Aptamer-mediated EXPARs-molecular beacon fluorescent biosensor as well as preparation method and application thereof

Abstract

The invention discloses an aptamer-mediated EXPARs-molecular beacon fluorescent biosensor as well as a preparation method and application of the aptamer-mediated EXPARs-molecular beacon fluorescent biosensor. The construction and detection process of the sensor comprises three key steps: protein binding aptamer, exponential isothermal amplification (EXPAR) and molecular beacon fluorescence resonance energy transfer (FRET). Through the ingenious design of the EXPAR system, the system realizes triple signal amplification, the detection sensitivity is remarkably improved, and Abeta42 and Abeta40 amyloid proteins as low as 100fM level can be detected. Besides, based on the sequence specificity of the aptamer and the highly specific design of each nucleic acid sequence involved in the detection system, the method can be used for simultaneously detecting the Abeta42 amyloid protein and the Abeta40 amyloid protein or other substances in the same system (such as a plasma sample), so that the detection efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease and the most common cause of dementia. Its onset is insidious, and diagnosis, especially early diagnosis, is difficult. In both developed and developing countries, Alzheimer's disease has a significant impact on individuals, caregivers, and society.

[0003] Aβ-amyloid protein, a polypeptide containing 39-42 amino acids, is a major cause of Alzheimer's disease due to its aggregation and deposition in brain tissue. Aβ42 and Aβ40 amyloid proteins, in particular, are key components. Pathologically, the accumulation of soluble Aβ42 and Aβ40 oligomers in brain tissue is a crucial factor in the formation of amyloid plaques, a characteristic pathological change in Alzheimer's disease. Furthermore, Aβ42 and Aβ40 are among the earliest biomarkers to show changes in their levels during the biobiogenesis of Alzheimer's disease, with changes occurring in cerebrospinal fluid and plasma (which permeates from cerebrospinal fluid) as early as 17-23 years into the preclinical stage. Aβ42 and Aβ40 are also among the most reliable diagnostic indicators at any stage, aiding in the differential diagnosis of AD or non-AD when patients enter the MCI or dementia stage. Therefore, detecting their concentrations in cerebrospinal fluid or plasma is of great significance for screening and diagnosing Alzheimer's disease. Currently, detection technologies for Aβ42 and Aβ40 are mainly divided into conventional and emerging technologies. Conventional technologies, such as ELISA, have low sensitivity and can only be used for qualitative detection. Chemiluminescent immunoassay, a more sensitive conventional technology, also faces problems such as high cost and interference from heterophile antibodies. Emerging technologies, such as single-molecule immunoassay arrays and microfluidic chips, while offering high sensitivity and accuracy, also suffer from high costs and implementation difficulties. The detection of Aβ42 and Aβ40 amyloid proteins primarily relies on cerebrospinal fluid (CSF) or plasma samples. Currently, plasma Aβ42 and Aβ40 amyloid protein detection is less invasive, and the predictive accuracy difference between plasma and CSF models is not statistically significant (p=0.44, AUC=0.93). Furthermore, plasma biomarker detection can also meet clinical predictive needs. Therefore, plasma Aβ42 and Aβ40 detection is gradually replacing the more invasive CSF detection. However, the levels of Aβ42 and Aβ40 in the plasma of Alzheimer's patients are extremely low, making conventional detection technologies often inadequate. Furthermore, due to the increasing prevalence of Alzheimer's disease, the pressure on screening is growing, making the use of emerging methods impractical. In addition to the absolute concentrations of Aβ42 and Aβ40, decreased cerebrospinal fluid and plasma Aβ42 / Aβ40 ratios are also associated with memory decline. In cerebrospinal fluid, this ratio is more significantly associated with amyloid protein load and cognitive decline than Aβ42 levels alone. In plasma, multiple studies have found significant differences in the Aβ42 / Aβ40 ratio between patients and cognitively normal individuals. Therefore, the Aβ42 / Aβ40 ratio is also a highly valuable indicator. However, the abundance of Aβ42 and Aβ40 in plasma is extremely low, highlighting the urgent need for rapid, sensitive methods that can simultaneously detect Aβ42 and Aβ40.Therefore, we currently need to develop a simple, highly sensitive, and highly specific method for detecting Aβ42 and Aβ40.

[0004] Aptamers are single-stranded DNA or RNA molecules obtained through artificial selection. They can bind to target molecules with high specificity and affinity and undergo specific spatial structural changes, playing a role in signal transduction in the detection of their target molecules. Since the Aβ42 and Aβ40 aptamers were identified, they have quickly gained public attention and play a very important role in the detection of Aβ42 and Aβ40. In recent years, isothermal amplification technology has been increasingly widely used. It can maintain a constant temperature during amplification without complex thermal cycling steps. Among them, EXPAR (exponential isothermal amplification) is a highly efficient isothermal amplification method, and its amplification efficiency can be further improved through flexible template design. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, its preparation method, and its application.

[0006] To achieve its objective, the present invention employs the following technical solution:

[0007] A first aspect of the present invention provides a method for preparing an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, comprising the following steps:

[0008] (1) Synthesis of aptamer complex: Aβ42 aptamer solution, Aβ42 primer solution, Aβ40 aptamer solution and Aβ40 primer solution were mixed and reacted to obtain Aβ42 / Aβ40 aptamer complex mixture;

[0009] (2) Magnetic bead coupling: After the magnetic separation of avidin-modified magnetic beads, the Aβ42 / Aβ40 aptamer complex mixture solution prepared in step (1) is added to them, and after thorough shaking and suspension, the mixture is incubated at room temperature for coupling.

[0010] (3) Primer replacement and separation: Take the oligomers of Aβ42 protein and Aβ40 protein standards or the test sample containing Aβ42 protein and Aβ40 protein and add them to the system obtained in step (2), and incubate at 25~45℃ for 5~25 minutes; after incubation, magnetically separate and separate the supernatant for later use;

[0011] (4) Preparation of EXPAR final product: Take Aβ40 template XXY, Aβ40 template YYZ, Aβ42 template XXY, Aβ42 template YYZ, dNTPs and the supernatant obtained in step (3) to prepare a reaction system. Add the enzyme and buffer required for isothermal amplification, perform isothermal amplification, carry out EXPAR reaction at 55~65℃, and then terminate the reaction to obtain EXPAR final product;

[0012] (5) Molecular beacon binding product: Aβ42 molecular beacon and Aβ40 molecular beacon are added to the EXPAR final product obtained in step (4), and mixed and incubated at 49~60℃ to form the fluorescent biosensor;

[0013] The nucleotide sequences of the Aβ42 aptamer, Aβ42 primer, Aβ40 aptamer, Aβ40 primer, Aβ42 template XXY, Aβ42 template YYZ, Aβ40 template XXY, Aβ40 template YYZ, Aβ42 molecular beacon, and Aβ40 molecular beacon are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0014] Preferably, the reaction conditions for step (1) are: denaturation at 95°C for 5 minutes, followed by annealing at 0.1°C / second to 25°C to form an Aβ42 / Aβ40 aptamer complex mixture.

[0015] Preferably, step (3) involves incubating at 25°C for 5 minutes.

[0016] Preferably, the reaction system in step (4) further contains: 10×NeBuffer r3.1, Vent (exo-) DNA polymerase, Nt.BstNBI nicking endonuclease, and 10×ThermoPol The reaction buffer was prepared, and the reaction was carried out at 55-60°C for 16-19 minutes, followed by incubation at 80°C for 15 minutes to terminate the reaction.

[0017] Preferably, in step (5), the mixture is incubated at 49°C for 8 to 15 minutes.

[0018] A second aspect of the present invention provides an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, prepared using any of the methods described above.

[0019] A third aspect of the invention provides the application of the above-described aptamer-mediated EXPAR-molecular beacon fluorescent biosensor in the detection of Aβ42 and Aβ40.

[0020] Furthermore, the application is as follows:

[0021] (1) Different concentrations of Aβ42 and Aβ40 protein standards were reacted in the fluorescent biosensor, and the fluorescence intensity of the fluorescent biosensor was tested to construct a standard curve and obtain a linear equation.

[0022] (2) The sample to be tested is reacted in the fluorescent biosensor, and its fluorescence signal is measured by a fluorescence spectrometer. According to the standard curve or linear equation, the concentration of Aβ42 and Aβ40 proteins in the sample to be tested is calculated to determine whether the sample to be tested contains Aβ42 and Aβ40 proteins and their concentration.

[0023] The sample to be tested is plasma.

[0024] In the application technology solution, the detection linear range is Aβ40: 1 pM-10 nM, Aβ42: 0.5 pM-5 nM.

[0025] The beneficial effects of this invention are:

[0026] This invention provides a fluorescent biosensor for the ultrasensitive detection of Aβ42 and Aβ40 in Alzheimer's disease plasma. It is based on a molecular beacon (MB)-assisted molecular beacon fluorescent biosensor mediated by specific aptamers of Aβ42 and Aβ40 amyloid-beta proteins, enabling ultrasensitive and convenient multiplex detection of Aβ42 and Aβ40 amyloid proteins in the plasma of Alzheimer's disease (AD) patients. To validate this strategy, we used Aβ42 and Aβ40 amyloid proteins for practical detection. In the presence of the target, the protein binds to the corresponding aptamer, altering its conformation and displacing the aptamer's complementary strand. This complementary strand serves as a primer to trigger EXPAR. We improved upon the conventional EXPAR dual-template design of "XY-YY" by adding an amplification site "X" to the first template to further improve EXPAR amplification efficiency; and adding a site "Z" to the second template to further enhance EXPAR amplification efficiency, achieving dual exponential amplification while reducing competition for binding sites with molecular beacons in the signal output stage. EXPAR triggering produces a large number of products, which activate their complementary molecular beacons, generating fluorescent signals. This enables rapid, sensitive, and convenient detection of Aβ42 and Aβ40. Furthermore, the specificity of the detection method is improved by leveraging the sequence specificity between different target aptamers and the specific design of each sequence in the detection system for different targets. Through flexible design of each sequence in the system, convenient multiplex detection of different targets within the same system is achieved simultaneously, and it can also be used for the detection of other biomarkers. The proposed detection platform possesses excellent characteristics such as low cost, ease of operation, high sensitivity and specificity, short detection time, and good versatility, showing great potential in the screening and diagnosis of Alzheimer's disease.

[0027] The detection process of the sensor of this invention includes three key steps: protein-binding aptamer, exponential isothermal amplification (EXPAR), and molecular beacon fluorescence resonance energy transfer (FRET). Through ingenious design of the EXPAR system, the system achieves triple signal amplification, significantly improving detection sensitivity and enabling the detection of Aβ42 and Aβ40 amyloid proteins down to the 100 fM level. Furthermore, based on the sequence specificity of the aptamer and the highly specific design for each nucleic acid sequence involved in the detection system, this method can simultaneously detect Aβ42 amyloid protein, Aβ40 amyloid protein, or other substances in the same system (such as plasma samples), thereby improving detection efficiency. Notably, this sensor has demonstrated good reliability in actual clinical plasma samples and has been successfully applied to the detection of Aβ42 and Aβ40 amyloid proteins in clinical plasma samples.

[0028] (1) The construction of the fluorescent biosensor of the present invention achieves dual signal amplification, thereby significantly improving the detection sensitivity.

[0029] (2) The fluorescent biosensor of the present invention utilizes the characteristic that the concentration of template 1 is often much higher than that of template 2 in the dual-template EXPAR system. A Z site is added to the final product generation template (original YY template), so that the sequence of molecular beacon binding is the sequence generated by the Z site, which reduces the sites competing with the molecular beacon for binding to the final product and further improves the detection sensitivity.

[0030] (3) The fluorescent biosensor of the present invention uses aptamers with high sequence specificity to different targets as signal converters and designs each sequence in the detection system for different targets with high specificity, thereby significantly improving the detection specificity.

[0031] (4) The construction of the fluorescent biosensor of the present invention, through the flexible design of each sequence in the system, enables multiple detection of different targets in the same system at the same time, thereby significantly improving the detection efficiency.

[0032] (5) The fluorescent biosensor of the present invention is programmable and can detect other biomarkers based on the characteristics described in (2) and (3) above. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the construction principle of the fluorescent biosensor of this invention.

[0034] Figure 2Figure 1 shows the feasibility analysis and optimization results of replacing the aptamer complementary strand with Aβ42 and Aβ40 amyloid proteins: (a) Fluorescence spectrophotometer results and spectra of the optimal incubation temperature and time for replacing the aptamer complementary strand with Aβ42 amyloid proteins; (b) Temperature fixed at 25℃; (c) Temperature fixed at 37℃; (d) Temperature fixed at 45℃; (e) Fluorescence spectrophotometer results; (f) Temperature fixed at 25℃; (g) Temperature fixed at 37℃; (h) Temperature fixed at 45℃.

[0035] Figure 3 The following figures show the preliminary characterization, feasibility analysis, and optimization results of the Vent (exo-) DNA polymerase and Nt.BstNBI nicking endonuclease dosages for the Aβ42 and Aβ40 EXPAR systems: (a) 12% polyacrylamide gel electrophoresis image of the Aβ42 EXPAR system for feasibility analysis and preliminary characterization: M: 25-500bp DNA Marker; Lane A: Aβ42 XXY template; Lane B: Aβ42 YYZ template; Lane C: No polymerase added; Lane D: Complete EXPAR system; Lane E: No enzymes added; Lane F: No Nt.BstNBI added; (b) 12% polyacrylamide gel electrophoresis image of the Aβ40 EXPAR system for feasibility analysis and preliminary characterization: M: 25-500bp DNA Marker; Lane A: Aβ40 XXY template; Lane B: Aβ40 Y-YZ template; Lane C: Aβ40 XXY template + Aβ40 primer; Lane D: complete EXPAR system; Lane E: no polymerase added; Lane F: no Nt.BstNBI added; Lane G: neither enzyme added; (c) Electrophoresis band of Aβ42 template XX-Y + Aβ42 primer (lane A); (de) qPCR amplification curve (d) and results (e) with optimized Vent (exo-) DNA polymerase dosage; (fg) qPCR amplification curve (f) and results (g) with optimized Nt.BstNBI nicking restriction enzyme dosage.

[0036] Figure 4Figures show the optimized results of the dosage of ThermoPol Buffer and NeBuffer r3.1: (a) qPCR amplification curve and results with optimized ThermoPol Buffer concentration; (b) qPCR amplification curve and results with optimized NeBuffer r3.1 concentration.

[0037] Figure 5 The following is a graph showing the optimized dosage and ratio of XXY and YYZ templates: (ad) qPCR amplification curves and results of optimized XXY and YYZ concentrations and ratios: (ab) XXY concentration is fixed at 100 nM; (cd) YYZ concentration is fixed at 10 nM.

[0038] Figure 6 The results of DMSO dosage optimization and EXPAR effect verification of Aβ40 protein detection system are shown in the figure: (a) qPCR amplification curve and results of DMSO concentration optimization; (b) qPCR amplification curve and results of Aβ40 EXPAR system.

[0039] Figure 7 These are the fluorescence results from the qPCR instrument used to characterize the Aβ42 detection system.

[0040] Figure 8 These are the fluorescence results from the qPCR instrument used to characterize the Aβ40 detection system.

[0041] Figure 9 The results of optimizing the incubation temperature for the molecular beacon and product in the Aβ42 and Aβ40 detection systems are shown in the figure: (a) qPCR fluorescence results after optimizing the incubation temperature of the Aβ42 molecular beacon and complementary strand; (b) qPCR fluorescence results after optimizing the incubation temperature of the Aβ40 molecular beacon and complementary strand.

[0042] Figure 10 The results of optimizing the molecular beacon concentrations of the Aβ42 and Aβ40 detection systems are shown in the figure: (a) Optimization of Aβ42 molecular beacon concentration; (b) Optimization of Aβ40 molecular beacon concentration.

[0043] Figure 11 The following are the comparison results of the detection effects of four detection systems for Aβ42 and Aβ40: (a) qPCR fluorescence results of the four detection systems for Aβ42; (b) qPCR fluorescence results of the four detection systems for Aβ40.

[0044] Figure 12 These are schematic diagrams illustrating the principles of the XXZ / ZZ, XZZ, and XZ / ZZ systems.

[0045] Figure 13 The following are the sensitivity test results for the Aβ42 and Aβ40 detection systems: (a) qPCR fluorescence results for sensitivity testing of the Aβ42 detection system; (b) qPCR fluorescence results for sensitivity testing of the Aβ40 detection system.

[0046] Figure 14 The following are the repeatability test results for the Aβ42 and Aβ40 detection systems: (a) qPCR fluorescence results for repeatability testing of the Aβ42 detection system; (b) qPCR fluorescence results for repeatability testing of the Aβ40 detection system.

[0047] Figure 15 The following are the specific detection results of the Aβ42 and Aβ40 detection systems: (a) qPCR fluorescence results of the Aβ42 detection system; (b) qPCR fluorescence results of the Aβ40 detection system.

[0048] Figure 16 This is a graph showing the results of measuring the Aβ42 / Aβ40 concentration ratio in 31 clinical plasma samples. Detailed Implementation

[0049] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0051] The nucleotide sequences involved in the embodiments of this invention are shown in Table 1:

[0052] Table 1

[0053]

[0054] Aβ42 amyloid protein sequence (SEQ ID NO.19): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.

[0055] Aβ40 amyloid protein (SEQ ID NO.20): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.

[0056] The main reagents and their sources in the examples are as follows:

[0057] Nt.BstNBI nicking endonuclease (NEB, USA), Vent (exo-) DNA polymerase (NEB, USA), 10×ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Triton® X-100 pH 8.8 (NEB, USA), 10×NeBuffer r3.1 (1M NaCl, 500 mM Tris-HCl, 100 mM MgCl2, 1 mg / ml Recombinant Albumin, pH 7.9 @ ​​25℃) (NEB, USA), 1M pH 7.4 Tris-HCl (Beyotime, Shanghai), KCl (Sangon Biotech, Shanghai), Streptavidin magnetic beads (MCE, USA), dNTPs mixture (Lanjieke, Hefei), Hexafluoroisopropanol (HFIP) (Maclean, Shanghai), Dimethyl sulfoxide (DMSO) (Solepro, Beijing), 100×BL1439A protease inhibitor (Lanjieke, Hefei), SYBR GreenI (Lanjieke, Hefei), Lysozyme (Solepro, Beijing), Bovine Serum Albumin (BSA) (BIOAGRIO, Australia), Carcinoembryonic Antigen (CEA) (Sangon Biotech, Shanghai), 30% Acrylamide / Diacrylamide Solution (29:1) (Sangon Biotech, Shanghai), 5×Tris-Borate-EDTA Buffer (TBE) (Sangon Biotech, Shanghai), 1×Tris-Borate-EDTA Buffer (TBE) (Sangon Biotech, Shanghai), N,N,N',N'-Tetramethylethylenediamine (VETEC, Germany), 10,000×Gelred (BBI, Shanghai), 25-500 bp DNA Marker (BBI, Shanghai), 6×Loading Buffer (Takara, Dalian).

[0058] The Binding Buffer used for aptamer complex synthesis in Example 1 was prepared with ddH2O and contained 50 mM Tris-HCl, 100 mM KCl, and pH 7.4.

[0059] Example 1: Aptamer-mediated EXPAR-molecular beacon fluorescent biosensor of the present invention

[0060] Preparation of the aptamer-mediated EXPAR-molecular beacon fluorescent biosensor of the present invention ( Figure 1 (This is a schematic diagram of the construction process). To perform the detection, follow these steps. The nucleotide sequences used in the preparation steps are shown in Table 1:

[0061] (1) Synthesis of aptamer complex:

[0062] Dilute the aptamers and their complementary strands in Table 1 with Binding Buffer. Take 2 μL of Aβ42 aptamer (40 μM), 2 μL of Aβ42 aptamer complementary strand (40 μM), 2 μL of Aβ40 aptamer (40 μM), and 2 μL of Aβ40 aptamer complementary strand (40 μM) and mix them. Place the mixture in a PCR instrument for denaturation (95℃, 5 min) and annealing (0.1℃ / s, 25℃) to form an Aβ42 / Aβ40 aptamer complex mixture.

[0063] (2) Magnetic bead coupling: Vortex the magnetic beads for 20 seconds to fully suspend them. Take 5 μL of magnetic beads (10 mg / mL) into a 200 μL PCR tube, place it on a magnetic rack, and magnetically separate them. Discard the supernatant. Repeat the magnetic separation and supernatant disposal steps once. Add 50 μL of Binding Buffer and wash the magnetic beads thoroughly. After adding Binding Buffer to the PCR tube, cap the tube, vortex the magnetic beads for 15 seconds, and magnetically separate them. Discard the supernatant. Perform the above steps 3 times to obtain magnetically separated magnetic beads. Then add 8 μL of the Aβ42 / Aβ40 aptamer complex mixture solution prepared in step (1) to it, vortex to fully suspend it, and incubate at room temperature for 30 minutes.

[0064] (3) Treat Aβ42 and Aβ40 protein monomers into oligomers: Since only the oligomeric form of Aβ42 and Aβ40 protein can bind to its specific DNA aptamer and replace the complementary strand, the Aβ protein monomers need to be prepared into oligomers before conducting the experiment. Aβ42 and Aβ40 proteins were treated separately: Hexafluoroisopropanol (HFIP) was pre-chilled on ice for 5-10 minutes. Taking the treatment of a 20 μg protein sample as an example, 4.43 μL of HFIP was added to the 20 μg Aβ42 protein sample to dissolve Aβ42 (4.62 μL for the Aβ40 protein sample), ensuring that all Aβ42 was in monomeric state. The solution was placed at room temperature for 30 minutes to ensure that the solution was colorless and clear. This clear solution was then left uncapped in a fume hood overnight to allow the HFIP to evaporate completely. Then, 0.89 μL of dimethyl sulfoxide (DMSO), which had been pre-chilled on ice for 5-10 minutes, was added to bring the protein concentration to 5 mM. Finally, 44.5 μL of 1×PBS, which had been pre-chilled on ice for 5-10 minutes, was added to dilute to 100 μM. The mixture was stirred for 15 seconds and incubated at 4°C for 24 hours to obtain protein oligomers. A small portion of these oligomers was then taken out and diluted with 1×PBS to the required concentration for use.

[0065] (4) Replacement and separation of primers (i.e., complementary strands of aptamers): Take 3 μL of Aβ42 protein oligomer solution (0.5, 5, 25, 250, 1000, 5000 pM) and 3 μL of Aβ40 protein oligomer solution (1, 5, 50, 500, 1000, 10000 pM) prepared in step (3) and add them to 13 μL of the system obtained in step (2). Then add 1 μL of Binding Buffer to bring the system to 20 μL and incubate at 25°C for 5 minutes. Magnetic separation is performed to separate the supernatant for subsequent use.

[0066] (5) Product generation: EXPAR was divided into two parts, A and B. Part A was prepared as follows: 0.5 μL Aβ40 template XXY (2 μM), 0.5 μL Aβ40 template YYZ (200 nM), 0.5 μL Aβ42 template XXY (2 μM), 0.5 μL Aβ42 template YYZ (200 nM), 2 μL dNTPs mixture (10 mM), and 1 μL 10×NeBuffer r3.1. Part B was prepared as follows: 0.3 μL Vent (exo-) DNA polymerase (2 U / μL), 0.8 μL Nt.BstNBI nicking endonuclease (10 U / μL), and 2 μL 10×ThermoPol The reaction buffer and 1.9 μL ddH2O were prepared. After mixing the two parts A and B, the supernatant (10 μL) obtained in step (4) was added as a primer to the 10 μL mixture of the two parts A and B, and the total volume was 20 μL.

[0067] The prepared reaction system was subjected to isothermal amplification: 55℃ for 17.5 minutes (35 cycles, 30 seconds per cycle, with fluorescence signal acquisition at the end of each cycle), followed by incubation at 80℃ for 15 minutes to terminate the reaction, yielding the EXPAR final product. During the reaction, the free Aβ42 aptamer complementary strand and Aβ40 aptamer complementary strand in the reaction system acted as specific primers, binding to the first "X" site of their corresponding XXY templates. Under the action of Vent (exo-) DNA polymerase, Nt.BstNBI nicking endonuclease, and dNTPs, target-specific EXPAR amplification was initiated in the PCR instrument, generating the first round of amplification products. The first round of amplification products cleaved by Nt.BstNBI nicking endonuclease then bound to the first "X" site of more XXY templates, generating more first round of amplification products; the abundance of these cleaved products by Nt.BstNBI nicking endonuclease... The significantly amplified first-round amplification product binds to the second "X" site of the XXY template, generating the corresponding second-round amplification product. The second-round product, cleaved by the Nt.BstNBI nicking restriction enzyme, binds to the first "Y" site of the YYZ template, producing a third-round amplification product. This third-round amplification product, again cleaved by the Nt.BstNBI nicking restriction enzyme, binds to the first "Y" site of more YYZ templates, generating even more third-round amplification products. Finally, these exponentially amplified third-round products, cleaved by the Nt.BstNBI nicking restriction enzyme, bind to the second "Y" site of the YYZ template, generating the fourth-round product, the EXPAR final product. The entire process involves multiple cycles to achieve exponential signal amplification.

[0068] (6) Molecular beacon binding product: Molecular beacons are added after the EXPAR reaction is terminated. This is because the optimal hybridization temperature of the molecular beacon and the EXPAR final product is different from the temperature required for EXPAR amplification, and the stepwise addition helps to reduce the complexity of the EXPAR reaction system. 2.5 μL of Aβ42 molecular beacon (2 μM) and 2.5 μL of Aβ40 molecular beacon (2 μM) are added to 20 μL of the EXPAR final product obtained in step (5), for a total volume of 25 μL. The mixture is then incubated at 49 °C for 10 minutes in a qPCR instrument.

[0069] (7) Acquiring fluorescence signals: When the molecular beacon is not bound to the product, it is in a closed state. The quencher group at the 3' end is close to the fluorophore at the 5' end, and the fluorophore is quenched and cannot emit any fluorescence. When the molecular beacon is bound to the product, it is opened, and the quencher group at the 3' end is away from the fluorophore at the 5' end, and the fluorophore releases fluorescence again. The fluorescence quantification of the system in step (6) is performed using a qPCR instrument with CY5 and FAM dual channels. The fluorescence quantification time lasts for one to two cycles (30 seconds per cycle).

[0070] (8) Clinical plasma sample testing: Plasma samples were collected from 7 patients with early-stage Alzheimer's disease, 11 patients with mid-to-late-stage Alzheimer's disease, and 14 healthy controls. All samples were obtained from the Fourth People's Hospital of Yibin City. The blood collection tubes were EDTA anticoagulant tubes (purple-headed tubes). After blood collection, the plasma was separated by centrifugation at 4°C and 2000g for 15 minutes. The plasma was then slowly thawed at 4°C. After thawing, the plasma was immediately placed on ice. 1 ml of plasma was aliquoted into 10 tubes, 100 μL per tube. 1 μL of the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) was added to each tube. One tube was used for testing, and the rest were stored at -80°C for long-term storage. One sample was randomly selected and aliquoted into two tubes, one with a volume of 41 μL and the other with a volume of 60 μL (for plasma sample spike recovery determination). Before use, the plasma was centrifuged at 2000g for 15 minutes at 4°C in a refrigerated centrifuge to remove the precipitate, and the supernatant was used for testing. 6 μL of plasma sample was added to the system of step (2) to replace the Aβ42 and Aβ40 amyloid protein standard mixture in step (4) for subsequent testing.

[0071] For plasma sample spike recovery testing, aliquots of 60 μL plasma sample were divided into three 20 μL tubes. 1 μL of a mixture of Aβ42 and Aβ40 protein standards was added to each of the three 20 μL plasma sample tubes. For subsequent measurements, 6.3 μL of the mixture (with 0.7 μL of binding buffer added to maintain a total volume of 20 μL) was used for plasma sample spike recovery testing (to avoid the influence of sample dilution caused by the added protein standards). The different concentration gradients of the Aβ42 and Aβ40 protein standard mixtures were: 20 pM Aβ42 and 200 pM Aβ40, 200 pM Aβ42 and 2000 pM Aβ40, and 2000 pM Aβ42 and 20000 pM Aβ40. The plasma sample spike recovery testing method was the standard addition method. The plasma sample before adding the protein standards was prepared... (RFU) Target -RFU Blank Substitute the values ​​into the standard curve to obtain the protein concentration in the mixed system, and then add the protein standard. Substitute the protein concentration into the standard curve to obtain the protein concentration in the mixed system. Subtract the protein concentration measured before adding the protein standard from the protein concentration measured after adding the protein standard, and divide the difference by the original concentration of the added protein standard to obtain the recovery rate of the protein in the plasma sample.

[0072] Example 2: Feasibility analysis and condition optimization of replacing aptamer complementary strands with Aβ42 and Aβ40 amyloid proteins.

[0073] Since the replacement of the aptamer complementary strand with a protein is a signal conversion step in the entire detection process, its feasibility and efficiency determine the feasibility and detection efficiency of the entire sensor. In this embodiment, a feasibility analysis and condition optimization were performed on step (4) in Example 1, aiming to determine its feasibility while improving its efficiency. The fluorescence resonance energy transfer (FRET) phenomenon was used to perform a feasibility analysis and condition optimization on the replacement of the aptamer complementary strand with Aβ42 and Aβ40 proteins.

[0074] The 3' end of the Aβ42 aptamer (5 μL, 20 μM) was labeled with BHQ3, the 5' end of the complementary strand of the Aβ42 aptamer (5 μL, 20 μM) was labeled with CY5, the 3' end of the Aβ40 aptamer (5 μL, 20 μM) was labeled with BHQ1, and the 5' end of the Aβ40 aptamer (5 μL, 20 μM) was labeled with FAM (all labeled strands were synthesized by Qingke Biotechnology). The four were mixed with Binding Buffer (20 μL). A blank control group and an experimental group were set up. The blank control group was mixed with 10 μL of Binding Buffer and incubated. The experimental group was mixed with Aβ42 (5 μL, 25 μM) and Aβ40 (5 μL, 25 μM) and incubated. The blank control group and experimental group were each set up with three large groups, each containing three smaller groups. The incubation temperature for the first large group was 25℃, the second large group was 37℃, and the third large group was 45℃. The incubation time for the first smaller group was 5 minutes, the second smaller group was 15 minutes, and the third smaller group was 25 minutes. When the aptamer binds to its complementary strand, the quencher group at the 3' end of the aptamer is close to the fluorophore at the 5' end of the complementary strand, and the fluorophore is quenched and cannot emit any fluorescence. When the protein is added, the complementary strand of the aptamer is displaced from the aptamer by the protein, and the quencher group at the 3' end of the aptamer moves away from the fluorophore at the 5' end, and the fluorophore re-emits fluorescence. The fluorescence emission spectra of each group were measured at room temperature using a fluorescence spectrophotometer and a quartz cuvette with an optical path length of 1.0 cm (the excitation slit width and the emission slit width were both 5 nm). The FAM fluorescence emission spectrum was recorded in the range of 510 to 550 nm, with an excitation wavelength of 492 nm and an emission wavelength of 518 nm. The maximum fluorescence emission intensity occurred at 518 nm. The CY5 fluorescence emission spectrum was recorded in the range of 660 to 700 nm, with an excitation wavelength of 643 nm and an emission wavelength of 667 nm. The maximum fluorescence emission intensity occurred at 667 nm.

[0075] from Figure 2 It can be seen that replacing the complementary strands of the aptamers for Aβ42 and Aβ40 proteins is feasible, and the optimal conditions for replacing the complementary strands of the aptamers for Aβ42 and Aβ40 proteins are an incubation temperature of 25°C and an incubation time of 5 minutes.

[0076] Example 3: Feasibility analysis, preliminary characterization, and condition optimization of the EXPAR reaction system

[0077] Since EXPAR is the signal amplification step in the entire detection process, its feasibility and efficiency determine the efficiency of the entire sensor. In this embodiment, the feasibility analysis, preliminary characterization and condition optimization of step (5) in embodiment 1 are carried out to prove that the EXPAR system we constructed is feasible and improve its efficiency.

[0078] I. Feasibility analysis and preliminary system characterization of the EXPAR fraction in the Aβ42 detection system using 12% polyacrylamide gel electrophoresis.

[0079] The polyacrylamide gel was prepared from the following components: 5.9 mL double-distilled water, 6 mL 30% acrylamide / bisacrylamide solution (29:1), 3 mL 5×Tris-borate-EDTA buffer, 110 μL 10% ammonium persulfate, and 10 μL N,N,N',N'-tetramethylethylenediamine. The inner chamber (cathode) of the electrophoresis tank was filled with 1×TBE buffer, and the outer chamber (anode) was filled to one-quarter capacity with 1×TBE buffer. Electrophoresis was then performed at a constant voltage of 120 V for 40 minutes. After electrophoresis, the gel was carefully peeled off and transferred to a staining cassette. Double-distilled water was added to cover the gel, and 2 μL of 10,000×Gelred nucleic acid dye was added. The gel was then stained on a shaker for 15 minutes and finally analyzed using an imaging system.

[0080] Two main groups were set up: the first group was Aβ42, and the second group was Aβ40. Each group was further divided into eight subgroups: the first subgroup was template XXY; the second subgroup was template YYZ; the third subgroup was template XX-Y+ primer; the fourth subgroup was the complete EXPAR system; the fifth subgroup was the EXPAR product without Vent(exo-); the sixth subgroup was the EXPAR product without Nt.BstNBI nicking endonuclease; and the seventh subgroup was the EXPAR product without either enzyme.

[0081] from Figure 3 As shown in diagram a, lane A contains the Aβ42 template XXY, lane B contains the Aβ42 template YYZ, lane C contains the EXPAR product without the addition of Vent(exo-), lane D contains the EXPAR product of the complete system, lane E contains the EXPAR product without the addition of Nt.BstNBI nicking endonuclease, and lane F contains the EXPAR product without the addition of either enzyme. Figure 3 Lane A of c is an Aβ42 template with XX-Y+Aβ42 primers. Figure 3In lane a, almost no bands were generated in lanes C, E, and F, while a small amount of amplification products were generated in lane D. This indicates that the EXPAR part of the Aβ42 detection system is feasible and that amplification can only occur correctly when both enzymes are present.

[0082] from Figure 3 As shown in b, lane A is the Aβ40 template XXY, lane B is the Aβ40 template YYZ, lane C is the Aβ40 template XX-Y+Aβ40 primer, lane D is the EXPAR product of the complete system, lane E is the EXPAR product without the addition of Vent(exo-), lane F is the EXPAR product without the addition of Nt.BstNBI nicking endonuclease, and lane G is the EXPAR product without the addition of either enzyme. Figure 3 In b, lanes E, F, and G produced almost no bands, while lane D produced a small amount of amplification products. This indicates that the EXPAR component in the Aβ40 detection system is feasible and that amplification can only occur correctly when both enzymes are present.

[0083] To improve the sensitivity and accuracy of Aβ42 and Aβ40 detection, it is necessary to ensure high amplification efficiency and high signal-to-noise ratio of EXPAR, and to ensure that the blank control group begins to amplify just as the extremely high concentration of the target group reaches the amplification plateau (indicated by the fluorescence signal generated by SYBR Green I dye). This would result in a positive correlation between the final fluorescence intensity at detection and the logarithm of the target concentration. We optimized the Aβ42 detection system using a primer concentration of 10 nM.

[0084] II. Optimization of Vent (exo-) DNA polymerase concentration

[0085] The sensor was prepared using the same method as in Example 1, except that the concentrations of Vent (exo-) DNA polymerase used were 0.03 U / μL, 0.05 U / μL, 0.07 U / μL, and 0.09 U / μL, respectively. The amplification efficiency was finally indicated by the Sybr green I fluorescence intensity, and the results are shown in [Figure 1]. Figure 3 de. Figure 3 d represents the amplification curves for each group. Figure 3 e represents the Sybr green I fluorescence intensity of the experimental group and the blank group when the experimental group of each Vent(exo-) DNA polymerase concentration group just reaches the amplification plateau phase. It can be seen that when the Vent(exo-) DNA polymerase concentration is 0.03 U / μL, the Sybr green I fluorescence intensity and signal-to-noise ratio have reached a very high level, which is not much different from the other three higher concentration groups, and the blank group has begun to produce fluorescence signals.

[0086] III. Optimization of the concentration of Nt.BstNBI nicking endonuclease

[0087] The sensor was prepared using the same method as in Example 1, except that the concentrations of the Nt.BstNBI nicking endonuclease used were 0.2 U / μL, 0.4 U / μL, 0.6 U / μL, and 0.8 U / μL, respectively. The amplification efficiency was finally indicated by the Sybr green I fluorescence intensity, and the results are shown in [Figure 1]. Figure 3 fg. Figure 3 f represents the amplification curves for each group. Figure 3 g represents the Sybr green I fluorescence intensity of the experimental group and the blank group when the experimental group of each Nt.BstNBI nicking endonuclease concentration group just reaches the amplification plateau phase. It can be seen that when the Nt.BstNBI nicking endonuclease concentration is 0.4 U / μL, the Sybr green I fluorescence intensity has reached a very high level, which is not much different from the other three groups, and the signal-to-noise ratio is the highest among the four groups. The blank group has also begun to produce fluorescence signals.

[0088] IV. Optimization of ThermoPol Buffer Concentration

[0089] The sensor was prepared using the same method as in Example 1, except that the concentrations of ThermoPol Buffer used were 0×, 0.3×, 0.5×, and 1×. The amplification efficiency was ultimately indicated by the Sybr green I fluorescence intensity. The results are shown in [Figure 1]. Figure 4 a. When the ThermoPol Buffer concentration was 1×, Sybr greenⅠ fluorescence intensity, signal-to-noise ratio, and repeatability were the highest, and the blank group had also begun to produce fluorescence signals.

[0090] V. Optimization of NeBuffer r3.1 Concentration

[0091] The sensor was prepared using the same method as in Example 1, except that the concentrations of NeBuffer r3.1 were 0×, 0.3×, 0.5×, and 1×. The amplification efficiency was ultimately indicated by the Sybr green I fluorescence intensity, and the results are shown in [Figure 1]. Figure 4 b. When the concentration of NeBuffer 3.1 is 0.5×, the fluorescence intensity, signal-to-noise ratio and repeatability of Sybr greenⅠ are the highest, and the blank group has also begun to produce fluorescence signals.

[0092] VI. Optimization of the concentration ratio of template 1 to template 2

[0093] Given that previous literature has mentioned that in dual-template EXPAR, the concentration of template 1 is preferably higher than that of template 2, we optimized the ratio of template 1 to template 2 concentrations.

[0094] 1. Optimization of the ratio of Aβ42 template 1 (Aβ42 template XXY) concentration to Aβ42 template 2 (Aβ42 template YYZ) concentration. The template 1 concentration was fixed. The sensor fabrication method was the same as in Example 1, except that the template 1:template 2 concentration ratios were 0.5:1, 1:1, 2:1, and 5:1, and the template 1 concentration was fixed at 100 nM. The amplification efficiency was finally indicated by Sybr green I fluorescence intensity. The results are shown in [Figure 1]. Figure 5 a.

[0095] 2. Optimization of the ratio of template 1 concentration to template 2 concentration (with template 1 concentration fixed)

[0096] The sensor was prepared using the same method as in Example 1. The difference was that the ratio of template 1 concentration to template 2 concentration was 10:1, 15:1, 20:1, and 25:1, respectively, and the template 1 concentration was fixed at 100 nM. Finally, the Sybr green I fluorescence intensity was used to indicate the amplification efficiency. The results are shown in [Figure 1]. Figure 5 b.

[0097] 3. Optimization of the ratio of template 1 concentration to template 2 concentration (with template 2 concentration fixed)

[0098] The sensor was prepared using the same method as in Example 1, except that the ratio of template 1 concentration to template 2 concentration was 0.5:1, 1:1, 2:1, and 5:1, and the template 2 concentration was fixed at 10 nM. The amplification efficiency was finally indicated by Sybr green I fluorescence intensity, and the results are shown in [Figure 1]. Figure 5 c.

[0099] 4. Optimization of the ratio of template 1 concentration to template 2 concentration (with template 2 concentration fixed)

[0100] The sensor was prepared using the same method as in Example 1. The difference was that the ratio of template 1 concentration to template 2 concentration was 10:1, 15:1, 20:1, and 25:1, respectively, and the template 2 concentration was fixed at 10 nM. Finally, the Sybr green I fluorescence intensity was used to indicate the amplification efficiency. The results are shown in [Figure 1]. Figure 5 d.

[0101] from Figure 5 The results show that when the template 1:template 2 concentration ratio is 10:1 (template 1 concentration 100 nM, template 2 concentration 10 nM), the Sybr green I fluorescence intensity is the highest, and the blank group has also begun to produce fluorescence signals. (This concentration refers to the concentration of Aβ42 template in its target-only detection system. If it is the final Aβ42 and Aβ40 multiplex detection system, then the template 1 concentration is 50 nM and the template 2 concentration is 5 nM).

[0102] VII. DMSO Concentration Optimization

[0103] Given that the appropriate concentration of DMSO can improve the signal-to-noise ratio of EXPAR as mentioned in previous literature, we optimized the concentration of added DMSO.

[0104] The sensor preparation method differed from Example 1 in that different concentrations of DMSO were added: 0%, 2%, 3%, and 5%. The amplification efficiency was ultimately indicated by Sybr green I fluorescence intensity, and the results are shown below. Figure 6 a. When the added DMSO concentration was 0%, the Sybr green I fluorescence intensity and signal-to-noise ratio were both the highest. This may be due to the differences in the templates and template combinations in our designed sensors.

[0105] VIII. Validation of the EXPAR component of the Aβ40 protein detection system

[0106] After optimizing the EXPAR section of the Aβ42 protein detection system, to verify whether the optimized conditions were also effective for the EXPAR section of the Aβ40 protein detection system, the optimized conditions were applied to the EXPAR section of the Aβ40 protein detection system. Sybr green I fluorescence intensity was used to indicate amplification efficiency. The results are shown in [Figure number missing]. Figure 6 b. The optimized conditions of the EXPAR part of the Aβ42 detection system were applied to the EXPAR part of the Aβ40 protein detection system, and the effect was still good. This is because the detection systems we designed for the two targets are the same except for the template and primer sequences used.

[0107] Since SYBR Green I fluorescence intensity indicates amplification efficiency, based on the above results, we determined the final reaction conditions as follows: Vent (exo-) DNA polymerase: 0.03 U / μL; Nt.BstNBI nick endonuclease: 0.4 U / μL; ThermoPol buffer: 1×; NeBuffer r3.1: 0.5×; The concentrations of XXY template and YYZ template in their respective detection systems were 100 nM and 10 nM, respectively.

[0108] Example 4

[0109] Since the fluorescence generated by the product opening the molecular beacon is the signal output step in the entire detection process, its feasibility and efficiency determine the overall efficiency of the sensor. Therefore, to further improve the detection efficiency of Aβ42 and Aβ40, we optimized the conditions of the molecular beacon FRET fluorescence output system based on the optimized EXPAR conditions. From this point on, we will no longer use 10 nM primers for the Aβ42 or Aβ40 detection systems individually, but will instead perform multiplex detection of Aβ42 and Aβ40 proteins. Before this, we first need to characterize the Aβ42 and Aβ40 detection systems to demonstrate that the detection systems of the two targets do not interfere with each other, thus proving that the multiplex detection of the two targets is feasible. Next, we will use... (RFU) Target -RFU Blank This indicates the amount of molecular beacon activated by the product. Unless otherwise specified, when characterizing a system for one target, only the molecular beacon corresponding to that target is added, and removing the template only removes the template corresponding to that target, without affecting the template of the other target.

[0110] I. Characterization of the Aβ42 detection system

[0111] To characterize the accuracy of the Aβ42 detection system, different reaction components were added to different groups. Group 1 did not add Vent(exo-) DNA polymerase; Group 2 did not add Nt.BstNBI nicking endonuclease; Group 3 did not add either enzyme; Group 4 did not add Aβ42 template XXY; Group 5 did not add Aβ42 template YYZ; Group 6 did not add either template; Group 7 used the Aβ40 aptamer to recognize Aβ42; Group 8 used the molecular beacon of the Aβ42 detection system to detect Aβ40. Results are shown below. Figure 7 The Aβ42 detection system produced almost no results in any of these groups. Only when the system is complete and correct will obvious results be produced. .

[0112] II. Characterization of the Aβ40 detection system

[0113] To characterize the accuracy of the Aβ40 detection system, different reaction components were added to different groups. Group 1 did not add Vent(exo-) DNA polymerase; Group 2 did not add Nt.BstNBI nicking endonuclease; Group 3 did not add either enzyme; Group 4 did not add Aβ40 template XXY; Group 5 did not add Aβ40 template YYZ; Group 6 did not add either template; Group 7 used the Aβ42 aptamer to recognize Aβ40; Group 8 used the molecular beacon of the Aβ40 detection system to detect Aβ42. Results are shown in […]. Figure 8 The Aβ40 detection system produced almost no emissions in these groups. Only when the system is complete and correct will obvious results be produced. .

[0114] comprehensive Figure 7 , Figure 8 The results show that there is almost no mutual interference between the Aβ42 and Aβ40 detection systems, and multiple detection of Aβ42 and Aβ40 is feasible.

[0115] III. Optimization of the incubation temperature for mixing molecular beacons and products

[0116] Previous literature indicates that the optimal incubation temperature for hybridization between the product and molecular beacons should be 7-10°C below the stem Tm value. Choosing this temperature range is crucial because both excessively low and high temperatures present significant drawbacks: at low temperatures, molecular motion slows down, reducing the probability of collisions between molecular beacons and products, and decreasing binding efficiency. Although most molecular beacons remain closed (low background fluorescence), the product makes it extremely difficult to unfold them. Furthermore, low temperatures weaken the "strictness" of nucleic acid hybridization; even weak interactions with partially complementary sequences containing mismatches can force the stem to open, generating a fluorescent signal, leading to false positives and an inability to distinguish between perfectly matched and mismatched targets. Conversely, while excessively high temperatures increase molecular motion and collision probability, stable hybrids are difficult to form, and even if binding occurs, dissociation is likely. More seriously, excessive molecular beacons can spontaneously open, generating high background fluorescence signals. To avoid these phenomena and maximize the efficiency of opening the molecular beacon with its complementary strand, we optimized the temperature for mixing and incubating the molecular beacon with its complementary strand (product). Since the stem melting temperature (Tm) of the molecular beacon was predicted to be 57.5°C by the IDT OligoAnalyzer tool (the stem sequences of the two molecular beacons are the same), we set the starting point for temperature optimization at 49°C.

[0117] The sensor was prepared using the same method as in Example 1, except that molecular beacons of Aβ40 and Aβ42 were added, and incubation temperatures of 49°C, 55°C, and 60°C were used, respectively. The results are shown in [Figure 1]. Figure 9 ab, when the molecular beacon and product are mixed and incubated at 49°C Highest.

[0118] IV. Optimization of Molecular Beacon Concentration

[0119] The sensor was prepared using the same method as in Example 1, except that the concentrations of the Aβ40 and Aβ42 molecular beacons in the detection system were 2 nM, 20 nM, 200 nM, and 2000 nM, respectively. The results are shown in [Figure 1]. Figure 10 ab, when the molecular beacon concentration is 200 nM Highest.

[0120] V. Fluorescence Comparison of Four EXPAR Reaction Systems

[0121] Detailed sequences of the templates for each system are shown in Sequence Listing 1. System 1: XXZ / ZZ system. System 2: XZZ system. System 3: XXY / YYZ system. System 4: XZ / ZZ system ("Z" is equivalent to "Y" in the traditional EXPAR template combination, representing the amplification site that produces the final product; its sequence is the same as "Z" in the "XXY / YYZ" template combination).

[0122] The sensor was prepared using the same method as in Example 1, except that the reaction systems used were the first system, the second system, the third system, and the fourth system, respectively. A comparison was made... The optimal reaction system was determined, and the results are shown in [the table below]. Figure 11 ab, XXY / YYZ system The highest level yields the best results. The principles of the XXZ / ZZ system, XZZ system, and XZ / ZZ system are as follows: Figure 12 As shown.

[0123] Because we adopt To indicate the amount of molecular beacons opened by the product, based on the above results, we finally determined the optimal conditions as follows: the molecular beacon and the complementary strand are incubated at 49°C, the molecular beacon concentration is 200 nM, and the “XXY / YYZ” EXPAR template combination is used.

[0124] Example 5: Sensor Performance Verification

[0125] To verify the performance of our designed sensor, we tested and evaluated its sensitivity, repeatability, and specificity.

[0126] I. Sensitivity Testing

[0127] To assess the sensitivity of the Aβ42 and Aβ40 detection systems, and to determine the optimal linear range and lowest detection limit, this study aims to validate... To determine whether a strong positive correlation exists between Aβ42 and target concentration, different concentrations (0.5, 5, 25, 250, 1000, 5000 pM) of Aβ42 and (1, 5, 50, 500, 1000, 10000 pM) of Aβ40 amyloid protein were added to observe the final molecular beacon. The results are shown Figure 13 The sensor has good sensitivity. The optimal linear range for the Aβ42 detection system is 0.5 pM–5 nM, proportional to the logarithm of the target concentration, y = 2574.45656 + 2459.42092 lg x (R2 =0.9894), the optimal linear range of the Aβ40 detection system is 1 pM-10 nM, y=782.0674+128.48976 lg x (R 2 =0.9855), There is a good positive correlation between the logarithm of the target concentration and the LOD (limit of detection) value. - blank +3×S blank The detection limit of the Aβ42 detection system was found to be 0.11 pM, and the detection limit of the Aβ40 detection system was found to be 0.3 pM.

[0128] II. Repeatability Testing of the Aβ42 Detection System

[0129] To verify the repeatability of the Aβ42 detection system, five measurements were performed at three target concentrations: 5 pM, 250 pM, and 5 nM. ), calculate the coefficient of variation (CV) of the target detection results for each concentration, and the results are as follows Figure 14 a. When the Aβ42 detection system detects a 5pM target, the CV is 4.64%; when detecting a 250pM target, the CV is 8.85%; and when detecting a 5nM target, the CV is 6.73%. All CVs are less than 10%, indicating good repeatability.

[0130] III. Repeatability Testing of the Aβ40 Detection System

[0131] To verify the repeatability of the Aβ40 detection system, five measurements were performed for each of the three targets: 5 pM, 500 pM, and 10 nM. ), calculate the coefficient of variation (CV) of the target detection results for each concentration, and the results are as follows Figure 14 b. When the Aβ40 detection system detects a 5pM target, the CV is 6.4%; when detecting a 500pM target, the CV is 5.48%; and when detecting a 10nM target, the CV is 4.42%. All CVs are less than 10%, indicating good repeatability.

[0132] IV. Specificity detection of the Aβ42 detection system.

[0133] To verify the specificity of the Aβ42 detection system, Lysozyme, CEA (carcinoembryonic antigen), BSA (bovine serum albumin), and Aβ40 were detected using the Aβ42 detection system. The results are as follows: Figure 15a. When detecting CEA, BSA, and Lysozyme, the target is directly added to the Aβ42 and Aβ40 multiplex detection system. When detecting targets of the opposing detection system (e.g., Aβ40), the target is added to a detection system that only detects its own target (e.g., Aβ42). The detection system for the opposing target is supplemented with ddH2O. Figure 15 As shown in a, the Aβ42 detection system failed to produce significant results when detecting any of the four substances mentioned above. It has good specificity.

[0134] V. Specificity Detection of Aβ40 Detection System

[0135] To verify the specificity of the Aβ40 detection system, Lysozyme, CEA, BSA, and Aβ42 were detected using the Aβ40 detection system. The results are as follows: Figure 15 b. The Aβ40 detection system failed to produce any significant results when detecting the above four substances. It has good specificity.

[0136] Because we adopt The amount of molecular beacons activated by the product is used to indicate the level of these beacons. Based on the above results, we conclude that this sensor has good detection performance. Table 2 compares the detection capabilities of the sensor of this invention with other detection methods.

[0137] Table 2 Comparison of the sensor detection of the present invention with other detection methods

[0138]

[0139] Example 6: Clinical plasma sample testing

[0140] I. Determination of Aβ42 / Aβ40 concentration ratio in clinical plasma samples

[0141] To verify whether our sensor can withstand various interferences in complex samples, we measured the concentrations of Aβ42 and Aβ40 in clinical plasma samples and calculated the Aβ42 / Aβ40 concentration ratio. The results are as follows: Figure 16 The average Aβ42 / Aβ40 concentration ratio was highest in the healthy control group (0.24); lower in early-stage patients (0.11); and lowest in mid-to-late-stage patients (0.06), consistent with general patterns and serving as a marker for distinguishing between healthy individuals and Alzheimer's patients. Three early-stage patients met the cognitive impairment assessment criteria but were not diagnosed with Alzheimer's disease (the hospital does not know which three cases these were).

[0142] II. Determination of plasma sample spiked recovery rate

[0143] To evaluate the reliability of our sensor for measuring the concentrations of Aβ42 and Aβ40 in clinical plasma samples, we determined the recovery rate of plasma samples by adding 1 pM, 10 pM, and 100 pM Aβ42 standards and 10 pM, 100 pM, and 1000 pM Aβ40 standards to the plasma samples. The results are shown in Tables 3 and 4.

[0144] Table 3: Recovery rate of Aβ42 amyloid protein in clinical plasma samples (3 replicates)

[0145]

[0146] Table 4: Recovery rate of Aβ40 amyloid protein in clinical plasma samples (3 replicates)

[0147]

[0148] As shown in Tables 3 and 4, the recovery rates of Aβ42 and Aβ40 measured by this sensor in clinical plasma samples are quite satisfactory. The recovery rate of Aβ42 is in the range of 104.67%-109.3%, with a relative standard deviation of 6.84%-9.61%; the recovery rate of Aβ40 is in the range of 104.2%-106.93%, with a relative standard deviation of 4.88%-7.42%. Therefore, the results of the concentration determination of Aβ42 and Aβ40 in clinical plasma samples are reliable.

[0149] In summary, the results of the above demonstrate that the sensor of the present invention is reliable in detecting the concentrations of Aβ42 and Aβ40 amyloid proteins in actual clinical plasma samples, and can distinguish between healthy individuals and Alzheimer's patients by calculating the measured Aβ42 / Aβ40 concentration ratio.

Claims

1. A method for preparing an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, characterized in that, Includes the following steps: (1) Synthesis of aptamer complex: Aβ42 aptamer solution, Aβ42 primer solution, Aβ40 aptamer solution and Aβ40 primer solution were mixed and reacted to obtain Aβ42 / Aβ40 aptamer complex mixture; (2) Magnetic bead coupling: After the magnetic separation of avidin-modified magnetic beads, the Aβ42 / Aβ40 aptamer complex mixture solution prepared in step (1) is added to them, and after thorough shaking and suspension, the mixture is incubated at room temperature for coupling. (3) Primer replacement and separation: Take the oligomers of Aβ42 protein and Aβ40 protein standards or the test sample containing Aβ42 protein and Aβ40 protein and add them to the system obtained in step (2), and incubate at 25~45℃ for 5~25 minutes; after incubation, magnetically separate and separate the supernatant for later use; (4) Preparation of EXPAR final product: Take Aβ40 template XXY, Aβ40 template YYZ, Aβ42 template XXY, Aβ42 template YYZ, dNTPs and the supernatant obtained in step (3) to prepare a reaction system. Add the enzyme and buffer required for isothermal amplification, perform isothermal amplification, carry out EXPAR reaction at 55~65℃, and then terminate the reaction to obtain EXPAR final product; (5) Molecular beacon binding product: Aβ42 molecular beacon and Aβ40 molecular beacon are added to the EXPAR final product obtained in step (4), and mixed and incubated at 49~60℃ to form the fluorescent biosensor; The nucleotide sequences of the Aβ42 aptamer, Aβ42 primer, Aβ40 aptamer, Aβ40 primer, Aβ42 template XXY, Aβ42 template YYZ, Aβ40 template XXY, Aβ40 template YYZ, Aβ42 molecular beacon, and Aβ40 molecular beacon are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

2. The preparation method according to claim 1, characterized in that: The reaction conditions for step (1) are: denaturation at 95°C for 5 minutes, followed by annealing at 0.1°C / second to 25°C to form a mixture of Aβ42 / Aβ40 aptamer complex.

3. The preparation method according to claim 1, characterized in that: Step (3) Incubate at 25°C for 5 minutes.

4. The preparation method according to claim 1, characterized in that: The reaction system in step (4) also contains: 10×NeBuffer r3.1, Vent (exo-) DNA polymerase, Nt.BstNBI nicking endonuclease, and 10×ThermoPol reaction buffer; the reaction is carried out at 55~60℃ for 16~19 minutes, and then incubated at 80℃ for 15 minutes to terminate the reaction.

5. The preparation method according to claim 1, characterized in that: In step (5), mix and incubate at 49°C for 8-15 minutes.

6. An aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the aptamer-mediated EXPAR-molecular beacon fluorescent biosensor of claim 6 in the detection of Aβ42 and Aβ40.

8. The application according to claim 7, characterized in that, The application is as follows: (1) Different concentrations of Aβ42 and Aβ40 protein standards were reacted in the fluorescent biosensor, and the fluorescence intensity of the fluorescent biosensor was tested to construct a standard curve and obtain a linear equation. (2) The sample to be tested is reacted in the fluorescent biosensor, and its fluorescence signal is measured by a fluorescence spectrometer. According to the standard curve or linear equation, the concentration of Aβ42 and Aβ40 proteins in the sample to be tested is calculated to determine whether the sample to be tested contains Aβ42 and Aβ40 proteins and their concentration.

9. The application according to claim 8, characterized in that: The sample to be tested was plasma.

10. The application according to claim 7, characterized in that: The detection linear range is Aβ40: 1 pM-10 nM, Aβ42: 0.5 pM-5 nM.

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