A biosensor for detecting two markers in the plasma of patients with alzheimer's disease

Abstract

This invention discloses a biosensor for detecting dual-labeled biomarkers in the plasma of Alzheimer's disease patients, belonging to the field of biosensor technology. The biosensor comprises an electrode modified with a ZnS / CdS / Bi2Se3 complex, an S1:C1:M triplet, an S2:C2:M triplet, and a DNA molecule system. Using a ZnS / CdS / Bi2Se3 ternary composite material as the sensing substrate and a PtPd / CeO2 heterojunction nanozyme as the signal regulation element, the biosensor combines aptamer-mediated specific recognition and fulcrum-triggered strand displacement reaction to achieve simultaneous dual-mode detection of the biomarkers β-amyloid 1-42 and β-amyloid 1-40 in the plasma of Alzheimer's disease patients. This is suitable for early screening and clinical diagnosis of Alzheimer's disease and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a biosensor for detecting dual-labeled substances in the plasma of Alzheimer's disease patients. Background Technology

[0002] Alzheimer's disease is an irreversible, progressive neurodegenerative disease. Its main pathological feature is the metabolic disorder and accumulation of β-amyloid protein (Aβ). Among them, Aβ1-42 and Aβ1-40 are the most important subtypes. The ratio of the two is a core biomarker for diagnosing Alzheimer's disease, which has higher stability, accuracy and anti-interference ability compared with a single biomarker.

[0003] Currently, the detection methods for Alzheimer's disease biomarkers are mainly single-mode detection methods, including electrochemical, fluorescence, and surface-enhanced Raman scattering. While these methods are simple to operate, their sensitivity and anti-interference capabilities are insufficient, making it difficult to meet the stringent requirements of early diagnosis. Dual-mode detection strategies, combining two complementary single-mode technologies, can achieve synergistic performance improvements. Among them, electrochemiluminescence / photoelectrochemical (ECL / PEC) dual-mode detection combines the high sensitivity of ECL with the ease of operation of PEC, becoming a research hotspot in the field of biosensing. However, existing ECL / PEC dual-mode sensors have a core problem: it is difficult to construct a universal probe that simultaneously modulates both signals. The requirements of PEC for band structure and light absorption contradict those of ECL for electron transfer efficiency and luminescence quantum yield, easily leading to problems such as signal crosstalk and poor modal compatibility.

[0004] Nanozymes, with their artificially mimicked enzyme properties, can effectively bridge the reaction mechanisms of two detection modes, thus providing a feasible approach to overcoming the signal modulation bottleneck of dual-mode sensors. However, traditional single-component or simple composite nanozymes often face limitations such as limited catalytic activity and insufficient substrate specificity. Heterojunction nanozymes, on the other hand, can significantly enhance catalytic performance through the synergistic effect of the built-in electric field and interfacial electron transfer at the heterojunction interface, making them ideal materials for achieving efficient signal transduction. Furthermore, improving detection sensitivity through specific recognition and effective signal amplification is also key to constructing highly sensitive sensors for Alzheimer's disease biomarkers. Summary of the Invention

[0005] The purpose of this invention is to provide a biosensor for detecting dual-labeled substances in the plasma of Alzheimer's disease patients, thereby addressing the problems existing in the prior art. The biosensor of this invention has advantages such as high accuracy, high sensitivity, and good specificity, and can be applied to the sensitive determination of Aβ1-42 and Aβ1-40 in Alzheimer's disease plasma, thus possessing significant application value.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a biosensor for detecting dual-labeled substances in the plasma of Alzheimer's disease patients. The biosensor includes an electrode modified with a ZnS / CdS / Bi2Se3 complex, an S1:C1:M triplet, an S2:C2:M triplet, and a DNA molecule system. The ZnS / CdS / Bi2Se3 composite is a ternary composite nanomaterial formed by sequentially loading CdS and ZnS onto the surface of Bi2Se3 nanowires. The nucleotide sequence of the S1 chain in the S1:C1:M triplet is shown in SEQ ID NO.1, the nucleotide sequence of the C1 chain is shown in SEQ ID NO.3, and the nucleotide sequence of the M chain is shown in SEQ ID NO.5. The nucleotide sequence of the S2 chain in the S2:C2:M triplet is shown in SEQ ID NO.2, the nucleotide sequence of the C2 chain is shown in SEQ ID NO.4, and the nucleotide sequence of the M chain is shown in SEQ ID NO.5. The DNA molecular system includes aptamer 1 strand, T1 strand, probe 1-PtPd / CeO2, aptamer 2 strand, T2 strand, and probe 2-PtPd / CeO2. The nucleotide sequence of the aptamer 1 chain is shown in SEQ ID NO. 6; The nucleotide sequence of the T1 chain is shown in SEQ ID NO.7; The nucleotide sequence of the aptamer 2 chain is shown in SEQ ID NO. 8; The nucleotide sequence of the T2 chain is shown in SEQ ID NO.9; The probe 1-PtPd / CeO2 is obtained by heterojunction of probe 1 chain with PtPd / CeO2; the nucleotide sequence of probe 1 chain is shown in SEQ ID NO.10; The probe 2-PtPd / CeO2 is obtained by heterojunction of probe 2 chain with PtPd / CeO2; the nucleotide sequence of probe 2 chain is shown in SEQ ID NO.11; The dual markers are β-amyloid protein 1-42 and β-amyloid protein 1-40.

[0007] Furthermore, the electrode is an indium tin oxide electrode.

[0008] Furthermore, the preparation method of the ZnS / CdS / Bi2Se3 composite includes the following steps: using Se nanowires as templates, Bi2Se3 nanowires are grown in situ, and then CdS and ZnS are sequentially loaded on the surface of the Bi2Se3 nanowires by a stepwise epitaxial growth method, and the residual Se phase is removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite.

[0009] Furthermore, the method for in-situ growth of the Bi2Se3 nanowires includes: mixing NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, SeO2 and bismuth acetate and reacting them with ultrasound to obtain Se / Bi2Se3 nanowires, and calcining to remove the Se phase to obtain the Bi2Se3 nanowires.

[0010] Further, the method for loading the CdS and the ZnS includes: reacting the Bi2Se3 nanowires with NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, thioacetamide and cadmium acetate to obtain CdS / Bi2Se3; The CdS / Bi2Se3 was mixed with NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, thioacetamide and zinc sulfate, and the Se phase was removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite.

[0011] Furthermore, the probe 1 chain and the probe 2 chain are coupled to the PtPd / CeO2 heterojunction via thiol groups, respectively.

[0012] Furthermore, the PtPd / CeO2 heterojunction is a heterostructure formed by loading PtPd bimetallic nanoparticles onto the surface of CeO2 nanorods.

[0013] The present invention also provides a method for preparing the above-mentioned biosensor, comprising the following steps: Bi2Se3 nanowires were grown in situ using Se nanowires as templates. CdS and ZnS were then loaded onto the surface of the Bi2Se3 nanowires sequentially using a stepwise epitaxial growth method. The residual Se phase was removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite. The ZnS / CdS / Bi2Se3 composite chitosan was mixed and then drop-coated onto the electrode surface to obtain the electrode modified with the ZnS / CdS / Bi2Se3 composite. The probe 1 chain is coupled to the PtPd / CeO2 heterojunction to obtain the probe 1-PtPd / CeO2; The probe 2 chain is coupled to the PtPd / CeO2 heterojunction to obtain the probe 2-PtPd / CeO2.

[0014] The present invention also provides the application of the above-described biosensor in the preparation of products for detecting dual markers in the plasma of Alzheimer's disease patients, wherein the dual markers are β-amyloid 1-42 and β-amyloid 1-40.

[0015] The present invention also provides a product for detecting dual markers in the plasma of Alzheimer's disease patients, comprising the above-mentioned biosensor; The dual markers are β-amyloid protein 1-42 and β-amyloid protein 1-40.

[0016] The present invention discloses the following technical effects: This invention constructs an ECL / PEC dual-mode biosensor, enabling precise detection of Aβ1-42 and Aβ1-40 levels in the serum of Alzheimer's disease patients. A schematic diagram of the biosensor's fabrication process and detection mechanism is shown below. Figure 1 As shown, this invention uses a ZnS / CdS / Bi2Se3 ternary composite material as the ECL and PEC dual-signal sensing substrate, and a PtPd / CeO2 heterojunction nanozyme as the signal regulation element. By combining aptamer-specific recognition and fulcrum-mediated chain displacement reaction, it achieves simultaneous dual-mode detection of the biomarkers β-amyloid protein 1-42 (Aβ1-42) and β-amyloid protein 1-40 (Aβ1-40) in Alzheimer's disease plasma.

[0017] This invention improves photon capture efficiency and optimizes interfacial charge transport by modulating the structures of ZnS, CdS, and Bi2Se3 to achieve synergistic matching, thus enabling the ZnS / CdS / Bi2Se3 substrate to simultaneously possess excellent ECL and PEC response performance. Utilizing the electronic structure optimization effect of PtPd alloys and the oxygen vacancy regulation effect of CeO2, PtPd / CeO2 heterojunction nanozymes exhibit highly efficient catalytic reaction rates and precise control, achieving cascade amplification of target signals. Based on aptamer recognition and fulcrum-mediated chain substitution reactions, the diversity of substrate selection and detection sensitivity can be improved. By integrating the ECL and PEC signal output systems, cross-validation and complementary amplification of signals can be achieved, enhancing the accuracy and reliability of detection results.

[0018] The biosensor prepared by this invention has advantages such as high accuracy, high sensitivity, and good specificity. It can be applied to the simultaneous and sensitive determination of biomarkers Aβ1-42 and Aβ1-40 in the plasma of Alzheimer's disease patients, and has important application value in the clinical diagnosis and drug research of Alzheimer's disease. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The diagrams below illustrate the fabrication process and detection mechanism of the biosensor of the present invention; wherein, (A) is a schematic diagram of the target recognition and trigger release mechanism; (B) is a schematic diagram of the sensor construction and signal amplification path in ECL mode; (C) is a schematic diagram of the sensor construction and signal amplification path in PEC mode; (D) is a schematic diagram of the detection mechanism in ECL mode; and (E) is a schematic diagram of the detection mechanism in PEC mode. Figure 2 Figure 1 shows the structural characterization results of the ZnS / CdS / Bi2Se3 composite; (A) is a scanning electron microscope (SEM) image of Bi2Se3 nanowires; (B) is a scanning electron microscope (SEM) image of ZnS / CdS / Bi2Se3; (C) is a transmission electron microscope (TEM) image of ZnS / CdS / Bi2Se3; and (D) is an X-ray photoelectron spectroscopy (XPS) image of ZnS / CdS / Bi2Se3. Figure 3 Figure 1 shows the structural characterization and catalytic verification results of PtPd / CeO2 heterojunction nanozymes. (A) is a transmission electron microscope image of PtPd / CeO2; (B) is an X-ray photoelectron spectrum of PtPd / CeO2; (C) is an oxidase-like catalytic verification image of PtPd / CeO2 catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine to blue diimine radical cations via H2O2; (D) is the UV-Vis absorption spectrum of 3,3′,5,5′-tetramethylbenzidine solutions with and without PtPd / CeO2 (curve a) and with PtPd / CeO2 (curve b). Figure 4 Figure 1 shows the verification results of the sensor substrate interface construction process. (A) is the ECL-time response curve, where a represents ZnS / CdS / Bi2Se3 / ITO, b represents S1:C1:M / ZnS / CdS / Bi2Se3 / ITO, c represents b after incubation with the Aβ1-42-dependent T1 chain, and d represents c reacting with probe 1-PtPd / CeO2. (B) is the PEC mode response curve, where a represents ZnS / CdS / Bi2Se3 / ITO, b represents S2:C2:M / ZnS / CdS / Bi2Se3 / ITO, c represents b after incubation with the Aβ1-40-dependent T2 chain, and d represents c reacting with probe 2-PtPd / CeO2. Figure 5 The figure shows the results of fluorescence spectral verification of the feasibility of the fulcrum-mediated chain substitution reaction; where a represents probe 1-FAM, b represents the mixture of S1:C1:M-Au NPs complex and probe 1-FAM, and c represents the mixture of S1:C1:M-Au NPs complex, Aβ142-dependent T1 and probe 1-FAM. Figure 6ECL mode detection graphs for different concentrations of Aβ1-42 are shown; (A) is the ECL-time response curve, where ag corresponds to concentrations of 0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 5 ng / mL and 10 ng / mL, respectively; (B) is the linear relationship between ΔECL and the logarithm of Aβ1-42 concentration. Figure 7 PEC mode detection graphs for different concentrations of Aβ1-40 are shown; (A) is the photocurrent-time response curve, where ag corresponds to concentrations of 0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 5 ng / mL and 10 ng / mL, respectively; (B) is the linear relationship between Δphotocurrent and the logarithm of Aβ1-40 concentration. Figure 8 This is a graph showing the specific detection results of the biosensor of the present invention; Figure 9 A graph showing the results of detecting the Aβ1-42 / Aβ1-40 ratio in the plasma of Alzheimer's disease patients and healthy individuals; Figure 10 This is a comparison chart showing the detection results of the Aβ1-42 / Aβ1-40 ratio in the plasma of Alzheimer's disease patients using the biosensor of this invention and the ELISA method. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to 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 stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said 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.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The sequence information involved in this invention is as follows: S1 chain: 5′-TTTTTTTTTTTTTTCCCTATATTGTACTGGTCCCC-3′ (SEQ ID NO. 1); S2 chain: 5′-TTTTTTTTTTTTTTCCCTATACGTTCGCGCCCCCT-3′ (SEQ ID NO. 2); C1 chain: 5′-SH-AGCCGGTGGGGGACCAGTACAATATAGGGTAGACTGCTAACTCG-3′ (SEQ IDNO.3); C2 chain: 5′-SH-GTGGCTGGGAGGGGGCGCGAACGTATAGGGTAGACTGCTAACTCG-3′ (SEQ IDNO.4); M chain: 5′-CGAGTTAGCAGTCTA-3′ (SEQ ID NO.5); Aptamer 1 chain: 5′-TCAGCGGGGAGGAAGCCGGTGGGGGACCAGTACAAAAGTGGGT AGGGCGGGTTGGAAAA-3′ (SEQ ID NO. 6); T1 chain: 5′-TTGTACTGGTCCCCCACCGGCT-3′ (SEQ ID NO.7); Aptamer 2 chain: 5′-SH-GGTGGCTGGAGGGGGCGCGAACG-3′ (SEQ ID NO. 8); T2 chain: 5′-CGTTCGGCCCCCTCCAGCCAC-SH-3′ (SEQ ID NO.9); Probe 1 chain: 5′-AGCAGTCTACCCTATATTGTACTGGTCCCC-SH-3′ (SEQ ID NO.10); Probe 2 chain: 5′-AGCAGTCTACCCTATACGTTCGCGCCCCCT-SH-3′ (SEQ ID NO.11).

[0027] Example 1

[0028] This embodiment provides a biosensor for detecting dual-labeled substances in the plasma of Alzheimer's disease patients, comprising a sensing substrate and a DNA molecule system. Its construction process is as follows: S1. The indium tin oxide (ITO) electrode was ultrasonically cleaned sequentially with acetone, ethanol, and deionized water for 5 min, and then dried with nitrogen. An equal volume of 0.5 mg / mL ZnS / CdS / Bi2Se3 composite dispersion was mixed with 1 wt% chitosan, and 20 μL was drop-coated onto the working area of ​​the ITO glass electrode. After drying with an infrared lamp, the ZnS / CdS / Bi2Se3 / ITO electrode was obtained.

[0029] The preparation method of the ZnS / CdS / Bi2Se3 complex is as follows: 1 mL NaOH (0.1 M), 1 mL hexadecyltrimethylammonium bromide (0.1 M), 1 mL ascorbic acid (0.1 M), 0.4 mL SeO2 (0.01 M), and 0.1 mL bismuth acetate (0.01 M) were added to 5 mL deionized water, sonicated for 10 min, and then transferred to a vacuum oven at 90 °C for 8 h. After the reaction, the mixture was centrifuged at 5500 rpm for 5 min and washed three times with deionized water to obtain Se / Bi2Se3 nanowires. The Se / Bi2Se3 nanowires were calcined in a muffle furnace at 230 °C for 20 min to remove the Se phase, yielding pure Bi2Se3 nanowires. These nanowires were redispersed in deionized water to obtain a Bi2Se3 nanowire dispersion for later use. 1 mL NaOH (0.05 M), 1 mL hexadecyltrimethylammonium bromide (0.1 M), 1 mL ascorbic acid (0.1 M), 0.3 mL thioacetamide (0.01 M), and 0.25 mL cadmium acetate (0.01 M) were added to 5 mL deionized water. Add M) to 5 mL of Bi2Se3 nanowire dispersion, mix well, react at 90℃ for 8 h, centrifuge and wash to obtain Cd / Bi2Se3, redisperse in deionized water to obtain Cd / Bi2Se3 dispersion; then add 1 mL NaOH (0.05 M), 1 mL hexadecyltrimethylammonium bromide (0.1 M), 1 mL ascorbic acid (0.1 M), 0.3 mL thioacetamide (0.01 M) and 0.25 mL zinc sulfate (0.01 M) to 5 mL of Cd / Bi2Se3 dispersion, react at 90℃ for 8 h; after the reaction, calcine at 230℃ for 30 min to remove residual Se phase, centrifuge and wash 3 times to obtain ZnS / CdS / Bi2Se3 complex.

[0030] S2. The ZnS / CdS / Bi2Se3 / ITO electrode was immersed in 100 μL of a 2.5 μM S1:C1:M triplet solution and incubated at 4 °C for 3 h to obtain the sensing substrate S1:C1:M / ZnS / CdS / Bi2Se3 / ITO. Before use, the C1 chain needs to be incubated with 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride at 4 °C for 1 h to break the disulfide bonds. The S1:C1:M triplet was prepared by mixing the S1 chain, C1 chain and M chain in an equimolar ratio and reacting for 1 h.

[0031] The ZnS / CdS / Bi2Se3 / ITO electrode was immersed in 100 μL of a 2.5 μM S2:C2:M triplet solution and incubated at 4 °C for 3 h to obtain the sensing substrate S2:C2:M / ZnS / CdS / Bi2Se3 / ITO. The C2 chain required incubation with 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride at 4 °C for 1 h before use to break disulfide bonds. The S2:C2:M triplet was prepared by mixing the S2, C2, and M chains in an equimolar ratio and reacting for 1 h.

[0032] S3. The DNA molecular system includes aptamer 1 strand, T1 strand, probe 1-PtPd / CeO2, aptamer 2 strand, T2 strand, and probe 2-PtPd / CeO2. The preparation method of probe 1-PtPd / CeO2 is as follows: 50 μL of thiol-labeled 5 μM probe 1 chain is mixed with an equal volume of PtPd / CeO2 heterojunction (1 mg / mL) and reacted for 2 h to obtain probe 1-PtPd / CeO2.

[0033] The preparation method of probe 2-PtPd / CeO2 is as follows: 50 μL of thiol-labeled 5 μM probe 2 chain is mixed with an equal volume of PtPd / CeO2 heterojunction (1 mg / mL) and reacted for 2 h to obtain probe 2-PtPd / CeO2.

[0034] The preparation method of PtPd / CeO2 heterojunction is as follows: 1.52 g Ce(NO3)2·6H2O was dissolved in 30 mL of deionized water to obtain solution A; 9.6 g NaOH was dissolved in 40 mL of deionized water to obtain solution B; solution B was added dropwise to solution A, and the mixture was continuously magnetically stirred for 20 min to form a homogeneous slurry; the slurry was transferred to a 100 mL polytetrafluoroethylene high-pressure reactor and reacted at 100 °C for 24 h. After naturally cooling to room temperature, the mixture was centrifuged and washed until neutral, and then vacuum dried to obtain CeO2 nanorods; 50 mg of CeO2 nanorods were dispersed in 10 mL of deionized water and magnetically stirred until homogeneous. 0.40 mL of H2PtCl6 (19.4 mM) and 0.40 mL of H2PdCl4 (100 mM) were added sequentially, and the mixture was mixed for 10 min. Then, 0.80 mL of ascorbic acid (0.1 M) was added, and the mixture was magnetically stirred and incubated for 12 hours in the dark. h; After the reaction was completed, the mixture was centrifuged and washed three times to obtain PtPd / CeO2 heterojunctions, which were then dispersed in deionized water for later use.

[0035] The method of using the biosensor constructed above is as follows: 1. Methods for detecting Aβ1-42: N1. The plasma sample from an Alzheimer's disease patient to be tested was mixed with aptamer 1:T1 modified magnetic beads, reacted in a shaker at 25°C for 2 h, and then magnetically washed 3 times to obtain the Aβ1-42-dependent T1 chain. This step can realize the signal transduction of Aβ1-42 in the plasma of Alzheimer's disease patients.

[0036] Preparation method of aptamer 1:T1 modified magnetic beads: 100 μL of carboxyl-activated magnetic beads (1 mg / mL) was mixed with 100 μL of 5'-NH2 modified aptamer 1 (concentration 3 μM), and the mixture was reacted in a shaker at 25℃ for 2 h. After magnetic washing 3 times, the aptamer 1 / magnetic bead complex was obtained. The obtained aptamer 1 / magnetic bead complex was further mixed with 100 μL of T1 chain solution (concentration 3 μM), and the mixture was reacted in a shaker at 25℃ for 1 h. After magnetic washing 3 times, the aptamer 1:T1 modified magnetic beads were obtained.

[0037] N2. The sensing substrate S1:C1:M / ZnS / CdS / Bi2Se3 / ITO was reacted with 100 μL of a mixed solution containing an Aβ1-42-dependent T1 chain and a 2.5 μM probe 1-PtPd / CeO2 at 37 °C for 50 min to activate the fulcrum-mediated chain substitution reaction on the surface of the sensing substrate. Then, the reacted sensing substrate was used as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system. The ECL signal was detected in a buffer solution containing 50 mM H2O2. The Aβ1-42 content in the plasma of Alzheimer's disease patients could be calculated based on the standard curve between ΔECL (the difference in ECL signal with and without Aβ1-42) and the logarithm of the Aβ1-42 concentration.

[0038] 2. Methods for detecting Aβ1-40: N1. Plasma samples from Alzheimer's disease patients were mixed with aptamer 2:T2 modified magnetic beads and reacted in a shaker at 25°C for 2 h. After magnetic washing three times, the Aβ1-40-dependent T2 chain was obtained. This step enables the signal transduction of Aβ1-40 in the plasma of Alzheimer's disease patients.

[0039] Preparation method of aptamer 2:T2 modified magnetic beads: 100 μL of carboxyl-activated magnetic beads (1 mg / mL) was mixed with 100 μL of 5'-NH2 modified aptamer 2 (concentration 3 μM), and the mixture was reacted in a shaker at 25℃ for 2 h. After magnetic washing 3 times, the aptamer 2 / magnetic bead complex was obtained. The obtained aptamer 2 / magnetic bead complex was further mixed with 100 μL of T2 chain solution (concentration 3 μM), and the mixture was reacted in a shaker at 25℃ for 1 h. After magnetic washing 3 times, the aptamer 2:T2 modified magnetic beads were obtained.

[0040] N2. The sensing substrate S2:C2:M / ZnS / CdS / Bi2Se3 / ITO was reacted with 100 μL of a mixed solution containing Aβ1-40-dependent T2 chains and 2.5 μM probe 2-PtPd / CeO2 at 37 °C for 50 min to activate the fulcrum-mediated chain substitution reaction on the surface of the sensing substrate. Then, the reacted sensing substrate was used as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system. The sensing substrate was incubated in a mixed solution containing 3,3'-diaminobenzidine and H2O2, and then placed in a buffer solution containing ascorbic acid. The PEC response was detected under visible light irradiation. The Aβ1-40 content in the plasma of Alzheimer's disease patients could be calculated based on the standard curve between ΔPEC (the difference in photocurrent with and without Aβ1-40) and the logarithm of the Aβ1-40 concentration.

[0041] Example 2

[0042] This embodiment tests the performance of the biosensor prepared in Example 1. The specific process is as follows: (1) Characterization of the ZnS / CdS / Bi2Se3 ternary complex The biosensor of this invention utilizes the unique photoelectric properties, good biocompatibility, and excellent electron transport efficiency of the ZnS / CdS / Bi2Se3 ternary composite material to improve the ECL and PEC signal responses. To verify the successful synthesis of the ZnS / CdS / Bi2Se3 ternary composite material, the ZnS / CdS / Bi2Se3 prepared in Example 1 needs to be characterized. First, scanning electron microscopy was used to observe that the Bi2Se3 nanowires exhibited a clear linear morphology, and the surface was smooth. (See details...) Figure 2 (A) For example Figure 2 As shown in (B)-(C), when CdS and ZnS nanoparticles are grown in situ on the surface of Bi₂Se₃ nanowires, a rough and tightly bound heterojunction interface is formed in scanning electron microscopy and transmission electron microscopy. Furthermore, X-ray photoelectron spectroscopy is used to analyze the ZnS / CdS / Bi₂Se₃ structure, as shown in (B)-(C). Figure 2 As shown in (D), this material contains Se, Bi, S, Cd, and Zn elements. The above results indicate the successful synthesis of ZnS / CdS / Bi₂Se₃.

[0043] (2) Characterization of PtPd / CeO2 heterojunction nanozymes The biosensor of this invention utilizes the bimetallic synergistic effect, carrier-interface interaction, and enzyme-like catalytic activity of the PtPd / CeO2 heterojunction to achieve simultaneous regulation of two signals. To verify the successful synthesis of the PtPd / CeO2 heterojunction, the structure of the PtPd / CeO2 prepared in Example 1 needs to be characterized. First, the structure of PtPd / CeO2 was observed using transmission electron microscopy. Figure 3 Image (A) shows a large number of rod-like structures with densely distributed fine nanoparticles on its surface; secondly, X-ray photoelectron spectroscopy was used to analyze PtPd / CeO2, such as... Figure 3 As shown in (B), this material contains Pt, Pd, O, and Ce elements. The above results indicate the successful synthesis of the PtPd / CeO2 heterojunction.

[0044] Benefiting from the synergistic effect of the heterojunction interface, PtPd / CeO2 can trigger a Fenton-like reaction, promoting the homolytic cleavage of H2O2. After adsorption and activation on the PtPd surface, H2O2 molecules gain electrons and undergo reductive cleavage, generating hydroxyl radicals (·OH). Figure 3As shown in (C), the PtPd / CeO2 heterojunction exhibits peroxidase-like activity, oxidizing colorless 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2 to generate a blue diimine radical cation, which shows a strong UV-Vis absorption peak at 630 nm. See [link to details]. Figure 3 (D) demonstrates the peroxidase-like activity of the PtPd / CeO2 heterojunction.

[0045] (3) Characterization of the sensing substrate The biosensor of this invention uses ZnS / CdS / Bi2Se3 as the sensing substrate and amplifies the signal through a fulcrum-mediated chain substitution reaction catalyzed by a PtPd / CeO2 heterojunction, thereby improving detection sensitivity. Therefore, the construction process of the sensing interface, the synergistic regulation of the PtPd / CeO2 heterojunction, and the feasibility of the fulcrum-mediated chain substitution reaction are monitored and verified.

[0046] like Figure 4 As shown, the construction of the sensing interface was characterized using ECL and PEC techniques. When ZnS / CdS / Bi₂Se₃ was assembled onto the ITO surface, the resulting ZnS / CdS / Bi₂Se₃ / ITO electrode exhibited an ECL signal of 6830 au and a corresponding photocurrent of 3.67 μA. Figure 4 Curve a in (B) indicates that the ZnS / CdS / Bi2Se3 ternary composite is a high-performance ECL and PEC substrate material. The S1:C1:M triple chain was assembled sequentially (4322 au in ECL mode). Figure 4 Curve b) of (A) or the S2:C2:M triplet (3.04 μA in PEC mode), Figure 4 Curve b in (B), and the Aβ1-42-dependent T1 chain (3510a.u. in ECL mode), Figure 4 The curve in (A) c) or the Aβ1-40-dependent T2 chain (2.62 μA in PEC mode) Figure 4 Following curve c) in (B), both ECL intensity and photocurrent decreased due to the insulating effect of the DNA molecular layer. Subsequently, probe 1-PtPd / CeO2 (or probe 2-PtPd / CeO2) was incubated on the surface of the modified electrode, and the ECL and PEC signals showed opposite trends (in ECL mode). Figure 4 In (A), the curve d is 8650 au, under PEC mode. Figure 4(Curve d in (B) is 1.58 μA). During ECL detection, the PtPd / CeO2 heterojunction nanozyme catalyzes the decomposition of H2O2 to generate ·OH, promoting the luminescence reaction and enhancing the ECL emission of ZnS / CdS / Bi2Se3. In PEC mode, the peroxidase-like activity of PtPd / CeO2 catalyzes the H2O2-mediated oxidation of 3,3'-diaminobenzidine. The resulting non-conductive precipitate layer creates a steric hindrance effect, blocking the electron transport pathway and leading to a significant decrease in photocurrent response. These results validate the successful construction of this biosensor and the feasibility of simultaneously detecting Aβ1-42 and Aβ1-40.

[0047] To verify the feasibility of an Aβ1-42-dependent T1 chain-triggered fulcrum-mediated chain substitution reaction, Figure 5 Fluorescence spectroscopy based on fluorescence resonance energy transfer was employed. The system used 5(6)-carboxyfluorescein succinimide ester (FAM) as the energy donor and gold nanospheres as the energy acceptor. The FAM-labeled single-stranded probe DNA (probe 1-FAM) exhibited a significant fluorescence signal at its characteristic emission wavelength of 527 nm, with an intensity of approximately 2055 au ( ). Figure 5 (Curve a). After adding the S1:C1:M-AuNPs complex to probe 1-FAM, the fluorescence intensity only decreased slightly. Figure 5 The fluorescence signal (b, 1866 au) is attributed to static quenching in the reaction system. Upon further addition of the Aβ1-42-dependent T1 chain, the fluorescence signal sharply decreased to 181 au. Figure 5 In curve c), the Aβ1-42-dependent T1 chain shortens the spatial distance between the FAM donor and the gold nanosphere acceptor. This energy transfer between the FAM and the gold nanosphere results in significant fluorescence quenching. These results confirm that the Aβ1-42-dependent T1 chain is crucial for the fulcrum-mediated chain substitution reaction.

[0048] (4) Validation of the detection performance of the biosensor The detection performance of the biosensor was verified using different concentrations of Aβ1-42 and Aβ1-40 (0, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 5 ng / mL and 10 ng / mL) according to the usage method described in Example 1.

[0049] ECL-time response curves of different concentrations of Aβ1-42 are shown below. Figure 6 As shown in (A), it can be seen that the ECL intensity gradually increases with the increase of Aβ1-42 concentration. The linear relationship between the ECL signal change (ΔECL) and the logarithm of the Aβ1-42 concentration is as follows: Figure 6As shown in (B), the linear regression equation is ΔECL = 1418.1 lgc (pg / mL) + 1100.8 (R²). 2 =0.995), and the detection limit is 0.25 pg / mL (S / N=3).

[0050] Similarly, the PEC response curves for different concentrations of Aβ1-40 are as follows: Figure 7 As shown in (A), it can be seen that the photocurrent intensity gradually increases with the increase of Aβ1-40 concentration. The linear relationship between the change in photocurrent intensity (Δphotocurrent intensity) and the logarithm of the Aβ1-40 concentration is as follows: Figure 7 As shown in (B), the linear regression equation is Δphotocurrent intensity = 0.287 lgc (pg / mL) + 0.298 (Rc). 2 =0.996), and the detection limit was 0.32 pg / mL (S / N=3).

[0051] Furthermore, bovine serum albumin, β-amyloid oligomers, and β-amyloid monomers were used as interfering agents to detect the specificity of the biosensor. The results are as follows: Figure 8 As shown, when bovine serum albumin, β-amyloid oligomers, and β-amyloid monomers replaced the targets Aβ1-42 and Aβ1-40, the signal changes in their ECL and PEC responses were negligible compared to the blank buffer solution control, demonstrating that the biosensor has high specificity for the detection of Aβ1-42 and Aβ1-40.

[0052] Example 3

[0053] This embodiment uses plasma samples from 6 Alzheimer's disease patients and 6 healthy volunteers to validate the detection performance of the biosensor in Example 1. The blood samples in this embodiment were obtained from volunteers recruited at a hospital in Xuzhou City, and all subjects signed informed consent forms. Sample management complied with the World Health Organization (WHO) blood collection guidelines (WHO publication ISBN 13: 978-92-4-159922-1, 2010), and the research protocol was approved by the ethics committee.

[0054] The sample processing procedure is as follows: Blood samples were placed in K2EDTA anticoagulant tubes and gently inverted 10 times to mix. To avoid aspiration of the white film layer or red blood cells, blood samples were centrifuged at 1800×g for 10 min at room temperature within 4 h of collection. Subsequently, plasma samples were transferred to low-protein adsorption tubes to reduce protein adsorption and Aβ loss. Aliquoted samples were stored at -80℃ for 24 h, avoiding repeated freeze-thaw cycles (≤2 cycles). On the day of testing, plasma samples were completely thawed on ice and centrifuged at 10000×g for 5 min at 4℃ to remove insoluble impurities. The supernatant was collected for subsequent testing. Then, the Aβ1-42 / Aβ1-40 ratio was detected according to the method described in Example 1. The Aβ1-42 / Aβ1-40 ratio in the plasma sample was calculated using the linear regression equation from Example 2, and the results are as follows. Figure 9 As shown, the Aβ1-42 / Aβ1-40 ratio in the plasma samples of Alzheimer's disease patients was significantly lower than the corresponding ratio in the plasma of healthy volunteers, showing a significant statistical difference (P<0.001).

[0055] In addition, the Aβ1-42 / Aβ1-40 ratio was detected using a standard ELISA commercial kit (purchased from Shanghai Sangon Biotech Co., Ltd.), and compared with the detection results of the biosensor of this invention. The results are as follows: Figure 10 As shown, the detection ratio error between the two methods is less than 5%. This result demonstrates that the biosensor of this invention has good detection capabilities and applications even for complex clinical plasma samples.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A biosensor for detecting dual-labeled substances in the plasma of Alzheimer's disease patients, characterized in that, The biosensor includes an electrode modified with a ZnS / CdS / Bi2Se3 complex, an S1:C1:M triplet, an S2:C2:M triplet, and a DNA molecule system; The ZnS / CdS / Bi2Se3 composite is a ternary composite nanomaterial formed by sequentially loading CdS and ZnS onto the surface of Bi2Se3 nanowires. The nucleotide sequence of the S1 chain in the S1:C1:M triplet is shown in SEQ ID NO.1, the nucleotide sequence of the C1 chain is shown in SEQ ID NO.3, and the nucleotide sequence of the M chain is shown in SEQ ID NO.

5. The nucleotide sequence of the S2 chain in the S2:C2:M triplet is shown in SEQ ID NO.2, the nucleotide sequence of the C2 chain is shown in SEQ ID NO.4, and the nucleotide sequence of the M chain is shown in SEQ ID NO.

5. The DNA molecular system includes aptamer 1 strand, T1 strand, probe 1-PtPd / CeO2, aptamer 2 strand, T2 strand, and probe 2-PtPd / CeO2. The nucleotide sequence of the aptamer 1 chain is shown in SEQ ID NO. 6; The nucleotide sequence of the T1 chain is shown in SEQ ID NO.7; The nucleotide sequence of the aptamer 2 chain is shown in SEQ ID NO. 8; The nucleotide sequence of the T2 chain is shown in SEQ ID NO.9; The probe 1-PtPd / CeO2 is obtained by heterojunction of probe 1 chain with PtPd / CeO2; the nucleotide sequence of probe 1 chain is shown in SEQ ID NO.10; The probe 2-PtPd / CeO2 is obtained by heterojunction of probe 2 chain with PtPd / CeO2; the nucleotide sequence of probe 2 chain is shown in SEQ ID NO.11; The dual markers are β-amyloid protein 1-42 and β-amyloid protein 1-40.

2. The biosensor according to claim 1, characterized in that, The electrode is an indium tin oxide electrode.

3. The biosensor according to claim 1, characterized in that, The preparation method of the ZnS / CdS / Bi2Se3 composite includes the following steps: using Se nanowires as templates, Bi2Se3 nanowires are grown in situ, and then CdS and ZnS are sequentially loaded on the surface of the Bi2Se3 nanowires by a stepwise epitaxial growth method. The residual Se phase is removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite.

4. The biosensor according to claim 3, characterized in that, The method for in-situ growth of the Bi2Se3 nanowires includes: mixing NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, SeO2 and bismuth acetate and reacting them with ultrasound to obtain Se / Bi2Se3 nanowires, and calcining to remove the Se phase to obtain the Bi2Se3 nanowires.

5. The biosensor according to claim 3, characterized in that, The method for loading the CdS and the ZnS includes: reacting the Bi2Se3 nanowires with NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, thioacetamide and cadmium acetate to obtain CdS / Bi2Se3; The CdS / Bi2Se3 was mixed with NaOH, hexadecyltrimethylammonium bromide, ascorbic acid, thioacetamide and zinc sulfate, and the Se phase was removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite.

6. The biosensor according to claim 1, characterized in that, The probe 1 chain and the probe 2 chain are coupled to the PtPd / CeO2 heterojunction via thiol groups, respectively.

7. The biosensor according to claim 1, characterized in that, The PtPd / CeO2 heterojunction is a heterostructure formed by loading PtPd bimetallic nanoparticles onto the surface of CeO2 nanorods.

8. A method for preparing a biosensor as described in any one of claims 1-7, characterized in that, Includes the following steps: Bi2Se3 nanowires were grown in situ using Se nanowires as templates. CdS and ZnS were then loaded onto the surface of the Bi2Se3 nanowires sequentially using a stepwise epitaxial growth method. The residual Se phase was removed by calcination to obtain the ZnS / CdS / Bi2Se3 composite. The ZnS / CdS / Bi2Se3 composite chitosan was mixed and then drop-coated onto the electrode surface to obtain the electrode modified with the ZnS / CdS / Bi2Se3 composite. The probe 1 chain is coupled to the PtPd / CeO2 heterojunction to obtain the probe 1-PtPd / CeO2; The probe 2 chain is coupled to the PtPd / CeO2 heterojunction to obtain the probe 2-PtPd / CeO2.

9. The use of a biosensor as described in any one of claims 1-7 in the preparation of a product for detecting dual-labeled substances in the plasma of Alzheimer's disease patients, characterized in that, The dual markers are β-amyloid protein 1-42 and β-amyloid protein 1-40.

10. A product for detecting dual markers in the plasma of Alzheimer's disease patients, characterized in that, Including the biosensor as described in any one of claims 1-7; The dual markers are β-amyloid protein 1-42 and β-amyloid protein 1-40.

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