Kit for detecting A beta oligomer

Abstract

The invention discloses a kit for detecting an A beta oligomer. The kit comprises a nucleic acid aptamer-activator modified streptavidin magnetic bead, a methylene blue nucleic acid signal probe modified streptavidin magnetic bead, a Cas12a-crRNA compound and a synthetic gold nanocluster probe modified glassy carbon electrode. According to the invention, the beta-CD-AuNCs probe with high quantum yield is used as an electrochemiluminescence material, methylene blue is used as an electrochemiluminescence quencher, and through combination with CRISPR / Cas technology, signal change of an ECL reaction system is effectively regulated and controlled, and AbetaO detection is realized. The method has the advantages of strong detection specificity, good selectivity, high sensitivity and strong anti-interference capability on AbetaO, and has good market value.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and biosensor technology, and in particular relates to a kit for detecting Aβ oligomers. Background Art

[0002] Alzheimer's disease (AD) is currently the most prevalent neurodegenerative disorder worldwide. Aβ peptides in the brain can aggregate into Aβ oligomers (AβOs), which then aggregate into Aβ fibrils (AβFs), ultimately leading to the formation of plaques. AβOs are considered the most neurotoxic species. Therefore, accurate quantification of AβO levels can provide valuable insights into disease status and progression, making AβOs an important biomarker for the diagnosis and research of AD. Currently, methods for detecting AβOs include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and surface-enhanced Raman spectroscopy (SERS). While these methods offer the advantages of high selectivity and sensitivity, they require expensive instrumentation, cumbersome procedures, and specialized technicians. Therefore, the development of a highly sensitive and selective biosensor detection platform is crucial for the early diagnosis and treatment of AD patients.

[0003] Electrochemiluminescence (ECL), also known as electrochemiluminescence (ECL), is the luminescence phenomenon produced by the redox reaction of a luminescent substance on the electrode surface, which forms an excited state and then returns to the ground state, using energy provided by an electrode. Due to its advantages such as low background noise, high sensitivity, wide linear range, and simple equipment, ECL analytical technology has been widely used in various fields such as environmental monitoring, bioanalysis, and clinical diagnosis. Gold nanoclusters (AuNCs) are ultrasmall nanomaterials composed of a few to hundreds of gold atoms, typically ranging in size from 1 to 3 nanometers. Due to their unique optical and electrochemical properties, catalytic activity, electronic properties, and excellent biocompatibility, they have attracted widespread attention in fields such as biomedicine, catalysis, and sensing. In particular, the application of AuNCs in ECL and fluorescence sensing provides a new tool for highly sensitive and selective biomarker detection.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR / Cas) and its associated proteins (CRISPR / Cas) have attracted significant attention as a novel technology in the life sciences. The CRISPR / Cas12a system is one of the most widely used CRISPR / Cas systems in biosensing. Guided by crRNA, this system can specifically recognize and cleave target single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), a process known as cis-cleavage activity. Furthermore, upon target binding, the CRISPR / Cas12a system exhibits unique trans-cleavage activity, cleaving surrounding nonspecific single-stranded DNA (ssDNA) with an extremely high catalytic turnover rate. Due to its exceptional sequence specificity and high sensitivity, the CRISPR / Cas12a system holds great promise for molecular diagnostics and biosensing. However, the application of the CRISPR / Cas12a system in the detection of AβO remains underexplored. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a kit for detecting Aβ oligomers based on the CRISPR / Cas12a system combined with gold nanocluster electrochemiluminescence technology. This kit should have the advantages of simple operation, low cost, high detection sensitivity and good specificity.

[0006] The technical solution adopted by the present invention is as follows: a kit for detecting Aβ oligomers, comprising nucleic acid aptamer-activator modified streptavidin magnetic beads (Aptamer-Activator / MBs), methylene blue nucleic acid signal probe modified streptavidin magnetic beads (DNA-MB / MBs), Cas12a-crRNA complex, synthetic gold nanocluster probe ( β -CD-AuNCs) modified glassy carbon electrode ( β -CD-AuNC / GCE).

[0007] Furthermore, the kit also includes a co-reactant.

[0008] The aptamer-activator modified streptavidin magnetic beads (Aptamer-Activator / MBs) are prepared as follows: Preparation of Aptamer-Activator: Mix 50 μL of 100 μM Aptamer, 50 μL of 100 μM Activator sense strand, and 50 μL of 100 μM Activator antisense strand, react at 95°C for 5 min, and cool naturally to room temperature to obtain Aptamer-Activator. Preparation of Aptamer-Activator / MBs: Take 10 μL of 10 mg / mL streptavidin magnetic beads, wash them three times with trimethylamine hydrochloride (Tris-HCl) buffer solution containing 0.1% Tween-20, add 100 μL of 0.1-1.5 μM Aptamer-Activator and mix. Incubate with shaking at 300-500 rpm at room temperature for 0.5-3 h, then wash them five times with Tris-HCl buffer solution containing 0.1% Tween-20 to remove unbound Aptamer-Activator, and disperse them in 100 μL of ultrapure water to obtain Aptamer-Activator / MBs.

[0009] The methylene blue nucleic acid signal probe modified streptavidin magnetic beads (DNA-MB / MBs) are prepared as follows: 10 μL of 10 mg / mL streptavidin magnetic beads are washed three times with a Tris-HCl buffer solution containing 0.1% Tween-20, 100 μL of 0.5-2.5 μM methylene blue nucleic acid signal probe DNA-MB is added and mixed, and the mixture is incubated at room temperature with shaking at 300-500 rpm for 0.5-3 h. Subsequently, the beads are washed five times with a Tris-HCl buffer solution containing 0.1% Tween-20 to remove unbound DNA-MB, and the beads are dispersed in 100 μL of ultrapure water to obtain DNA-MB / MBs.

[0010] The nuclease 12a-guide RNA (Cas12a-crRNA) complex was prepared by adding 10 μL of 500 nM guide RNA (crRNA) and 5 μL of 1 μM nuclease 12a (Cas12a) to a reaction buffer containing 15 μL of diethyl pyrocarbonate (DEPC)-treated water, 10 μL of 50 mM sodium chloride, 10 mM tris hydrochloride, 10 mM magnesium chloride and 100 μg / ml recombinant albumin and incubating at 25°C.

[0011] The synthesized gold nanocluster probe modified glassy carbon electrode ( β -CD-AuNC / GCE) was prepared as follows: Dissolve NaHCO3 in H2O. β -cyclodextrin ( β -CD) solution was dissolved in NaHCO3 aqueous solution to prepare β -CD alkaline reaction solution. At room temperature, add NaHCO3 aqueous solution to chloroauric acid aqueous solution containing NaHCO3 (1 M). Under vigorous stirring, add β-CD alkaline reaction solution. Add NaHCO3 aqueous solution and heat at 80℃. After the reaction is completed, filter and add to ultrafiltration centrifuge tube to obtain the product. Centrifuge and wash the product to obtain the synthesized gold nanocluster probe. β -CD-AuNCs; 4 μL β -CD-AuNCs solution was mixed with 1 μL 1% chitosan and then dropped onto the pre-treated glassy carbon electrode (GCE) surface and dried at 37 °C to obtain β -CD-AuNC / GCE.

[0012] The coreactant is prepared as follows: N,N-diisopropylethylamine is added to a PBS buffer solution at a final concentration of 0.1 M, and the pH is adjusted to 11.5 with sodium hydroxide to obtain the coreactant.

[0013] The base sequence of the probe involved in the above scheme is as follows: (1) DNA-MB: 5'-biotin-AAAAAAAAAAAAAAAAAAAAA-MB-3'; (2) Aptamer: 5'-GCCTGTGGTGTTGGGGCGGGTGCG-biotin-3'; (3) crRNA: 5'-UAAUUUCUACUAAGUGUAGUATCGCGCCCCACTGCGTTCT-3'; (4) Activator sense strand: 5'-ACTTTGTATCGCGCCCCACTGCGTTCTTCGTTGACGGCGTAA-3'; (5) Activator antisense strand: 5'-AACACCACAGGCTTACGCCGTCAACGAAGAACGCAGTGGGGCGCGATACAAAGT-3'.

[0014] When the present invention is used to detect AβO, the steps are as follows: (1) Add 100 μL of AβO at different concentrations to Aptamer-Activator / MBs and incubate at room temperature with shaking at 300-500 rpm for 0.5-3 h. Collect the supernatant containing Activator by magnetic separation.

[0015] (2) Take 50 μL of the supernatant containing Activator and mix it with the Cas12a-crRNA complex. After reacting at 37°C for 5-10 minutes, add it to the DNA-MB / MBs and incubate it at 37°C at a speed of 300-500 rpm for 0.5-3 hours. Then, take the supernatant containing the cut MB by magnetic separation; (3) Electrochemiluminescence test was performed using the step pulse method. β -CD-AuNC / GCE was used as the working electrode, platinum wire electrode as the counter electrode, Ag / AgCl as the reference electrode, DIPEA as the co-reactant, the initial potential was 0 V, the termination potential was 0.8 V, the pulse time was 1 s, the scan rate was 0.1 V / s, and the photomultiplier tube high voltage was set to 550 V. The initial ECL signal intensity was measured. After removing the electrode, the excess material on the electrode surface was removed. Subsequently, 5 μL MB was added dropwise to the prepared β -CD-AuNC / GCE reacts for 1~20min, MB can be quenched by host-guest recognition β -CD-AuNCs ECL signal, after removing the electrode, remove the excess material on the electrode surface, measure the ECL signal intensity after the reaction, and achieve ultra-high sensitivity detection of AβO through the difference between the initial ECL signal intensity and the ECL signal intensity after the reaction.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention has high quantum yield β The CD-AuNCs probe is an electrochemiluminescent material with methylene blue as an electrochemiluminescence quencher. Aptamer-activator-modified streptavidin magnetic beads (Aptamer-Activator / MBs) and methylene blue nucleic acid signal probe-modified streptavidin magnetic beads (DNA-MB / MBs) are combined with CRISPR / Cas technology to effectively regulate the signal changes in the ECL reaction system. The difference in signal changes enables the detection of AβO. The present invention has strong specificity, good selectivity, high sensitivity, and strong anti-interference ability for the detection of AβO. Furthermore, the present invention is low-cost, simple to manufacture, stable, sensitive, and has a wide linear range (1.0×10 -8 ~1.0×10 -1 μg / mL), with a low detection limit (0.2 fg / mL), and has good market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 for β -Electrochemiluminescence-time curve of CD-AuNCs modified glassy carbon electrode.

[0018] Figure 2Methylene blue / β -Electrochemiluminescence-time curve of CD-AuNCs modified glassy carbon electrode.

[0019] Figure 3 This is the electrochemiluminescence-time curve of the present invention for detecting AβO (AβO concentration is 0.1 μg / mL).

[0020] Figure 4 This is a linear relationship diagram between the change in electrochemiluminescence intensity and the logarithmic value of AβO concentration. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0022] The β-Amyloid (1-40), human oligomers (Qiangyao (Shanghai) Biotechnology Co., Ltd.), streptavidin magnetic beads (Suzhou Weidu Biotechnology Co., Ltd.), EnGen® Lba Cas12a (Cpf1) (New England Biolabs), and nucleic acid probes (Sangon Biotech (Shanghai) Co., Ltd.) used in the present invention are products of the prior art. Example

[0023] Take 8 mL of 25 mg / mL β -SH-CD and 0.16 μL 300 mg / mL chloroauric acid solution were added to 90 mL 8 mg / mL sodium bicarbonate aqueous solution, mixed and placed in a constant temperature water bath at 80 ℃ for 12 hours. After the reaction was completed, it was filtered with a 200 mesh water filter (0.22 μm) and the reaction solution was centrifuged at 7000 rpm using an ultrafiltration centrifuge tube with a molecular weight of 10,000 to obtain the purified β -CD-AuNCs were stored in a refrigerator at 4°C away from light. Example

[0024] A glassy carbon electrode with a diameter of 3 mm was polished with 1.0 μm, 0.3 μm, and 0.05 μm Al2O3 powders in sequence to a smooth mirror surface. It was then ultrasonically cleaned in HNO3 solution, anhydrous ethanol, and deionized water for 3 minutes and dried with N2. 4 μL of the solution prepared in Example 1 was taken. β -CD-AuNCs were mixed with 1 μL 1% chitosan and added dropwise on the treated glassy carbon electrode surface and dried at 37 °C to obtain β -CD-AuNCs modified glassy carbon electrode. A three-electrode system was used to βA glassy carbon electrode modified with -CD-AuNCs was used as the working electrode, a platinum wire electrode was used as the counter electrode, and Ag / AgCl was used as the reference electrode. These electrodes were inserted into a 0.1 mol / L phosphate buffer solution with a pH of 7.4 containing 0.1 mol / L N,N-diisopropylethylamine. Electrochemiluminescence (ECL) was measured using a step pulse method with an initial potential of 0 V, a termination potential of 0.8 V, a pulse time of 1 s, a scan rate of 0.1 V / s, and a photomultiplier tube high voltage of 550 V. The ECL signal generated on the working electrode surface was detected and obtained (see [1]). Figure 1 ). Example

[0025] A glassy carbon electrode with a diameter of 3 mm was polished with 1.0 μm, 0.3 μm, and 0.05 μm Al2O3 powders in sequence to a smooth mirror surface. It was then ultrasonically cleaned in HNO3 solution, anhydrous ethanol, and deionized water for 3 minutes and dried with N2. 4 μL of the solution prepared in Example 1 was taken. β -CD-AuNCs were mixed with 1 μL 1% chitosan and added dropwise on the treated glassy carbon electrode surface and dried at 37 °C to obtain β -CD-AuNCs modified glassy carbon electrode, take 5 μL of methylene blue and add it dropwise β After 5 min of reaction on the CD-AuNCs modified glassy carbon electrode, the excess material on the electrode surface was removed and a three-electrode system was used with methylene blue / β A glassy carbon electrode modified with -CD-AuNCs was used as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl reference electrode. These electrodes were inserted into a 0.1 mol / L phosphate buffer solution (pH 7.4) containing 0.1 mol / L N,N-diisopropylethylamine. Electrochemiluminescence (ECL) was measured using a step pulse method with an initial potential of 0 V, a termination potential of 0.8 V, a pulse time of 1 s, a scan rate of 0.1 V / s, and a photomultiplier tube high voltage set to 550 V. The ECL signal generated on the working electrode surface was detected, and a significant decrease in the ECL signal was observed (see Figure 2). Figure 2 ). Example

[0026] Mix 50 μL of 100 μM Aptamer, 50 μL of 100 μM Activator sense strand, and 50 μL of 100 μM Activator antisense strand, react at 95°C for 5 min, and cool naturally to room temperature to obtain Aptamer-Activator. Example

[0027] 10 μL of 10 mg / mL streptavidin magnetic beads were washed three times with a Tris-HCl buffer solution containing 0.1% Tween-20, 100 μL of 1 μM Aptamer-Activator prepared in Example 4 was added and mixed, and the mixture was incubated at room temperature with shaking at 500 rpm for 2 h. Subsequently, the beads were washed five times with a Tris-HCl buffer solution containing 0.1% Tween-20 to remove unbound Aptamer-Activator, and dispersed in 100 μL of ultrapure water to obtain Aptamer-Activator / MBs. Example

[0028] Take 10 μL of 10 mg / mL streptavidin magnetic beads, wash them three times with Tris-HCl buffer solution containing 0.1% Tween-20, add 100 μL of 1 μM DNA-MB and mix them. Incubate them at room temperature with shaking at 500 rpm for 2 h, then wash them five times with Tris-HCl buffer solution containing 0.1% Tween-20 to remove unbound DNA-MB, and disperse them in 100 μL of ultrapure water to obtain DNA-MB / MBs. Example

[0029] In a reaction buffer containing 15 μL of DEPC water and 10 μL of 1×NEBuffer 2.1, 10 μL of 500nM crRNA and 5 μL of 1 μM Cas12a were added and incubated at 25°C to prepare the Cas12a-crRNA complex. Example

[0030] 100 μL of 0.1 μg / mL AβO was added to the Aptamer-Activator / MBs prepared in Example 6, and incubated at room temperature at a speed of 500 rpm for 2 h. After magnetic bead separation, 50 μL of the supernatant containing Activator was mixed with the Cas12a-crRNA complex prepared in Example 7. After reacting at 37 ° C for 10 min, it was added to the DNA-MB / MBs prepared in Example 6. After incubation at a speed of 500 rpm at 37 ° C for 1 h, the MB solution was obtained by magnetic separation; 5 μL of MB solution was added dropwise to β After 5 min of reaction on the glassy carbon electrode modified with -CD-AuNCs, the excess substances on the electrode surface were removed and a three-electrode system was used with MB solution / βA glassy carbon electrode modified with AβO-CD-AuNCs was used as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl reference electrode. These electrodes were inserted into a 0.1 mol / L phosphate buffer solution (pH 7.4) containing 0.1 mol / L N,N-diisopropylethylamine. Electrochemiluminescence (ECL) was measured using a step pulse method with an initial potential of 0 V, a termination potential of 0.8 V, a pulse duration of 1 s, a scan rate of 0.1 V / s, and a photomultiplier tube high voltage setting of 550 V. The ECL signal generated on the working electrode surface was significantly reduced compared to that obtained in the solution without AβO (see Figure 2). Figure 3 ). Example

[0031] 100 μL of AβO at different concentrations was added to the Aptamer-Activator / MBs prepared in Example 6, and incubated at room temperature at a speed of 500 rpm for 2 h. After magnetic bead separation, 50 μL of the supernatant containing Activator was mixed with the Cas12a-crRNA complex prepared in Example 7. After reacting at 37 ° C for 10 min, it was added to the DNA-MB / MBs prepared in Example 6. After incubation at a speed of 500 rpm at 37 ° C for 1 h, the MB solution was obtained by magnetic separation; 5 μL of MB solution was added dropwise to β After 5 min of reaction on the glassy carbon electrode modified with -CD-AuNCs, the excess substances on the electrode surface were removed and a three-electrode system was used with MB solution / β A glassy carbon electrode modified with -CD-AuNCs was used as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl reference electrode. These electrodes were immersed in a 0.1 mol / L phosphate buffer solution (pH 7.4) containing 0.1 mol / L N,N-diisopropylethylamine. Electrochemiluminescence (ECL) was measured using a step pulse method with an initial potential of 0 V, a termination potential of 0.8 V, a pulse duration of 1 s, a scan rate of 0.1 V / s, and a photomultiplier tube high voltage set to 550 V. The ECL signal generated on the working electrode surface was detected at an AβO concentration of 1.0×10 -8 ~1.0×10 -1 The logarithmic value of AβO showed a good linear relationship with the difference of signal change in the range of μg / mL, and the detection limit was 0.2 fg / mL (see Figure 4 ).

[0032] The above description is merely a typical embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for detecting Aβ oligomers, comprising aptamer-activator modified streptavidin magnetic beads Aptamer-Activator / MBs, methylene blue nucleic acid signal probe modified streptavidin magnetic beads DNA-MB / MBs, Cas12a-crRNA complex, and synthetic gold nanocluster probe β -CD-AuNCs modified glassy carbon electrode β -CD-AuNC / GCE.

2. The kit for detecting Aβ oligomers according to claim 1, wherein: Also included are co-reactants.

3. The kit for detecting Aβ oligomers according to claim 1 or 2, wherein: The aptamer-activator modified streptavidin magnetic beads Aptamer-Activator / MBs are prepared as follows: 1) Preparation of Aptamer-Activator: Mix 50 μL of 100 μM Aptamer, 50 μL of 100 μM Activator sense strand, and 50 μL of 100 μM Activator antisense strand, react at 95°C for 5 min, and cool naturally to room temperature to obtain Aptamer-Activator. 2) Preparation of Aptamer-Activator / MBs: Wash 10 μL of 10 mg / mL streptavidin magnetic beads three times with trimethylamine hydrochloride (Tris-HCl) buffer containing 0.1% Tween-20. Add 100 μL of 0.1-1.5 μM Aptamer-Activator and mix. Incubate at room temperature with shaking at 300-500 rpm for 0.5-3 h. Wash five times with Tris-HCl buffer containing 0.1% Tween-20 to remove unbound Aptamer-Activator. Disperse in 100 μL of ultrapure water to obtain Aptamer-Activator / MBs.

4. The kit for detecting Aβ oligomers according to claim 1 or 2, wherein: The methylene blue nucleic acid signal probe-modified streptavidin magnetic beads DNA-MB / MBs were prepared as follows: 10 μL of 10 mg / mL streptavidin magnetic beads were washed three times with a Tris-HCl buffer solution containing 0.1% Tween-20, 100 μL of 0.5-2.5 μM methylene blue nucleic acid signal probe DNA-MB was added and mixed, and the mixture was incubated at room temperature with shaking at a speed of 300-500 rpm for 0.5-3 h. Subsequently, the beads were washed five times with a Tris-HCl buffer solution containing 0.1% Tween-20 to remove unbound DNA-MB, and the beads were dispersed in 100 μL of ultrapure water to obtain DNA-MB / MBs.

5. The kit for detecting Aβ oligomers according to claim 1 or 2, wherein: The nuclease 12a-guide RNA (Cas12a-crRNA) complex was prepared by adding 10 μL of 500 nM guide RNA (crRNA) and 5 μL of 1 μM nuclease 12a (Cas12a) to a reaction buffer containing 15 μL of diethyl pyrocarbonate (DEPC)-treated water, 10 μL of 50 mM sodium chloride, 10 mM tris hydrochloride, 10 mM magnesium chloride and 100 μg / ml recombinant albumin and incubating at 25°C.

6. The kit for detecting Aβ oligomers according to claim 1 or 2, wherein: The synthetic gold nanocluster probe modified glassy carbon electrode β -CD-AuNC / GCE was prepared as follows: 1) Dissolve NaHCO3 in H2O. β -cyclodextrin ( β -CD) solution was dissolved in NaHCO3 aqueous solution to prepare β -CD alkaline reaction solution; at room temperature, add the NaHCO3 aqueous solution to the chloroauric acid aqueous solution containing NaHCO3; under vigorous stirring, add β -CD alkaline reaction solution; add NaHCO3 aqueous solution, heat at 80 ℃, filter after the reaction is completed, add ultrafiltration centrifuge tube and centrifuge to obtain the product; The product is centrifuged and washed to obtain the synthesized gold nanocluster probe. β -CD-AuNCs; 2) 4 μL β -CD-AuNCs solution was mixed with 1 μL 1% chitosan and then dropped onto the pre-treated glassy carbon electrode (GCE) surface and dried at 37 °C to obtain β -CD-AuNC / GCE.

7. The kit for detecting Aβ oligomers according to claim 2, wherein: The coreactant is prepared as follows: N,N-diisopropylethylamine is added to a PBS buffer solution at a final concentration of 0.1 M, and the pH is adjusted to 11.5 with sodium hydroxide to obtain the coreactant.

8. The kit for detecting Aβ oligomers according to claim 3, wherein: The base sequences involved are as follows: Aptamer: 5'-GCCTGTGGTGTTGGGGCGGGTGCG-biotin-3'; Activator sense strand: 5'-ACTTTGTATCGCGCCCCACTGCGTTCTTCGTTGACGGCGTAA-3'; Activator antisense strand: 5'-AACACCACAGGCTTACGCCGTCAACGAAGAACGCAGTGGGGCGCGATACAAAGT-3'.

9. The kit for detecting Aβ oligomers according to claim 4, wherein: The base sequences involved are as follows: DNA-MB: 5'-biotin-AAAAAAAAAAAAAAAAAAAAA-MB-3'.

10. The kit for detecting Aβ oligomers according to claim 5, wherein: The base sequences involved are as follows: crRNA: 5'-UAAUUUCUACUAAGUGUAGUATCGCGCCCCACTGCGTTCT-3'.

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