Beta amyloid protein oligomer detection electrode, preparation method thereof and dual-signal amplification sensing chip

Abstract

The invention provides a beta amyloid protein oligomer detection electrode and a preparation method thereof and a dual-signal amplification sensing chip, the electrode comprises a first current collector, gold nanoparticles are attached to the surface of the first current collector, the gold nanoparticles are in bonding connection with complementary double-stranded DNA formed by complementation of cDNA-FAM and Apt-TAMRA, TAMRA is tetramethyl rhodamine, and the surface of the first current collector is in bonding connection with the complementary double-stranded DNA formed by complementation of cDNA-FAM and Apt-TAMRA. The Apt is an aptamer of a beta amyloid protein oligomer, the cDNA is complementary DNA of the Apt, and the FAM is carboxyl fluorescein. By combining fluorescence resonance energy transfer and electrochemical signals, efficient, sensitive and stable dual detection of AbetaO can be realized, the detection precision and the signal-to-noise ratio are improved, and the influence of single signal error is avoided.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence and electrochemical sensing technology, specifically relating to a detection electrode for β-amyloid oligomers (AβO) based on a fluorescence-electrochemical aptamer, its preparation method, and a dual-signal amplification sensing chip. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide, and its incidence is increasing year by year with the aging population. Its characteristic pathological marker is the abnormal deposition of β-amyloid (Aβ) plaques in the brain, especially Aβ oligomers (AβO), which are considered one of the key factors in the pathogenesis of AD. AβO aggregates between neurons, inducing neurotoxic responses, and early serum or cerebrospinal fluid AβO levels are closely related to the progression of AD; therefore, AβO has become a biomarker for the diagnosis and monitoring of AD.

[0003] Current methods for AβO detection mainly include traditional techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blotting. These methods have limitations in terms of detection sensitivity and selectivity, and are also complex, time-consuming, and costly. In recent years, fluorescence detection and electrochemical sensors, as emerging sensing technologies, have shown great potential in the detection of AD-related biomarkers. In particular, aptamer-based sensors have become a research hotspot due to their high selectivity, low cost, and ease of preparation. However, current research on aptamer-based single-signal sensors for AD detection only includes some studies. Single-signal sensors are susceptible to interference and errors, resulting in low reliability.

[0004] Therefore, developing a fluorescence-electrochemical dual-signal aptamer sensor with high sensitivity, high selectivity, and low cost for the detection of AβO has important theoretical and practical significance. Summary of the Invention

[0005] The purpose of this invention is to provide a β-amyloid oligomer detection electrode, its preparation method, and a dual-signal amplification sensing chip to solve the problem of low reliability of single-signal sensors in the prior art.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a β-amyloid oligomer detection electrode, comprising a first current collector, gold nanoparticles attached to the surface of the first current collector, the gold nanoparticles being bonded to complementary double-stranded DNA formed by cDNA-FAM and Apt-TAMRA, wherein TAMRA is tetramethylrhodamine, Apt is an aptamer for β-amyloid oligomers, cDNA is the complementary DNA of Apt, and FAM is carboxyfluorescein.

[0007] In a specific embodiment of the present invention, the sequence of the cDNA-FAM is 5`-SH-C6-CGTTCGCCCCCT-FAM-3`; the sequence of the Apt-TAMRA is 5`-TAMRA-GGTGGCTGGAGGGGGCGAACG-3`.

[0008] Secondly, the present invention provides a method for preparing a β-amyloid oligomer detection electrode, comprising: S1, Apt and cDNA are coupled with tetramethylrhodamine and carboxyfluorescein, respectively, to obtain cDNA-FAM and Apt-TAMRA; S2, deposit gold nanoparticles on the first current collector to obtain a gold nanoparticle-modified electrode; S3, cDNA-FAM is covalently coupled to gold nanoparticles on the electrode surface via Au-S bonds to obtain a cDNA-FAM modified electrode; S4. Apt-TAMRA is reacted with cDNA-FAM on the electrode to produce a DNA complementation reaction, thus obtaining a β-amyloid oligomer detection electrode.

[0009] In some preferred embodiments of the present invention, in S2, the first current collector includes a silver electrode and a carbon layer deposited on the surface of the silver electrode.

[0010] In some preferred embodiments of the present invention, in S2, a mixed electrolyte of chloroauric acid and sulfuric acid is used to deposit gold nanoparticles on the first current collector by constant current scanning, thereby obtaining an electrode modified with gold nanoparticles.

[0011] Specifically, the first current collector is inserted into an aqueous solution of chloroauric acid and sulfuric acid, and gold nanoparticles are deposited using a constant current. After rinsing and drying, an electrode modified with gold nanoparticles is obtained. The mass ratio of sulfuric acid, chloroauric acid and water is (98~295):(0.17~1.36):1000; the constant current is 0.2~0.6 A, and the time is 400~800 s.

[0012] In some preferred embodiments of the present invention, S3 specifically involves: adding cDNA-FAM solution to the gold nanoparticle-modified electrode, and coupling cDNA-FAM with gold nanoparticles through incubation; after incubation, rinsing with phosphate buffer to remove uncoupled cDNA-FAM, then immersing the electrode in mercaptohexanol solution to block non-specific binding sites, and then rinsing with phosphate buffer and drying to obtain the cDNA-FAM-modified electrode.

[0013] The concentration of the cDNA-FAM solution is 1~3 μM, the incubation temperature of the cDNA-FAM solution on the electrode is 0~25℃, and the incubation time is 5~10 h; the concentration of the mercaptohexanol solution is 1~2 mM, and the soaking and blocking time is 20~60 min.

[0014] In some preferred embodiments of the present invention, S4 specifically involves: adding an Apt-TAMRA solution to a cDNA-FAM modified electrode and incubating it to carry out a DNA complementation reaction; after incubation, rinsing with phosphate buffer to remove unpaired Apt-TAMRA to obtain a β-amyloid oligomer detection electrode.

[0015] The concentration of the Apt-TAMRA solution is 3~6 μM, the incubation temperature of the Apt-TAMRA solution on the electrode is 0~25℃, and the incubation time is 30~60 min.

[0016] Thirdly, the present invention provides a dual-signal amplification sensing chip, including a substrate and a working electrode, a counter electrode and a reference electrode disposed on the substrate, wherein the working electrode is the β-amyloid oligomer detection electrode.

[0017] Preferably, the counter electrode comprises a polyimide substrate and a carbon layer deposited on the polyimide substrate, and the reference electrode is a silver / silver chloride reference electrode.

[0018] Specifically, the fabrication method of the dual-signal amplification sensor chip of the present invention is as follows: (1) Using polyimide (PI) as a substrate, a first substrate electrode and a second substrate electrode are printed on the substrate, and carbon layers are modified on the first substrate electrode and the second substrate electrode respectively to obtain a first current collector and a second current collector; a silver / silver chloride reference electrode is prepared by cyclic voltammetry modification. (2) The β-amyloid oligomer detection electrode was prepared according to the above preparation method. (5) An integrated chip that integrates fluorescence and electrochemical sensors, namely a dual-signal amplification sensor chip, is used to detect β-amyloid oligomers in clinical samples. The working electrode is the β-amyloid oligomer detection electrode, the second current collector is the counter electrode, and the silver / silver chloride reference electrode is the reference electrode.

[0019] The working electrode is located between the counter electrode and the reference electrode, forming a straight line segment; the counter electrode and the reference electrode are located on both sides of the working electrode, forming a symmetrical arc shape.

[0020] In step (1), the printing process of the first and second base electrodes has a spacing of 0.1~0.3 mm, a feed rate of 70~200 mm / min, and a flow rate of 0.5~1.5 mm. The printed polyimide substrate is placed on a hot plate at 80~100 ℃, heated and cured, and then cooled to room temperature. The printing process of the carbon layer modification has a feed rate of 100~300 mm / min and a flow rate of 0.3~0.8 mm. The printed polyimide substrate is placed on a hot plate at 80~100 ℃, heated and cured, and then cooled to room temperature.

[0021] The method for fabricating a silver / silver chloride reference electrode includes: mixing potassium chloride, hydrochloric acid and water uniformly to obtain an electrolyte, and chlorinating the silver reference electrode by cyclic voltammetry; the mass ratio of potassium chloride, hydrochloric acid and water is (3.7~15):(0.70~2.8):1000; the scanning range of the cyclic voltammetry is -0.15~1.05 V, the scanning rate is 25~100 mV / s, and the number of scans is 5~10.

[0022] Fourthly, the present invention provides the application of the β-amyloid oligomer detection electrode or the dual-signal amplification sensing chip in the detection of β-amyloid oligomers.

[0023] In use, the solution to be tested is dropped onto the β-amyloid oligomer detection electrode, and the fluorescence signal and electrochemical signal are measured respectively to accurately quantify the β-amyloid oligomer.

[0024] Specifically: the test solution is added dropwise to the reaction zone of the β-amyloid oligomer detection electrode; the test solution reacts with double-stranded DNA in the reaction zone, Apt specifically captures AβO in the solution and detaches from the double-stranded DNA, FRET is broken, FAM fluorescence is restored, and the fluorescence intensity is measured under 480 nm excitation light; during the electrochemical detection process, the shedding of Apt-TAMRA leads to a weakening of the electrochemical signal, which is detected by DPV scanning of the electrochemical workstation; wherein, the amount of test solution added is 5-20 μL; the reaction time between the test solution and the double-stranded DNA is 10-15 min.

[0025] The present invention provides a β-amyloid oligomer detection electrode or a dual-signal amplification sensing chip that can be used to detect clinical samples from Alzheimer's disease patients.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention, based on the principle of fluorescence resonance energy transfer (FRET), designs a fluorescence-electrochemical dual-signal aptamer sensor for AβO detection. Specifically, the specific aptamer for AβO (Apt) is modified onto the fluorescent acceptor TAMRA, while the complementary DNA (cDNA) of Apt is modified onto the fluorescent donor FAM. The double-stranded DNA formed by the complementarity of Apt and cDNA is then modified onto the electrode surface to construct a dual-signal detection electrode. Figure 1 In this detection electrode, the fluorophore of double-stranded DNA undergoes energy transfer under FRET, leading to fluorescence quenching of the donor FAM. Simultaneously, due to TAMRA's electrochemically active molecule, its redox properties allow the detection electrode to respond to both fluorescence and electrochemical signals. Upon the addition of AβO, Apt specifically binds to AβO, detaching from the double-stranded DNA and interrupting the FRET process, restoring FAM fluorescence. Simultaneously, the release of Apt-TAMRA reduces the electrochemical signal of the system. Figure 1 Based on the changes in fluorescence intensity before and after the addition of AβO and the current response of the electrochemical signal, a quantitative detection curve for AβO was established, thereby achieving rapid, sensitive, and highly selective detection of AβO. This invention combines fluorescence resonance energy transfer (FRET) and electrochemical signals to achieve efficient, sensitive, and stable dual detection of AβO, improving detection accuracy and signal-to-noise ratio while avoiding the influence of single-signal errors. Based on the specificity of Apt for AβO, the sensor exhibits good anti-interference and reproducibility in practical applications, making it of significant practical value in the clinical disease detection industry. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the dual-signal amplification sensor chip for detecting AβO according to the present invention; Figure 2 The curves of fluorescence and electrochemical signals obtained in Example 1 of this invention are shown. Figure 3The fluorescence curve (A), electrochemical signal curve (C), fluorescence standard curve (B), and electrochemical standard curve (D) obtained in Example 3 of this invention are shown. Detailed Implementation

[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0030] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0031] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not intended to limit the order of the method steps or define the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0032] Example 1 (1) Preparation of fluorescent detection probe The fluorescent detection probe is a double-stranded DNA composed of an aptamer and a complementary sequence of a fluorescent signal molecule modified by chemical bonds. The fluorescent signal molecule needs to satisfy the FRET effect and electrochemical signal transduction function, and FAM and TAMRA are selected to modify the complementary sequence cDNA and the aptamer Apt, respectively.

[0033] The sequences of cDNA-FAM and Apt-TAMRA are as follows: cDNA: 5`-SH-C6-CGTTCGCCCCCT-FAM-3` Apt: 5`-TAMRA-GGTGGCTGGAGGGGGCGAACG-3` (2) Preparation of fluorescence-electrochemical three-electrode Based on conductive silver paste, the substrate electrode of the designed dual-signal amplification sensor chip is printed using a dispensing machine. The printing process is set with a through-pitch of 0.1 mm, a feed rate of 100 mm / min, and a flow rate of 1.0 mm. The polyimide PI substrate that has been dispensed and printed is placed on a 60 ℃ hot plate, heated and cured, and then cooled to room temperature to achieve the patterning of the substrate electrode.

[0034] Based on conductive carbon paste, a dispensing machine is used to print the designed counter electrode and working electrode structure patterns. The dispensing machine feed rate is set to 200 mm / min and the flow rate to 0.8 mm to achieve carbon layer modification of the first and second base electrodes. The polyimide PI substrate with completed positioning printing is placed on a 90 ℃ hot plate, heated and cured, and then cooled to room temperature to obtain the first current collector and the second current collector. The second current collector is used as the counter electrode.

[0035] Using an electrochemical workstation and commercially available counter and reference electrodes, the reference electrode was chlorinated in a potassium chloride / hydrochloric acid mixed electrolyte via cyclic voltammetry. The electrolyte was prepared by uniformly mixing potassium chloride, hydrochloric acid, and water at a mass ratio of 10.6:0.70:1000. Cyclic voltammetry was used to chlorinate the reference electrode in a scan range of -0.15 to 1.05 V, a scan rate of 50 mV / s, and 5 scans. The electrode was then rinsed with deionized water and dried to obtain a silver / silver chloride reference electrode.

[0036] (3) Modification of gold nanoparticles on the surface of the first current collector Using an electrochemical workstation and commercial counter and reference electrodes, sulfuric acid, chloroauric acid, and water were mixed uniformly at a mass ratio of 195:1.36:1000 to obtain an electrolyte. Gold nanoparticles were deposited on the surface of the first current collector in the chloroauric acid / sulfuric acid mixed electrolyte using a constant current electrodeposition method. The deposition current was 0.5 A and the deposition time was 600 s. After that, the gold nanoparticles were washed with deionized water and dried to obtain an electrode with a surface modified with gold nanoparticles.

[0037] (4) Constructing a fluorescence-electrochemical detection system 10 μL of thiol-containing cDNA-FAM (2 μM) was added dropwise to the surface of a gold nanoparticle-modified electrode, covalently coupled via Au-S bonds. The reaction was carried out at 4 °C, and the addition was repeated for a total of 10 h. Subsequently, uncoupled cDNA-FAM was removed by rinsing with phosphate buffer. The electrode was then immersed in mercaptohexanol solution (2 mM) for 30 min to block non-specific binding sites, followed by rinsing with phosphate buffer and air drying to obtain the cDNA-FAM-modified electrode.

[0038] 10 μL of Apt-TAMRA (5 μM) was added dropwise to the cDNA-FAM modified electrode surface, forming double-stranded DNA through base complementarity. The reaction was carried out at 4 °C, and the addition was repeated for a total of 40 min. Subsequently, the unpaired Apt-TAMRA was removed by rinsing with phosphate buffer, and the electrode was dried to obtain the double-stranded DNA modified electrode, which is the β-amyloid oligomer detection electrode and is used as the working electrode.

[0039] Similarly, 10 μL of Apt-TAMRA (10 μM) was added dropwise to the cDNA-FAM modified electrode surface, forming double-stranded DNA through base complementarity pairing. The reaction was carried out at 4°C, and the addition was repeated for a total of 40 min. Subsequently, the unpaired Apt-TAMRA was removed by rinsing with phosphate buffer, and the electrode was dried to obtain the double-stranded DNA modified electrode, which is the β-amyloid oligomer detection electrode and is used as the working electrode.

[0040] The cDNA-FAM modified electrode was used as a control.

[0041] The fluorescence and electrochemical signals of the fluorescence-electrochemical system in this embodiment were measured, and the results are as follows: Figure 2 First, the binding of the aptamer to the complementary strand was verified through the FRET effect, such as... Figure 2 As shown in Figure A, FRET was observed after different concentrations of Apt-TAMRA hybridized with cDNA-FAM on the electrode surface, with 10 μM Apt-TAMRA producing the strongest FRET signal. Therefore, 10 μM was subsequently selected as the modification concentration of Apt-TAMRA. The successful introduction of the electroactive tag TAMRA was then confirmed by cyclic voltammetry (CV). Figure 2 The CV curve of B showed the characteristic oxidation peak of TAMRA at 0.5 V, confirming the successful construction of the fluorescence-electrochemical system.

[0042] Example 2 AβO Specificity Experiment Take 10 μL of human serum simulant solution samples for testing. The samples contain AβO, Aβ monomer, Aβ cellulose, Tau protein, BSA, IL-6, catalase, lysozyme, malondialdehyde, α-glucosidase, H2O2, and Mg, respectively. 2+ Ca 2+ Fe 2+ Cl -The enzyme concentration was 50 ng / mL and the ion concentration was 100 μM. The sample was dropped onto the working electrode of the modified double-stranded DNA and reacted at 25℃ for 15 min. The fluorescence spectrum was measured (excitation wavelength 467 nm, emission wavelength range 500~640 nm). At the same time, the electrochemical signal was collected, the signal changes were observed, and the signal shift caused by each interfering substance was compared.

[0043] The results showed that adding samples containing AβO to the working electrode of modified double-stranded DNA caused a significant increase in fluorescence intensity and a marked decrease in electrochemical signal. Meanwhile, samples containing Aβ monomers, Aβ filaments, Tau protein, BSA, IL-6, catalase, lysozyme, malondialdehyde, α-glucosidase, H2O2, and Mg... 2+ Ca 2+ Fe 2+ Cl - The sample showed no significant effect on fluorescence intensity and electrochemical signal, indicating that the fluorescence-electrochemical system of this invention has a specific response to AβO.

[0044] Example 3 Fitting the AβO standard curve 10 μL of AβO human serum simulant samples at different concentrations (0, 1, 2, 4, 6, 10 ng / mL) were added to a working electrode modified with double-stranded DNA and reacted at 25 °C for 15 min. The fluorescence spectra were then measured (excitation wavelength 467 nm, emission wavelength range 500–600 nm). Figure 3 For samples A and B, a fluorescence standard curve was fitted using the fluorescence emission peak intensity values. Simultaneously, electrochemical signals were measured, and an electrochemical standard curve was fitted using the peak intensity values. Figure 3 (C and D in the middle).

[0045] Figure 3 Fluorescence results for A and B showed that the fluorescence emission peak intensity at 520 nm significantly increased with increasing AβO concentration. Within the concentration range of 0-10 ng / mL, the fluorescence intensity exhibited a good linear relationship with AβO concentration, with the linear regression equation being Y = 12976X + 489143 (R²). 2 = 0.99), the limit of detection (LOD) was calculated using 3σ / k, yielding an LOD of 0.38 ng / mL. Meanwhile, the electrochemical detection results ( Figure 3The reliability of the sensing system was further verified by steps A and B. As the AβO concentration increased, the conformational change of the aptamer caused TAMRA to move away from the electrode surface, and its characteristic oxidation peak current decreased accordingly. After taking the logarithm of the AβO concentration, in the range of 0-10 ng / mL, the current signal showed a significant linear correlation with the logarithm of the concentration, with the linear equation being Y = -0.7739X - 0.4109 (R0). 2 = 0.98), with a detection limit as low as 0.15 ng / mL.

[0046] In summary, the fluorescence-electrochemical dual-mode sensing system constructed in this invention exhibits high sensitivity and excellent linear response for the detection of AβO. The two detection methods corroborate each other, fully demonstrating the feasibility and reliability of this strategy in practical applications.

[0047] Example 4: Detection of AβO content in serum Serum samples were collected from three categories: healthy individuals, patients with early-stage Alzheimer's disease, and patients with severe dementia, with six samples from each category. The AβO levels in three of these samples were determined using a commercial AβO ELISA kit. The remaining three samples (10 μL each) were added to the working electrode modified with double-stranded DNA according to this invention and incubated at 25°C in the dark for 15 min. The fluorescence intensity and electrochemical signal were measured according to the method used in Example 3 for AβO determination, with each serum sample measured three times. Based on the fluorescence and electrochemical standard curves obtained in Example 3, the AβO content in the corresponding actual serum samples was calculated. The relative error and relative standard deviation of the results obtained from the dual-signal amplification sensor chip were compared with the actual AβO content in the serum samples to verify the accuracy and reliability of the aptamer fluorescence-electrochemical dual-signal method.

[0048] The results showed that the relative error and relative standard deviation between the AβO content obtained by the method of the present invention and the actual AβO content in serum samples were within acceptable ranges, and the errors were smaller than those measured by commercial AβO ELISA kits.

Claims

1. A detection electrode for β-amyloid oligomers, characterized in that, It includes a first current collector, on the surface of which gold nanoparticles are attached. The gold nanoparticles are bonded to a double-stranded DNA formed by the complementarity of cDNA-FAM and Apt-TAMRA, wherein TAMRA is tetramethylrhodamine, Apt is an aptamer of β-amyloid oligomer, cDNA is the complementary DNA of Apt, and FAM is carboxyfluorescein.

2. The β-amyloid oligomer detection electrode according to claim 1, characterized in that, The sequence of the cDNA-FAM is 5`-SH-C6-CGTTCGCCCCCT-FAM-3`; the sequence of the Apt-TAMRA is 5`-TAMRA-GGTGGCTGGAGGGGGCGAACG-3`.

3. The method for preparing the β-amyloid oligomer detection electrode according to claim 1 or 2, characterized in that, include: S1, Apt and cDNA are coupled with tetramethylrhodamine and carboxyfluorescein, respectively, to obtain cDNA-FAM and Apt-TAMRA; S2, deposit gold nanoparticles on the first current collector to obtain a gold nanoparticle-modified electrode; S3, cDNA-FAM is covalently coupled to gold nanoparticles on the electrode surface via Au-S bonds to obtain a cDNA-FAM modified electrode; S4. Apt-TAMRA is reacted with cDNA-FAM on the electrode to obtain a β-amyloid oligomer detection electrode.

4. The method for preparing the β-amyloid oligomer detection electrode according to claim 3, characterized in that, In S2, the first current collector includes a silver electrode and a carbon layer deposited on the surface of the silver electrode.

5. The method for preparing the β-amyloid oligomer detection electrode according to claim 1, characterized in that, In S2, a mixed electrolyte of chloroauric acid and sulfuric acid is used to deposit gold nanoparticles on the first current collector by constant current scanning, thereby obtaining an electrode modified with gold nanoparticles.

6. The method for preparing the β-amyloid oligomer detection electrode according to claim 1, characterized in that, S3 specifically involves: adding cDNA-FAM solution to the gold nanoparticle-modified electrode and incubating to couple cDNA-FAM with the gold nanoparticles; after incubation, rinsing with phosphate buffer to remove uncoupled cDNA-FAM, then immersing the electrode in mercaptohexanol solution to block non-specific binding sites, and finally rinsing with phosphate buffer and drying to obtain the cDNA-FAM-modified electrode.

7. The method for preparing the β-amyloid oligomer detection electrode according to claim 1, characterized in that, S4 specifically involves: adding Apt-TAMRA solution to the cDNA-FAM modified electrode and incubating it to carry out a DNA complementation reaction; after incubation, washing with phosphate buffer to remove unpaired Apt-TAMRA to obtain the β-amyloid oligomer detection electrode.

8. A dual-signal amplification sensor chip, characterized in that, It includes a substrate and a working electrode, a counter electrode, and a reference electrode disposed on the substrate, wherein the working electrode is the β-amyloid oligomer detection electrode as described in claim 1 or 2.

9. The application of the β-amyloid oligomer detection electrode according to claim 1 or 2 or the dual-signal amplification sensing chip according to claim 8 in the detection of β-amyloid oligomers.

10. The application according to claim 9, characterized in that, The test solution was added dropwise to the β-amyloid oligomer detection electrode, and the fluorescence signal and electrochemical signal were measured respectively.

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