Aptamer specifically recognizing alpha-amanitin and screening and application thereof

Abstract

The application discloses a nucleic acid aptamer specifically recognizing alpha-amatoxin and screening and application thereof, and belongs to the technical field of biology. The application discloses nucleic acid sequences of alpha-amatoxin (alpha-AMA) specific aptamers, namely, SEQ ID NO. 1 to SEQ ID NO. 18, and constructs an electrochemical and colorimetric multi-signal detection alpha-amatoxin platform based on the aptamer Apt-14 with the lowest Kd of 6.128nM, modifies the aptamer on a gold-plated glassy carbon electrode, releases the complementary strand of the aptamer by using the specific binding of the aptamer and the target, triggers HCR amplification to perform signal amplification, and the detection limit is 5ng / mL, and the effect is good. The method is simple in operation, and the enzyme-free amplification mode is more conducive to on-site detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nucleic acid aptamer that specifically recognizes α-amanita toxin and its screening and application. Background Technology

[0002] Amanitaphalloides is considered the most poisonous mushroom in the world, and most cases of mushroom poisoning are caused by accidental ingestion of Amanitaphalloides. Twenty-two amatoxins have been isolated and identified from Amanitaphalloides, mainly including amatoxins, phalotoxins, virotoxins, muscarine, botenic acid, isoxazole derivatives, as well as muscimol and coprine. Based on their amino acid composition and structure, these toxins can be classified into three main categories: amatoxins, phalotoxins, and virotoxins.

[0003] Amanitins are bicyclic octapeptide compounds. Based on the different substituent groups on their side chains, amatoxins can be classified into nine types, including α-amanitin (α-AMA), β-amanitin (β-AMA), and γ-amanitin (γ-AMA). Among them, α-AMA and β-AMA are the toxins in poisonous mushrooms that cause the vast majority of fatal effects. They are chemically stable, resistant to high temperatures, drying, and acids and alkalis; ordinary cooking does not destroy their toxicity. They are readily soluble in methanol, ethanol, and water, and have a molecular weight of 973-990 Da. Therefore, even after cooking, they can still cause a series of health hazards. Studies have shown that a 20g agaric mushroom often contains 5-8mg of amatoxins, an intake that is fatal to an adult. In cases of suspected accidental ingestion, a key diagnostic indicator for mushroom poisoning is the detection of amatoxins in bodily fluids such as blood plasma. However, because blood plasma can help eliminate toxins, patients who seek medical attention 30 hours after ingestion may test negative for amatoxins in their blood plasma, posing a significant risk to the treatment of those who have ingested the mushrooms.

[0004] α-AMA is a potent inhibitor of eukaryotic-dependent RNA polymerase II, blocking mRNA transcription and protein synthesis. Pharmacokinetics show that α-AMA can be transported to the liver via the enterohepatic circulation of bile acids and irreversibly bind to the RNA polymerase II site, inducing hepatocyte necrosis. The oral median lethal dose (LD50) of α-AMA is... 50The concentration of α-AMA in humans is approximately 0.1 mg / kg. Depending on the amount ingested, varying degrees of damage may occur to the liver, kidneys, and central nervous system. There is an incubation period of 6 to 12 hours before the onset of severe abdominal pain, vomiting, and diarrhea, followed by a significant recovery period lasting 48 to 72 hours. Afterward, patients experience a recurrence of symptoms, such as abdominal pain and bloody diarrhea, leading to rapid deterioration that can result in either recovery or death. Most patients with α-AMA poisoning die within 5 to 8 days from multiple organ failure affecting the liver, kidneys, heart, brain, lungs, and other organs. Therefore, establishing efficient, simple, and sensitive methods for detecting α-AMA is of great significance for preventing accidental ingestion of α-AMA from poisonous mushrooms.

[0005] Currently, α-AMA is mainly detected using liquid chromatography-mass spectrometry (LC-MS). While most detection methods can detect α-AMA, these methods have significant advantages and disadvantages. For example, large instruments offer high sensitivity but are expensive, time-consuming, and require specialized personnel. Real-time immunochromatography offers short detection times, and ELISA is simple to operate, but it is highly prone to false positives. Therefore, the identification of poisonous mushrooms and the establishment of rapid detection methods for α-AMA are particularly important. Aptamer-based detection sensors offer advantages such as rapid detection, simple operation, and low equipment cost.

[0006] Aptamers are single-stranded oligonucleotide molecules (ssDNA or RNA) that can be obtained through systematic evolution of ligands through exponential enrichment (SELEX) technology. Aptamers can specifically bind to targets such as metal ions, antibiotics, small molecules, proteins, and even whole cells or microorganisms such as bacteria, thus having broad applications in biochemical analysis, environmental monitoring, and food safety. Compared to antibodies, aptamers can be synthesized in vitro with high purity at a low cost. Furthermore, aptamers exhibit high thermal stability, low immunogenicity, chemical stability, and ease of labeling and modification.

[0007] After more than 30 years of development, many aptamer screening technologies have been developed. Based on different screening methods, they are divided into fixed library SELEX, fixed target SELEX, and homogeneous SELEX with neither fixed library nor target. These are further subdivided based on whether a fixation material is required, such as graphene oxide SELEX, magnetic bead SELEX, Capture SELEX, and capillary electrophoresis SELEX. Capture SELEX is a method for screening small molecule targets based on a fixed library. Graphene oxide SELEX utilizes the van der Waals forces, electrostatic attraction, and hydrogen bonds between single-stranded DNA and graphene oxide to adsorb the DNA onto the graphene oxide. When the target is present, the interaction force between the single-stranded nucleic acid and the target is greater than the interaction force with the graphene oxide. Graphene oxide SELEX is label-free and fixation-free, overcoming the limitation of Capture SELEX which requires a fixation step. Based on the SELEX method, nucleic acid aptamers for α-AMA were screened, and the nucleic acid sequence was used for the detection of α-AMA. Summary of the Invention

[0008] This invention aims to provide a nucleic acid aptamer that specifically recognizes α-AMA, as well as its screening and application. By combining magnetic beads-SELEX with graphene oxide-SELEX, a nucleic acid aptamer that specifically recognizes α-AMA is screened and obtained, which has higher affinity and specificity compared with existing aptamers.

[0009] A first aspect of the present invention provides a nucleic acid aptamer, Apt-14, that specifically recognizes α-AMA, the nucleic acid sequence of which is the sequence shown in SEQ ID NO.14.

[0010] Furthermore, the primary structure of the nucleic acid aptamer is as follows:

[0011] 5'-CATGCTTCCCCAGGGAGATGTTACCATCTGGCATAGGAGGTCAGGGTGCGTTTGGTATTGAGGAACATGC-3', with prominent stem and loop secondary structures, has a Gibbs free energy ΔG = -3.83 kcal / mol.

[0012] Furthermore, the 3' or 5' end of the nucleic acid aptamer is modified with a modifier.

[0013] Furthermore, the modifier is an electrochemical marker, specifically a carboxyl group.

[0014] A second aspect of the present invention provides a modified electrode containing the aforementioned nucleic acid aptamer Apt-14.

[0015] A third aspect of the present invention provides a method for preparing a detection sensor that specifically recognizes α-AMA, the method being as follows:

[0016] (1) Polish the glassy carbon electrode with alumina powder and clean it with ethanol and deionized water by ultrasonication.

[0017] (2) Gold is electrodeposited on the glassy carbon electrode surface in step (1);

[0018] (3) The complementary strand was mixed with the gold nanoparticle solution to obtain a DNA solution labeled with gold nanoparticles;

[0019] (4) The nucleic acid aptamer Apt-14 was dropped onto the glassy carbon electrode surface of step (2) and incubated for 12-16 h; then 6-mercapto-1-hexanol was added for incubation, rinsed and dried;

[0020] (5) The DNA solution labeled with gold nanoparticles from step (3) is dropped onto the glassy carbon electrode surface from step (4) for hybridization, washed with water and dried to obtain the detection sensor.

[0021] In one embodiment, in step (1), the glassy carbon electrode is polished to a mirror finish using 0.3 μm and 0.05 μm alumina powders in sequence.

[0022] In one embodiment, in step (2), the glassy carbon electrode is immersed in HAuCl4 solution and cyclic voltammetric scan is performed for 8 to 12 cycles in the potential range of -0.2 to 0.6V.

[0023] In one embodiment, in step (3), the nucleotide sequence of the complementary strand is shown in SEQ ID NO.19.

[0024] In one embodiment, in step (3), the complementary strand is mixed with the gold nanoparticle solution and reacted at 20℃~28℃ for 12~16h; PB buffer is added and shaken at room temperature for 0.5~1h; then PB buffer containing sodium chloride is added in batches and reacted at 20℃~28℃ for 12~16h; the DNA solution labeled with gold nanoparticles is obtained by centrifugation and washing 3~4 times with deionized water.

[0025] In one embodiment, in step (4), the mixture is incubated in 0.5–1.5 mM 6-mercapto-1-hexanol for 0.5–1.5 h.

[0026] In one embodiment, in step (5), 5–10 μL of a 0.5–1.5 μM DNA solution labeled with gold nanoparticles is dropped onto the surface of the glassy carbon electrode.

[0027] In one embodiment, in step (5), hybridization is performed at 35–38°C for 0.5–1.5 h.

[0028] The four aspects of the present invention provide a detection sensor for specifically recognizing α-AMA, said detection sensor being prepared according to the method described above.

[0029] A fifth aspect of the present invention provides a method for detecting α-AMA not for the purpose of diagnosing a disease, the method comprising using the detection sensor.

[0030] In one implementation, the method is as follows:

[0031] (1) The sample to be tested is dropped onto the detection sensor and incubated for a certain period of time;

[0032] (2) Clean the sensor by repeatedly blowing with deionized water and collect the cleaning solution;

[0033] (3) Add H1 solution and H2 solution to the cleaning solution, incubate for a certain period of time, and collect the reaction solution;

[0034] (4) Add the nano gold particle solution to the reaction solution, incubate at room temperature, add NaCl solution, let stand for 5-15 min, and measure the absorbance.

[0035] In one implementation, in step (1), the incubation time is 0.5 to 1.5 hours.

[0036] In one embodiment, in step (3), the nucleotide sequence of H1 is shown in SEQ ID NO.20, and the nucleotide sequence of H2 is shown in SEQ ID NO.21.

[0037] In one embodiment, in step (3), the final concentrations of H1 and H2 are 0.5–1.5 M.

[0038] In one embodiment, in step (3), the incubation is carried out at 35-38°C for 1.5-2.5 hours.

[0039] In one embodiment, in step (4), the reaction solution is reacted with the gold nanoparticles for 20 to 40 minutes.

[0040] In one embodiment, in step (4), the concentration of the NaCl solution is 0.1–0.5 M.

[0041] A sixth aspect of the present invention provides a method for screening nucleic acid aptamers that specifically recognize α-AMA, comprising the following steps:

[0042] S1: Construct the initial library, the sequence of which is shown below:

[0043] 5'-CATGCTTCCCCAGGGAGATG-N30-TTTGGTATTGAGGAACATGC-3', where N represents the bases A, G, C, or T, and N30 indicates that the length of the random region nucleotide is 30 bases;

[0044] S2: Incubate the initial library with streptavidin magnetic beads to label the initial library on the magnetic beads; bind the target to the labeled magnetic beads, remove non-specifically bound sequences using magnetic separation technology, and retain the supernatant to obtain the first enriched library; the target is α-AMA;

[0045] S3: Perform PCR amplification on the first enriched library to obtain the amplification product, purify it, and obtain the second enriched library.

[0046] S4: Replace the initial library in S2 with the second enriched library, and repeat steps S2 and S3 to obtain the third enriched library;

[0047] S5: Mix the third enriched library with α-AMA, incubate to obtain a mixture, mix the mixture with graphene oxide, incubate, centrifuge to obtain the supernatant, which is the fourth enriched library;

[0048] S6: Mix the fourth enriched library with α-AMA, incubate, then mix with graphene oxide and incubate. After centrifugation, retain the supernatant and perform PCR amplification to obtain the amplification product.

[0049] S7: The amplification product is purified to obtain an enriched library;

[0050] S8: Replace the third enriched library in S5 with the enriched library described above, and repeat steps S6 and S7 until nucleic acid aptamers with high affinity and strong specificity are screened out.

[0051] A seventh aspect of the invention provides the application of the nucleic acid aptamer, the modified electrode, or the detection sensor in the detection of α-AMA.

[0052] Beneficial effects:

[0053] The beneficial effects of this invention are as follows:

[0054] This invention designs a random single-stranded DNA library and screens to obtain the nucleic acid aptamer Apt.14, which has high affinity, strong specificity, and stable properties and can bind to α-AMA. Compared with the reported aptamers, this aptamer has a stronger binding ability to α-AMA, with the lowest Kd value of 6.128 nM, which is far lower than other aptamers screened in the same batch.

[0055] Furthermore, this invention constructs an electrochemical detection sensor based on this nucleic acid aptamer, which can directly detect α-AMA in urine and actual samples, with a detection limit of 5 ng / mL and a spiked recovery rate ranging from 90.86% to 110.68%. The detection method based on this aptamer does not require large and complex instruments or professional technicians, and the detection method has the advantages of simplicity and low cost. Attached Figure Description

[0056] To more clearly illustrate the specific solutions and technologies in this invention, the technologies and results used in this patent will be described below with accompanying drawings.

[0057] Figure 1 This is a schematic diagram of the experimental method for screening α-AMA proposed in this invention;

[0058] Figure 2 This is a schematic diagram of the secondary structure of SEQ ID NO.1-SEQ ID NO.9 in this invention;

[0059] Figure 3 This is a schematic diagram of the secondary structure of SEQ ID NO.10-SEQ ID NO.18 in this invention;

[0060] Figure 4 The figure shows the experimental results of the affinity between SEQ ID NO.14 of the present invention and α-AMA;

[0061] Figure 5 This is a schematic diagram illustrating the principle of α-AMA detection using a biosensor based on the present invention;

[0062] Figure 6 The detection limit results of the electrochemical sensor for α-AMA detection in the embodiments of the present invention are shown.

[0063] Figure 7 The specific detection structure for α-AMA detection by the electrochemical sensor in this embodiment of the invention is shown. Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0065] Example 1: Screening of nucleic acid aptamers

[0066] (1) Synthesize the random single-stranded DNA library and primers shown in the following sequences (synthesized by Shanghai Sangon Biotech Co., Ltd.):

[0067] Random ssDNA library:

[0068] 5'-CATGCTTCCCCAGGGAGATG-N30-TTTGGTATTGAGGAACATGC-3', where N30 represents a sequence consisting of 30 arbitrary nucleotide bases linked together.

[0069] 5' upstream primer: 5'-CATGCTTCCCCAGGGAGATG-3';

[0070] 5' Phosphorylation of downstream primer: 5'-P-GCATGTTCCTCAATACCAAA-3';

[0071] 5' Biotin immobilization primer: 5'-biotin-AAAAAAA-GCATGTTCCTCAATACCAAA-3'.

[0072] The random ssDNA library and the three primers were prepared into a 100 μM stock solution using BB buffer (Tris-HCl: 20 Mm, MgCl2: 50 mM, KCl: 5 mM, CaCl2: 2 Mm, pH 7.6) and stored at -20 °C.

[0073] (2) One cycle consists of target incubation, PCR amplification, and single-strand preparation. The library is immobilized with magnetic beads, and screening is performed using a target competitive binding method. After two rounds of magnetic bead screening, the prepared single strands are incubated with the target and then added to a graphene oxide solution for further screening. Seven rounds of graphene oxide screening are conducted, for a total of nine rounds. The screening process is detailed below. Figure 1 The specific filtering method is as follows:

[0074] (1) Random ssDNA libraries were denatured at 95℃ for 5 min and then hybridized with 5' biotin-fixed primers at 37℃ and 500 rpm for 2 h. PBS solution was used as a blank control group. The libraries were measured three times by NanoDrop and the mean value was calculated as the mass concentration of the libraries.

[0075] (2) After hybridization, the library and complementary primers (5' upstream primer and 5' phosphorylated downstream primer) were mixed (100 μL, with a final concentration of 5 μM) and placed in a PCR instrument with the following program: 95℃ for 5 min, slowly cooled to 60℃ at a rate of 0.1℃ / s; 60℃ for 1 min; slowly cooled to 25℃ at a rate of 0.1℃ / s, to obtain a mixture of library and complementary primers with good annealing properties.

[0076] (3) Take 25 μL of 10 mg / mL streptavidin magnetic bead stock solution and place it in a centrifuge tube. After magnetic separation, remove the supernatant and wash the magnetic beads three times with PBS to remove the protective solution. Then, add the mixed solution of the renaturated library and complementary primers from step (2), mix well, and incubate at 500 rpm for 2 h at room temperature. After magnetic separation, collect the magnetic beads immobilized with the library and recover the supernatant. Using PBS solution as a blank control group, measure the supernatant recovery solution three times using a NanoDrop micro-UV-Vis spectrophotometer and calculate the mean value as the mass concentration of the recovered library. Calculate the library immobilization efficiency.

[0077] (4) After washing the magnetic beads obtained in step (3) three times with PBS, add 100 μL of 5 μg / mL α-AMA and incubate in a metal bath at 37°C for 2 h. After magnetic separation, recover the supernatant and measure the mass concentration of the recovered DNA.

[0078] (5) Secondary library amplification. Using the supernatant recovered in step (4) as a template, 4 μL of the supernatant was amplified to obtain double-stranded DNA. The amplification system (50 μL) and PCR program are as follows:

[0079] system:

[0080] 2xTaqPCRMatsterMix 25μL upstream primer 1 μL (100 μM) Phosphorylation of downstream primers 1 μL (100 μM) Ultrapure water 19μL Template DNA 4μL

[0081] program:

[0082]

[0083] (6) Polyacrylamide gel electrophoresis verification: PCR products were electrophoresed on a 3% agarose gel. The gel imaging system was used to observe whether the electrophoretic bands were single and bright, and whether the bands were located at the 70bp position.

[0084] (7) Preparation of single-stranded libraries: The obtained PCR products were purified using a purification kit to remove other substances from the PCR reaction system. The concentration of purified nucleic acids was measured using a NanoDrop micro-UV-Vis spectrophotometer to determine the approximate digestion time. The purified product was taken, and 1 / 10 volume of digestion buffer and an appropriate amount of exonuclease were added and mixed thoroughly. The mixture was reacted at 37°C until digestion was complete. After digestion, the enzyme was inactivated at 65°C for 10 min to stop the digestion reaction. The digested product was purified by extraction with phenol / chloroform and precipitation with 70% ethanol. After drying in a 50°C oven, 50 μL of deionized water was added to dissolve the product as the ssDNA library for the next round of screening.

[0085] (8) Repeat the above steps once to perform the second round of magnetic bead screening to enrich aptamers with affinity, and then perform SELEX on graphene oxide.

[0086] (9) After denaturing the prepared 50 μL single-stranded DNA library, add 50 μL of α-AMA solution to a final concentration of 1 μg / mL, mix well, and incubate in a metal bath at 37℃ for 2 h. Take 1 mg / mL graphene oxide, centrifuge to remove the supernatant, reconstitute with 100 μL of the mixture, incubate, mix well, and incubate in a metal bath at 37℃ for 1 h. After incubation, centrifuge at 12000 rpm at 4℃ for 15 min, and recover the supernatant. Repeat centrifugation twice until no graphene oxide precipitate appears.

[0087] (10) Repeat steps (5) to (9) for 7 rounds. The purified PCR product from the seventh round of graphene oxide PCR was then sent to Shanghai Sangon Biotech for high-throughput sequencing. This resulted in the selection of 14 aptamer sequences. Figure 2 and Figure 3 These are schematic diagrams illustrating the secondary structure prediction of the sequences obtained from sequencing.

[0088] Table 1. Aptamers obtained through screening

[0089]

[0090]

[0091] Example 2: Affinity-Specificity Analysis of Nucleic Acid Aptamers

[0092] Affinity was analyzed using a colorimetric method with gold nanoparticles. The aptamers screened in Example 1 were dissolved and diluted. Different concentrations of aptamers (0, 50, 100, 200, 400, 800, 1600, and 3200 nM) were incubated with 1 μg / mL α-AMA at room temperature for 30 min. Then, 40 μL of gold nanoparticle (AuNPs) solution was added and incubated at room temperature for another 30 min. Finally, 1 M NaCl was added to bring the final concentration to 40 mM. The absorbance at 520 nm was measured using a UV spectrophotometer. The aptamer concentration was plotted on the x-axis, and (A′-A0) / A0 was used as the y-axis. Kd was calculated using the One site-Specific binding program in GraphPad Prism 8.0 software. A′ represents the absorbance at 520 nm for each aptamer concentration. A0 represents the absorbance at 520 nm when the aptamer concentration is 0. Based on the relationship between Kd and affinity, the lowest Kd for Apt.14 binding to α-AMA is 6.128 nM. The affinity effect is the best, and the affinity results are as follows... Figure 4 As shown. The Kd values ​​of the remaining screened aptamers that bind to α-AMA are all greater than 6.128 nM, with the aptamer having the weakest affinity at 68.01 nM, and its affinity is slightly worse than that of Apt.14.

[0093] Table 2 shows the affinity of the candidate sequences:

[0094] Kd 31.61 nM 36.25nM 40.58 nM 26.06 nM 62.61 nM 34.68 nM 37.11 nM 31.35nM 39.02nM aptamers Apt10 Apt11 Apt12 Apt13 Apt14 Apt15 Apt16 Apt17 Apt18 Kd 43.08nM 28.67 nM 47.42 nM 18.15nM 6.128nM 37.98nM 19.44 nM 37.96 nM 68.01nM

[0095] Example 3: Preparation of nucleic acid aptamers for detection sensors

[0096] (1) Preparation of an electrochemical sensor for specific detection of α-AMA

[0097] Apt.14, which has the best affinity for α-AMA and the lowest Kd, was selected. To evaluate the application performance of this aptamer, a dual-signal detection α-AMA platform was prepared, the principle of which is as follows: Figure 5 As shown.

[0098] Specifically as follows:

[0099] Gold plating was performed on a glassy carbon electrode (GCE): First, the glassy carbon electrode was polished to a mirror finish on a polishing cloth using 0.3 μm and 0.05 μm alumina powders, respectively. Then, it was ultrasonically treated with ethanol and deionized water for 60 seconds each to remove adsorbed impurities until the electrode surface was smooth and reflective, followed by drying. Subsequently, the glassy carbon electrode was immersed in a 3 mM HAuCl4 solution, and cyclic voltammetry was performed for 10 cycles within a potential range of -0.2 to 0.6 V to obtain a gold nanoparticle-modified glassy carbon electrode (Au / GCE).

[0100] Mix 20 μL of 90–100 mM complementary strand (SEQ ID NO. 19) with 1 mL of gold nanoparticle solution and react at 20–28 °C for 12–16 h. Add 113 μL of PB buffer and shake at room temperature for 0.5–1 h. Then add 90–100 μL of PB buffer (pH 7.4) containing 0.14–0.16 M sodium chloride in portions and react at 20–28 °C for 12–16 h. Wash 3–4 times with deionized water to obtain the gold nanoparticle-labeled DNA solution.

[0101] Using thiol-labeled Apt.14 as a capture probe, 10 μL of 50 nM Apt.14 solution was first uniformly added dropwise to Au / GCE. After storing overnight at room temperature, the electrode was immersed in 1 mM 6-mercapto-1-hexanol (MCH) for 1 h. After rinsing with deionized water and air-drying, 7 μL of 1 μM gold-labeled DNA solution was added to the electrode surface, and hybridization was carried out at 37 °C for 1 h. After rinsing the electrode with ultrapure water and air-drying, an electrochemical sensor based on the aptamer Apt.14 was obtained.

[0102] (2) Methods for detecting α-AMA

[0103] After incubating 10 μL of a sample containing α-AMA on the electrode surface for 1 h, 10 μL of deionized water was repeatedly pipetted onto the electrode surface for cleaning. The 10 μL was then transferred to a 200 μL centrifuge tube, and DNA hairpin structures (H1, H2, final concentration 1 μM) were added to initiate a hybridization chain reaction (HCR). The tube was incubated at 37 °C for 2 h to obtain the HCR product. The HCR product was detected using a gold nanoparticle colorimetric assay. 30 μL of the HCR product was added to 150 μL of AuNPs, incubated at room temperature for 30 min, followed by the addition of 20 μL of 0.3 M NaCl solution. After standing for 10 min, the absorbance of the system was measured at 520 nm.

[0104] In the presence of α-AMA, the aptamer on the electrode specifically binds to α-AMA, releasing a complementary strand (signal probe). The resulting double-stranded DNA structure exhibits strong negative charge, repelling the negatively charged gold nanoparticles in the solution. As the salt concentration increases, the stability of the gold nanoparticles continuously decreases, and aggregation occurs, causing the solution color to change from pink to purple. When α-AMA is absent from the system, the signal probe does not dissociate into the supernatant, and individual H1 and H2 do not undergo hybridization chain reactions, remaining stable in the solution. The sticky ends of single-stranded H1 and H2 adsorb onto the surface of the gold nanoparticles through electrostatic interactions, protecting the gold nanoparticles from aggregation caused by high salt concentrations, and the solution remains red.

[0105] Example 4: Detection of different concentrations of α-amatoxin using a nucleic acid aptamer-based detection sensor

[0106] α-AMA was diluted to different concentrations: 0.1 ng / mL, 0.5 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1 μg / mL. After incubating the electrode surface with each concentration of α-AMA for 1 hour, 10 μL of deionized water was pipetted onto the electrode surface and repeatedly rinsed. The 10 μL was then transferred to a 200 μL centrifuge tube, and DNA hairpin structures (H1, H2, final concentration 1 μM) were added to initiate a hybridization chain reaction (HCR). The tube was incubated at 37°C for 2 hours to obtain the HCR product, which was then detected colorimetrically using the same method as in Example 3. The current signal increased with increasing α-AMA concentration. A standard curve was plotted based on the α-AMA concentration and current signal, Y = 15.33X + 62.9(R²). 2 =0.992), the detection limit is 5 ng / mL, such as Figure 6 As shown.

[0107] Example 5: Specificity Validation of Nucleic Acid Aptamer-Based Detection Sensor

[0108] The following sample solutions were prepared: β-AMA and γ-AMA toxin concentrations of 50 μg / mL, α-AMA concentration of 1 μg / mL, and a blank sample. The method described in Example 3 was used to verify the specificity of the sensor. The colorimetric results are as follows: Figure 7 As shown. The electrochemical sensor prepared in Example 3 of this invention has good specificity and can be used in practical applications.

[0109] The performance of this electrochemical sensor in real samples was evaluated using standard spiking methods. α-AMA was detected in blank mushroom and urine samples using a spiked recovery method. Spiked concentrations were 100, 10, and 1 ng / mL, and the recoveries, ranging from 90.86% to 110.68%, are shown in the table below.

[0110] Table 3 Results of Spiked Recovery Experiment

[0111]

[0112]

[0113] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nucleic acid aptamer Apt-14 that specifically recognizes α-amanita toxin, characterized in that, The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

14.

2. The nucleic acid aptamer Apt-14 as described in claim 1, characterized in that, The 3' or 5' end of the nucleic acid aptamer Apt-14 is modified with a modifier.

3. A modified electrode, characterized in that, Includes the nucleic acid aptamer Apt-14 as described in claim 1 or 2.

4. A method for preparing a detection sensor that specifically recognizes α-amanita toxin, characterized in that, The method is as follows: (1) Polish the glassy carbon electrode to a mirror finish with alumina powder and then ultrasonically clean it with ethanol and deionized water; (2) Gold is electrodeposited on the glassy carbon electrode surface in step (1); (3) The complementary strand was mixed with the gold nanoparticle solution to obtain a DNA solution labeled with gold nanoparticles; (4) The nucleic acid aptamer Apt-14 described in claim 1 is dropped onto the glassy carbon electrode surface of step (2) and incubated for 12-16 h; then 6-mercapto-1-hexanol is added for incubation, rinsed and dried; (5) The DNA solution labeled with gold nanoparticles from step (3) is dropped onto the glassy carbon electrode surface from step (4) for hybridization, washed with water and dried to obtain the detection sensor; The nucleotide sequence of the complementary strand is shown in SEQ ID NO.

19.

5. The method according to claim 4, characterized in that, In step (2), the glassy carbon electrode is immersed in HAuCl4 solution and cyclic voltammetry is performed for 8 to 12 cycles in the potential range of -0.2 to 0.6V. In step (3), the complementary strand is mixed with the gold nanoparticle solution and reacted at 20℃ to 28℃ for 12 to 16 hours. PB buffer is added and shaken at room temperature for 0.5 to 1 hour. PB buffer containing sodium chloride is added in batches and reacted at 20℃ to 28℃ for 12 to 16 hours. The DNA solution labeled with gold nanoparticles is obtained by centrifugation and washing 3 to 4 times with deionized water.

6. The method according to claim 4 or 5, characterized in that, In step (4), the mixture is incubated in 0.5-1.5 mM 6-mercapto-1-hexanol for 0.5-1.5 h; in step (5), 5-10 μL of 0.5-1.5 μM labeled gold nanoparticle DNA solution is dropped onto the glassy carbon electrode surface.

7. A detection sensor specifically recognizing α-amanita toxin prepared by the method according to any one of claims 4 to 6.

8. A method for detecting α-amanita toxin not for the purpose of diagnosing a disease, characterized in that, The method involves using the detection sensor described in claim 7 for detection.

9. The method according to claim 8, characterized in that, The method is as follows: (1) The sample to be tested is dropped onto the detection sensor and incubated for a certain period of time; (2) Clean the sensor by repeatedly blowing with deionized water and collect the cleaning solution; (3) Add H1 solution and H2 solution to the cleaning solution, incubate for a certain period of time, and collect the reaction solution; Add nano-gold solution to the reaction solution, incubate at room temperature, add NaCl solution, let stand for 5-15 min, and measure absorbance; the nucleotide sequence of H1 is shown in SEQ ID NO.20, and the nucleotide sequence of H2 is shown in SEQ ID NO.21.

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