of Alzheimer's disease.

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

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

[0007] After the glassy carbon electrode was cleaned, the RuSi NPs@CS composite was added dropwise to its surface to obtain the RuSi NPs@CS / GCE electrode.

[0008] Immersing the RuSi NPs@CS / GCE electrode in a PHL:S double-strand solution for incubation reaction to obtain the biosensor;

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

[0010] The PHL:S double-stranded solution contains a PHL:S double-stranded chain obtained by incubating a carboxyl-activated PHL chain with an S chain;

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

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

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

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

[0015] Hexanol, Triton-X-100 and Ru(bpy)3 2+ The mixture was added to cyclohexane and stirred to react to form an oil-in-water system, followed by the addition of ethyl orthosilicate and NH3·H2O. After the reaction, a demulsifier was added to break the emulsion, and the mixture was collected by centrifugation to obtain ruthenium silicon nanoparticles. The ruthenium silicon nanoparticles and chitosan were mixed and reacted to obtain the RuSi NPs@CS complex.

[0016] Furthermore, 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 use of the above-mentioned biosensor in preparing a kit for detecting Aβ oligomers, a marker of Alzheimer's disease.

[0019] The present invention also provides a kit for detecting Aβ oligomers, a marker of Alzheimer's disease, comprising the above-mentioned biosensor.

[0020] Furthermore, the kit further comprises an M chain, an F chain, an H1-Fc chain, an H2-Fc chain, an H3-Fc chain, an H4-Fc chain, and an Aβ oligomer aptamer with a 3′-terminal amino group;

[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 also includes carboxylated magnetic beads.

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

[0030] This invention has constructed a dual-mode ratiometric biosensor that detects the content of Aβ oligomers, a marker of Alzheimer's disease. Based on magnetic bead separation technology and the specific binding of aptamers, this biosensor converts the detection of Aβ oligomers, a marker of Alzheimer's disease, into nucleic acid analysis. Ruthenium-silicon nanoparticles (RuSi NPs) serve as an electrochemiluminescent substrate, and ferrocene serves as a quencher and electrochemical beacon, enabling in situ quenching and electrochemical response of the electrochemiluminescent signal. Entropy-driven DNA cyclic activation of the hybridization chain reaction achieves cyclic amplification of the signal molecule. Based on the ratiometric response of the electrochemical and electrochemiluminescent dual-mode signals, this biosensor achieves highly sensitive and accurate detection of Aβ oligomers, a marker of Alzheimer's disease.

[0031] The present invention utilizes magnetic bead separation technology and aptamers to conduct signal transduction of Aβ oligomers, thereby improving the selectivity of the sensing system. RuSi NPs are used as electrochemiluminescence-active substrates, combined with in-situ quenching of ferrocene to reduce background signal interference and improve signal output and response. A signal amplification strategy based on entropy-driven DNA cyclic activation hybridization chain reaction enhances substrate selection diversity and detection sensitivity. The inverse change trends of the electrochemical and electrochemiluminescence dual modes improve the accuracy of the biosensor. Therefore, the biosensor prepared by the present invention has the advantages of high accuracy, strong sensitivity, and good selectivity, and can be applied to the accurate detection of Aβ oligomers, a marker of Alzheimer's disease, and has important application value in the clinical diagnosis and drug research of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Schematic diagram of the preparation process and sensing mechanism of the dual-mode ratiometric biosensor for Alzheimer's disease marker Aβ oligomers of the present invention;

[0034] Figure 2 Transmission electron microscopy (SEM) images of RuSi NPs;

[0035] Figure 3 X-ray photoelectron spectroscopy (XPS) diagram of RuSi NPs;

[0036] Figure 4 XPS graph of Ru 3d orbital electrons of RuSi NPs;

[0037] Figure 5 RuSi NPs and Ru(bpy)3 2+ Ultraviolet-visible (UV-vis) absorption spectrum;

[0038] Figure 6 The figure shows the feasibility of Aβ oligomer signal transduction and entropy-driven DNA recycling activation hybridization chain reaction. Lane 1 represents the aptamer of Aβ oligomer, lane 2 represents the mixture of aptamer and M, lane 3 represents the supernatant of the aptamer:M double chain after treatment with Aβ oligomer, lane 4 represents the PHL:S double chain, lane 5 represents the PHL:S double chain treated with the released M chain, lane 6 represents the F chain, lane 7 represents lane 5 further treated with the F chain, lane 8 represents the H1-Fc chain, lane 9 represents the reaction of lane 7 with the H1-Fc chain, and lane 10 represents the reaction of lane 7 with a mixture of the H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain.

[0039] Figure 7 Figure 3 is the electrochemical impedance spectroscopy (EIS) characterization diagram of the sensing substrate interface construction process; where a represents bare GCE, b represents RuSi NPs@CS / GCE, c represents PHL:S / RuSi NPs@CS / GCE, d represents the sensing substrate after c reacts with the M chain transduced by Aβ oligomers, e represents the sensing substrate after d reacts with the F chain, and f represents the sensing substrate after e further reacts with a mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain;

[0040] Figure 8 Figure 3 is the electrochemiluminescence-time response diagram of the sensing substrate interface construction process; where a represents RuSi NPs@CS / GCE, b represents PHL:S / RuSi NPs@CS / GCE, c represents the sensing substrate after b reacts with the M chain transduced by Aβ oligomers, d represents the sensing substrate after c reacts with the F chain, and e represents the sensing substrate after d further reacts with a mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain;

[0041] Figure 9 Electrochemical response diagrams of the constructed sensing system before and after the reaction with a 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 Figure 3 is the electrochemical and electrochemiluminescence response curves of Aβ oligomers at different concentrations; where a~i correspond to concentrations of 0, 50 fM, 100 fM, 500 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM, respectively;

[0043] Figure 11 is a linear relationship graph between the logarithm of the ratio of electrochemical peak current to electrochemiluminescence signal and the logarithm of Aβ oligomer concentration;

[0044] Figure 12 is the selective response histogram of the biosensor;

[0045] Figure 13 The figure compares the results of detecting Aβ oligomer content in blood samples of Alzheimer's patients (numbered 1 to 3) and healthy volunteers using a biosensor and ELISA method; wherein the constructed method refers to the method using the biosensor of the present invention, and the standard method refers to the ELISA method. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The sequence information involved in the present 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] The present invention converts the detection of Aβ oligomers, a marker of Alzheimer's disease, into nucleic acid analysis based on magnetic bead separation technology and the specific binding of aptamers. Ruthenium silicon nanoparticles (RuSi NPs) are used as electrochemiluminescence active substrates, and ferrocene is used as a quencher and electrochemical beacon to perform in situ quenching and electrochemical response of the electrochemiluminescence signal, significantly reducing the interference of background signals. Entropy-driven DNA cyclic activation of the hybridization chain reaction is used to achieve cyclic amplification of the signal molecules. Based on the ratiometric response of the electrochemical and electrochemiluminescence dual-mode signals, highly sensitive and accurate detection of Aβ oligomers, a marker of Alzheimer's disease, is achieved (see the schematic diagram of the sensing mechanism of the biosensor for details). Figure 1 ), which are detailed as follows:

[0062] Example 1

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

[0064] First, a glassy carbon electrode (GCE) was polished with 0.3 and 0.05 μm aluminum powder, then ultrasonically cleaned with ethanol and deionized water for 5 minutes and dried with nitrogen. Then, 10 μL of the RuSi NPs@CS composite was added to the surface to obtain a RuSi NPs@CS / GCE electrode.

[0065] Next, 100 μL of PHL chain (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 carboxyl-activated PHL chain. This was then incubated with an equal volume of 5.0 μM S chain at 37°C for 1 hour to obtain a PHL:S double-stranded solution. A RuSi NPs@CS / GCE electrode was immersed in the PHL:S double-stranded solution and incubated at 37°C for 60 minutes to obtain PHL:S / RuSi NPs@CS / GCE, the biosensor.

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

[0067] The biosensor is designed as follows: Aβ oligomers, a marker for Alzheimer's disease, are added to an aptamer:M / magnetic bead complex for signal transduction. Released M chains are then collected after magnetic separation. PHL:S / RuSi NPs@CS / GCE react with a solution of Aβ oligomer-dependent intermediate M chains and fuel F chains. The sensing substrate is then placed in a mixed solution containing H1-Fc chains, H2-Fc chains, H3-Fc chains, and H4-Fc chains. After incubation, the sensing substrate is collected. Finally, a three-electrode system is constructed, using the incubated sensing substrate as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode. The electrochemiluminescence signal is detected in tri-n-propylamine solution and in phosphate buffered saline under a nitrogen atmosphere. The electrochemiluminescence signal is detected using a multifunctional electrochemical and chemiluminescence instrument with a 700 V photomultiplier tube and a cycling voltage range of 0-1.4 V. The differential pulse voltammetry (DPV) signal was recorded on an electrochemical workstation with a voltage range of 0–0.4 V, a pulse amplitude of 25 mV, and a pulse period of 0.5 s. The details are as follows:

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

[0069] The specific procedure is as follows: 0.5 mg / mL carboxylated magnetic beads were added to a mixture containing N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL) and N-hydroxysuccinimide (10 mg / mL) for 1 hour at room temperature to obtain surface carboxyl-activated magnetic beads. These beads were then added to 100 μL of 3′-terminally amino-terminated Aβ oligomer aptamers (2.5 μM) and reacted at room temperature for 2 hours. The aptamers assembled onto the magnetic bead surface through amide bonds. The resulting aptamer / magnetic beads were separated by magnet and washed with buffer solution before being added to 100 μL of M chain (2.5 μM) and reacted at 37°C for 1 hour to form the aptamer:M / magnetic bead complex. After the complex was separated by magnet and washed with buffer solution, it was added to 100 μL of buffer solution containing different concentrations of Aβ oligomers and reacted at 37°C for 2 h. After separation by magnet, the Aβ oligomer signal transduction intermediate M chain was obtained.

[0070] (2) An entropy-driven DNA cyclic activation hybridization chain reaction was constructed at the PHL:S / RuSi NPs@CS / GCE electrode interface to obtain a sensing substrate assembled with long-chain DNA polymers.

[0071] The PHL:S / RuSi NPs@CS / GCE electrode was immersed in a 100 μL mixed solution containing the Aβ oligomer signaling intermediate M chain and 2.5 μM F chain. The solution was incubated at 37°C for 60 min to activate the entropy-driven DNA looping reaction, resulting in the formation of the PHL:F / RuSiNPs@CS / GCE. The PHL:F / RuSi NPs@CS / GCE was then placed in a 100 μL mixed solution containing the H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain. The reaction was incubated at 37°C for 1 h to initiate a hybridization chain reaction, forming long DNA polymers at the electrode interface and introducing a large amount of Fc. The final concentration of the H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain was 5 μM.

[0072] (3) The sensing substrate obtained in step (2) was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the auxiliary electrode to form a three-electrode system. The electrochemiluminescence signal was detected in a tri-n-propylamine solution and the electrochemical signal was detected in a phosphate buffer solution under a nitrogen atmosphere. The Aβ oligomer content in the blood sample of Alzheimer's patients was calculated based on the standard curve between the logarithm of the electrochemical-electrochemiluminescence signal ratio and the logarithm of the Aβ oligomer concentration. The electrochemiluminescence response was detected by a multifunctional electrochemical and chemiluminescence instrument, with a photomultiplier tube of 700 V and a cycling voltage range of 0-1.4 V. The differential pulse voltammetry (DPV) signal was recorded by an electrochemical workstation with a voltage range of 0-0.4 V, a pulse amplitude of 25 mV, and a pulse period of 0.5 s.

[0073] Example 2

[0074] This example tests the performance of the biosensor prepared in Example 1. The specific process is as follows:

[0075] (1) The biosensor of the present invention utilizes RuSi NPs to improve the electrochemiluminescence signal response, and ferrocene realizes in situ signal quenching. In order to verify the successful synthesis of RuSi NPs, the RuSi NPs prepared in Example 1 were characterized. Figure 2 It can be seen that RuSi NPs have a spherical structure and the particle size is about 90 nm. Figure 3 and Figure 4 It shows that the material contains Si, C, Ru, N and O elements. In addition, Figure 5 It is shown that RuSi NPs have the same 2+ The above results indicate that Ru(bpy)3 2+ Successful embedding of molecules into silica nanospheres.

[0076] (2) The biosensor of the present invention uses RuSi NPs as the sensing substrate, specifically recognizes Aβ oligomers through aptamers, and amplifies the signal through entropy-driven DNA cyclic activation hybridization chain reaction to improve the detection sensitivity. Therefore, the construction process of the sensing interface and the feasibility of entropy-driven DNA cyclic activation hybridization chain reaction are monitored and verified. Figure 6 As shown, lane 1 represents the aptamer chain in Aβ oligomers, lane 2 represents the mixture of aptamer and M, and the delayed new band represents the formation of the aptamer:M duplex. Lane 3 represents the supernatant after the aptamer:M duplex was treated with Aβ oligomers. A new, faster-migrating band appeared, indicating that aptamer recognition of Aβ oligomers and induction of M chain release are feasible. Lane 4 represents the PHL:S duplex, and lane 5 represents the PHL:S duplex treated with released M chain. Two new bands were observed, the slower band being attributed to the formation of the PHL:M duplex, and the other band indicating the release of the S chain. Lane 6 represents the F chain. When the PHL:M duplex was incubated with the F chain, two bands were observed in lane 7. The slower-migrating band indicates the formation of the PHL:F duplex, while the faster-migrating band is attributed to the release of the M chain. This suggests that the M chain can be displaced by the F chain, allowing the released M chain to re-enter the reaction, thus activating the upstream entropy-driven DNA loop. Lane 8 represents the H1-Fc chain. When lane 7 was reacted with the H1-Fc chain, a new lagging band was observed in lane 9, indicating that the H1-Fc chain could hybridize with the PHL chain to form an H1:PHL:F complex. When a mixture of H1-Fc, H2-Fc, H3-Fc, and H4-Fc chains was added to lane 7, a bright band clearly appeared at the top of lane 10, demonstrating the initiation of the downstream hybridization chain reaction and the formation of long DNA aggregates. These observations confirm the feasibility of the strategy of aptamer-specific recognition of Aβ oligomers and entropy-driven DNA loop activation of the hybridization chain reaction.

[0077] Then, electrochemical impedance spectroscopy, electrochemiluminescence and electrochemistry were used to characterize the construction of the sensing interface. Figure 7-Figure 9 As shown. Figure 7 As shown in curve a, on the bare GCE surface, the electron transfer impedance (R et ) is about 351.8 Ω. When RuSiNPs@CS is assembled on the GCE surface, the R et Increased to 1942.0 Ω ( Figure 7 At the same time, an obvious electrochemiluminescence signal of 14460 au ( Figure 8 When the modified electrode is sequentially connected with PHL:S double chain (2427.0 Ω, Figure 7 Curve c in Figure 2), released intermediate M chain (2787.0 Ω, Figure 7Curve d in), fuel F chain (3522.0 Ω, Figure 7 curve e) and a mixture containing H1-Fc chain, H2-Fc chain, H3-Fc chain, and H4-Fc chain (6139.0 Ω, Figure 7 Curve f) After incubation, R et The gradual increase is attributed to the negative charge of the DNA phosphate backbone and the interaction between [Fe(CN)6] 3- / 4- The electrostatic repulsion between the corresponding electrochemiluminescence intensity gradually decreased to 13210 au ( Figure 8 Curve b in ), 12490 au ( Figure 8 Curve c in ), 11482 au ( Figure 8 Curve d in ) and 5810 au ( Figure 8 The electron and energy transfer between RuSi NPs and ferrocene leads to the quenching of the electrochemiluminescence signal. At the same time, the introduction of ferrocene into the electrode interface leads to the appearance of an oxidation peak, with the current intensity reaching 278 nA at 0.17 V ( Figure 9 (b) ). Consequently, a reduced ECL signal and an enhanced electrochemical response were obtained, demonstrating the feasibility of a dual-mode ratiometric biosensor. The electrochemical impedance, ECL, and electrochemical results above effectively demonstrate the construction of the electrode sensing interface.

[0078] (3) The detection performance of the biosensor was verified using different concentrations of Aβ oligomers (0, 50 fM, 100 fM, 500 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM) according to the method described in Example 1.

[0079] The electrochemical peak current and electrochemiluminescence response curves of different concentrations of Alzheimer's disease marker Aβ oligomers are shown in Figure 2. Figure 10 As shown in Figure 2, 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 electrochemical to electrochemiluminescence signals and the logarithm of the Aβ oligomer concentration is as follows: Figure 11 As shown, the linear regression equation is log EC / ECL = 0.189 logc (M) + 0.416 (R 2 =0.996). Figure 12It was found that when the target Aβ oligomers were replaced by amyloid-β fibrils, amyloid-β monomers, immunoglobulin G, and L-cysteine, the electrochemical and electrochemiluminescent responses showed negligible signal changes compared to the blank control. However, at a concentration of 1 nM, when the interfering substances, amyloid-β fibrils, amyloid-β monomers, immunoglobulin G, and L-cysteine, were mixed with the target Aβ oligomers, the electrochemical and electrochemiluminescent signals were not significantly different from those of the target Aβ oligomers alone. These results indicate that the interfering substrates had no effect on the detection of Aβ oligomers. Due to the strong recognition ability of the aptamer for Aβ oligomers, satisfactory specificity for Aβ oligomer detection was achieved.

[0080] Example 3

[0081] This example uses blood samples from Alzheimer's patients (numbered AD patients 1-3) and healthy volunteers to verify the detection performance of the biosensor in Example 1. The specific process is as follows: Blood samples from three Alzheimer's patients (numbered AD patients 1-3) and one healthy volunteer were centrifuged after natural coagulation, and the supernatant was collected for further use. Aβ oligomer detection was then performed according to the method described in Example 1. The linear regression equation in Example 2 was used to calculate the concentration of Aβ oligomers in the blood samples. The results are shown in Figure 2. Figure 13 Compared with the results of standard commercial ELISA test kits, the concentration error is less than 5%, indicating that the biosensor designed in this invention has good detection capabilities and applications even for complex blood samples.

[0082] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

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

4.

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

3. The construction method according to claim 1, characterized in that The carboxyl-activated PHL chain is obtained by reacting the PHL chain 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 comprises the following steps: Hexanol, Triton-X-100 and Ru(bpy)3 2+ The mixture was added to cyclohexane and stirred to react to form an oil-in-water system, followed by the addition of ethyl orthosilicate and NH3·H2O. After the reaction, a demulsifier was added to break the emulsion, and the mixture was collected by centrifugation to obtain ruthenium silicon nanoparticles. The ruthenium silicon nanoparticles and chitosan were mixed and reacted to obtain the RuSi NPs@CS complex.

5. The construction method according to claim 4, characterized in that The demulsifier is acetone.

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

7. Use of the biosensor according to claim 6 in preparing a kit for detecting Aβ oligomers, a marker of Alzheimer's disease.

8. A kit for detecting Aβ oligomers, a marker of Alzheimer's disease, characterized in that: The biosensor according to claim 6.

9. The kit according to claim 8, characterized in that The kit further comprises an M chain, an F chain, an H1-Fc chain, an H2-Fc chain, an H3-Fc chain, an H4-Fc chain, and an Aβ oligomer aptamer with a 3′-terminal amino group; The nucleotide sequence of the Aβ oligomer aptamer is shown in SEQ ID NO.1; The nucleotide sequence of the M chain is shown in SEQ ID NO.2; The nucleotide sequence of the F chain is shown in SEQ ID NO.5; The nucleotide sequence of the H1-Fc chain is shown in SEQ ID NO.6; The nucleotide sequence of the H2-Fc chain is shown in SEQ ID NO.7; The nucleotide sequence of the H3-Fc chain is shown in SEQ ID NO.8; The nucleotide sequence of the H4-Fc chain is shown in SEQ ID NO.

9.

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

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