A method for detecting mycotoxin based on rca-hcr colorimetric aptamer sensor

Abstract

The present application relates to a kind of colorimetric aptamer sensor based on RCA-HCR The method for detecting mycotoxin, the method includes the preparation of Fe3O4@Au magnetic particle, the preparation of Fe3O4@Au@cDNA@APT magnetic probe, the preparation of ring template, Fe3O4@Au@cDNA@APT magnetic probe is incubated with mycotoxin standard solution or sample to be detected, RCA amplification, triggers HCR, the product obtained in step (6) is added with streptavidin-horseradish peroxidase, incubation, magnetic cleaning, then H2O2 And TMB are added, after reaction at room temperature in the dark, machine detection absorbance value is carried out.The method established in the present application has higher sensitivity, and the detection limit is as low as 0.02 ng / mL.The method established in the present application has good specific recognition ability to GT, and there is no obvious cross reaction between other possible non-target substances.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting mycotoxins based on an RCA-HCR colorimetric aptamer sensor. Background Technology

[0002] Aspergillus fumigatus ( Aspergillus fumigatus Aspergillosis can cause various diseases, including invasive aspergillosis (IA) and allergic aspergillosis. Invasive aspergillosis poses a fatal risk to immunocompromised patients and is a serious clinical challenge. Inhaled Aspergillus conidia initially attach to lung epithelial cells, then germinate, evolve into hyphae, and finally invade lung tissue. The development of antifungal drug resistance is a major cause of treatment failure and high mortality rates. Gliotoxin (GT) is a typical fungal secondary metabolite produced only by a few Aspergillus species, such as Aspergillus fumigatus. It is a polythiopiperazine dione compound with a relatively small molecular weight (326.4 Da) and possesses immunosuppressive, cytotoxic, and apoptosis-inducing biological activities. It can inhibit the phagocytic function of host macrophages and neutrophils, disrupt immune cell activity, and thus promote fungal colonization and spread in the body; simultaneously, it can inhibit angiogenesis and affect endothelial cell function, participating in the pathological damage of infected tissues.

[0003] As important metabolites and core virulence factors produced by Aspergillus fumigatus, mycotoxins and their derivatives can serve as important potential auxiliary diagnostic biomarkers. Mycotoxins can be detected in biological samples such as patient plasma, serum, and bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid, as a specimen directly exposed to lesions, may be an ideal specimen for detecting mycotoxins. The concentration level of mycotoxins can, to some extent, reflect the activity, virulence, and degree of infection of Aspergillus fumigatus in vivo, providing auxiliary diagnostic evidence for disease assessment.

[0004] As mentioned above, the detection of mycotoxins plays a crucial clinical role in the diagnosis and disease assessment of fungal diseases. However, traditional methods for diagnosing aspergillosis have many limitations. Fungal culture is time-consuming (3-7 days) and has a low positive rate; histopathological biopsy is an invasive procedure; and antigen detection suffers from cross-reactivity and false positives. While mycotoxin detection primarily relies on instrumental analysis methods such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), which, although highly sensitive, are complex to operate and expensive, making them unsuitable for rapid diagnosis. Furthermore, the concentration of mycotoxins in early-stage infection samples may be extremely low. Therefore, there is an urgent need to develop a highly sensitive, highly specific, and rapid sensor for the clinical detection of mycotoxins to assist in the early diagnosis and treatment monitoring of invasive aspergillosis.

[0005] Aptamers are short single-stranded DNA or RNA oligonucleotides obtained through iterative screening using exponentially enriched ligand system evolution techniques. Aptamers can bind to targets with high affinity and high selectivity, and are therefore often referred to as "chemical antibodies". Biosensors constructed using aptamers as biological recognition elements are called aptamer sensors. A typical device consists of a recognition unit (aptamer) and a signal transduction unit (electrochemical, fluorescence, colorimetric, etc.). Compared with traditional antibody sensors, aptamers have many advantages as novel recognition elements: (1) they are completely chemically synthesized, have low cost, and are easy to prepare on a large scale; (2) they have small molecular weights and are easy to modify chemically; (3) they have excellent stability and are easy to store and use; (4) they have a very wide target range, targeting ions, small molecules, proteins, cells, and even pathogens, thus helping aptamer sensors to achieve multi-scenario detection from medical diagnosis and environmental monitoring to food safety.

[0006] However, traditional aptamer sensors generally follow a "single target - single signal" response mode. Faced with the need to detect low-abundance biomarkers in biological samples, relying solely on aptamer recognition is insufficient to provide detectable signals that meet signal-to-noise ratio requirements. Isothermal amplification (RCA) refers to a class of methods that achieve efficient nucleic acid amplification at a constant temperature. Unlike traditional PCR, which requires thermal cycling equipment, RCA typically only requires a water bath or heating block to complete the reaction, making it ideal for on-site detection and biosensor integration. Rolling circle amplification (RCA) operates under isothermal conditions, using a small circular oligonucleotide as a template. Under the action of phi29 DNA polymerase, a single primer is continuously extended along a circular template (CT), generating long single-stranded DNA containing hundreds to thousands of repeat units. These ultra-long single strands also contain numerous repeat sites, serving as the "backbone" of signal carriers. By pre-designing the circular template sequence, the RCA product can be used to introduce desired functional fragments, such as G-quadruplexes and priming strands. During amplification, these functional fragments are replicated synchronously with the long-chain products, thereby achieving efficient signal amplification. The purpose of cascade amplification is to further increase the output signal by cascading different amplification techniques to integrate their respective advantages and produce a synergistic effect of "1 + 1 > 2". Hybridization chain reaction (HCR), as an enzyme-free, isothermal signal amplification technique, uses an amplification system consisting of two or more oligonucleotide probes designed with a "hairpin" structure. The initiation chain triggers the stable opening of the hairpin structure, leading to self-assembly.

[0007] This invention employs a cascaded signal amplification strategy combining RCA and HCR. By encoding a circular template, the amplified products of RCA carry numerous initiation sequences from hairpin probes, efficiently triggering the HCR reaction and generating a branched DNA scaffold. Because the hairpin probes are biotin-modified, the amplified branched products contain numerous avidin-binding sites. Upon coupling with streptavidin-modified horseradish peroxidase (HRP), these sites catalyze the colorimetric reaction of tetramethylbenzidine (TMB), thus constructing a colorimetric sensor for the detection of mycotoxins. Summary of the Invention

[0008] The purpose of this invention is to provide a method for detecting mycotoxins using a colorimetric aptamer sensor based on RCA-HCR.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a method for detecting mycotoxins using a colorimetric aptamer sensor based on RCA-HCR, which is roughly as follows: 1. Gold nanoparticles were deposited on the surface of Fe3O4 magnetic microparticles and modified with carboxyl groups. The aptamer was coupled to the magnetic microparticles via hybridization with amino-cDNA to form Fe3O4@Au@cDNA@APT magnetic probes.

[0010] 2. The magnetic probe is incubated with the target. The aptamer preferentially binds to the target and is released from the surface of the magnetic microparticle, exposing the cDNA.

[0011] 3. The RCA amplification system is added, where the circular template CT binds to cDNA and undergoes rolling circle amplification catalyzed by phi29 DNA polymerase, generating long DNA single strands on the surface of the magnetic microparticles. After magnetic washing, the magnetic microparticles are retained.

[0012] 4. Biotin-modified H1 and H2 are added to the product of step (3) to initiate HCR. After magnetic cleaning, the magnetic particles are retained.

[0013] 5. Add streptavidin-horseradish peroxidase to the product in step (4), perform magnetic cleaning, add H2O2 and TMB, react at room temperature in the dark, and then detect the absorbance value on an instrument.

[0014] More specifically, the method for detecting mycotoxins using an RCA-HCR-based colorimetric aptamer sensor provided by this invention includes the following more detailed steps: (1) Preparation of Fe3O4@Au magnetic microparticles: Gold nanoparticles were deposited on the surface of Fe3O4 magnetic microparticles and modified with carboxyl groups to obtain Fe3O4@Au magnetic microparticles; (2) Preparation of Fe3O4@Au@cDNA@APT magnetic probe: Fe3O4@Au magnetic microparticles were activated with EDC and NHS, incubated with amino-modified cDNA, and then aptamers were added to allow them to hybridize with cDNA to obtain the Fe3O4@Au@cDNA@APT magnetic probe. (3) Preparation of circular template: The ligation primer and the 5'-phosphorylated padlock probe were mixed and heat-treated, and then T4 DNA ligase and 1×T4 DNA ligase buffer were added and incubated; then exonuclease III and exonuclease I were added to react and obtain the circular DNA product, which is the circular template (CT). (4) The Fe3O4@Au@cDNA@APT magnetic probe is incubated with mycotoxin standard solution or the sample to be tested; (5) RCA amplification: RCA amplification is performed by adding the product obtained in step (4) to the RCA amplification system to obtain the RCA amplification product; wherein, the RCA amplification system includes phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer; after adding phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer to deionized water to 20 μL, the concentration of phi29 DNA polymerase in the RCA amplification system is 4 U, the concentration of dNTPs is 10 mM, and the concentration of circular template is 7.25 nM; (6) Initiating HCR: Add hairpin probes H1 and H2, both of which are modified with biotin at the ends, to the RCA amplification product obtained in step (5) for reaction; (7) Add streptavidin-horseradish peroxidase to the product obtained in step (6), incubate, wash magnetically, add H2O2 and TMB, react at room temperature in the dark, and then detect the absorbance value on an instrument. The sequences of the aptamer, cDNA, ligation primer, padlock probe, and hairpin probes H1 and H2 are as follows: Aptamer: 5'-TCCAGGGGAGGTACGACTGCGTAC-3' (SEQ ID NO.1) cDNA: 5'-GTACGCAGTC-3' (SEQ ID NO.2), Ligation primer: 5'-GACTGGGCCTCAGTCCGTTTCTCATGGTTC-3' (SEQ ID NO.3) Padlock probe: 5'-GACTGAGGCCCAGTCCTTGAATTTGACGACTGCGTACGAACCATGAGAAACG-3' (SEQ ID NO.4). H1: 5'-TGAATTTGACGACTGCGTACGAGTGTGTTCGTACGCAGTCGTC-3' (SEQ ID NO.5), H2: 5'-TCGTACGCAGTCGTCAAATTCAGACGACTGCGTACGAACACAC-3' (SEQ ID NO. 6).

[0015] The specific method for step (1) is as follows: Take a 1 mg / mL Fe3O4 solution and add 50 mM gold trichloride (AuCl3) solution, stirring for 10 min to ensure thorough mixing. After boiling the mixture, slowly add a 1% (w / w) sodium citrate solution to the Au... 3+ The gold nanoparticles were reduced to gold nanoparticles and deposited in situ on the surface of Fe3O4. The solution was kept boiling and stirred continuously for 30 min to ensure uniform dispersion of the gold nanoparticles. The solution was then slowly cooled to room temperature. The product was magnetically separated and washed three times with ultrapure water. After drying in a vacuum drying oven, Fe3O4@Au nanomaterials were initially obtained. The HS-PEG-COOH solution was slowly added dropwise to 100 μL of Fe3O4@Au dispersion to make the final concentration of the HS-PEG-COOH solution 5 mM. The mixture was incubated at room temperature for 2-4 hours. After the reaction, the mixed solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 3000 g for 20 min, and the supernatant was discarded after centrifugation. The supernatant was then resuspended in ultrapure water. The "centrifugation-discarding supernatant-resuspending" steps were repeated 3 times. 1 mL of deionized water was added to prepare Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL. The Fe3O4@Au dispersion was prepared by diluting the prepared Fe3O4@Au nanomaterials with deionized water to 10 mg / mL.

[0016] The specific method of step (2) is as follows: Take 1 mL of Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL and activate them with an equal volume of EDC and NHS; after activation, wash twice, add amino-modified cDNA, incubate at room temperature for 60 min, and then wash three times by magnetic separation; then add 50 μL of aptamer diluted to 10 μM with PBS, hybridize with cDNA for 60 min, wash and resuspend to obtain Fe3O4@Au@cDNA@APT magnetic probe.

[0017] The specific method of step (3) is as follows: Mix 100 μL of ligation primer and 100 μL of 5'-phosphorylated padlock probe and heat-treat at 95℃ for 5 min, then slowly cool to room temperature; add T4 DNA ligase (100 U) and 1×T4 DNA ligase buffer, and incubate at 25℃ overnight to promote ligation; after ligation, add exonuclease III (Exo III) (100 U) and exonuclease I (Exo I) (50 U) to digest the remaining single-stranded DNA and other byproducts; react at 37℃ for 2 h, and terminate the ligase activity by heating the reaction system to 80℃ for 10 min to obtain the circular DNA product, which is the circular template (CT).

[0018] The specific method of step (4) is as follows: Mix 10 μL of Fe3O4@Au@cDNA@APT magnetic probe with mycotoxin standard solution or sample to be tested, and incubate at room temperature for 30 min; after incubation, remove the supernatant by magnetic separation, retain the magnetic particles, and wash 3 times.

[0019] The specific method for step (5) is as follows: add the RCA amplification system to the product obtained in step (4) and react at 30℃ for 40 min.

[0020] The specific method of step (6) is as follows: add hairpin probes H1 and H2, both of which are modified with biotin at the ends, to the RCA amplification product obtained in step (5), and react at 30℃ for 90 min.

[0021] The specific method of step (7) is as follows: After the reaction in step (6) is completed, the system is placed on a magnetic rack and washed twice. Streptavidin-HRP is added and incubated for 10 min. After washing away the unbound HRP, TMB and H2O2 are added and incubated at room temperature in the dark for 10 min. Finally, dilute hydrochloric acid is added to terminate the reaction, and the absorbance at 450 nm is measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0022] The mycotoxin standard solution was obtained by mixing mycotoxin standard with PBS buffer; the concentrations of the mycotoxin standard solution were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 ng / mL; after the Fe3O4@Au@cDNA@APT magnetic probe was incubated with the mycotoxin standard solution, it was followed by steps (5) to (7) in sequence. After the reaction was completed, the absorbance value at 450 nm was detected, and the measured absorbance value and the concentration of the mycotoxin standard solution were imported into Origin software for fitting. It was found that in the range of 0.1-500 ng / mL, the logarithm of the absorbance value (y) and the logarithm of the mycotoxin concentration (x) showed a good linear correlation, and the linear regression equation was lg(y) = -0.8216 + 0.3196 lg(x), R 2 = 0.9830.

[0023] After the Fe3O4@Au@cDNA@APT magnetic probe is incubated with the sample to be tested, it is then processed sequentially through steps (5) to (7). After the reaction is completed, the absorbance value at 450 nm is detected. The obtained absorbance value is then substituted into the linear regression equation to obtain the concentration of mycotoxin, thus realizing the quantitative detection of mycotoxin in the sample to be tested.

[0024] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention uses a mycotoxin-specific aptamer as the recognition element and combines rolling circle amplification (RCA) and hybridization chain reaction (HCR) dual signal amplification strategies to construct a highly sensitive colorimetric aptamer sensor, which achieves rapid and specific detection of mycotoxins through colorimetric signal changes. The key components of this method include: aptamer, complementary sequence (cDNA), magnetic microparticles, circular template, phi29 DNA polymerase, H1 / H2, SA-HRP, and TMB.

[0025] (2) The method of the present invention has lower aptamer synthesis cost and is more stable. In addition, the detection time is much shorter than that of traditional culture methods and does not require large instruments.

[0026] (3) The method established in this invention has high sensitivity and a detection limit as low as 0.02 ng / mL.

[0027] (4) The method established in this invention has good specific recognition ability for GT and there is no obvious cross-reaction with other possible non-target substances.

[0028] (5) The method established in this invention has good stability in complex biological samples and can be used for quantitative analysis of actual samples.

[0029] (6) The recovery rate of the target established by the present invention is between 96.96% and 105.30%, and the relative standard deviation is less than 3.66%. The method has good accuracy and has certain application potential in clinical testing. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the principle of an RCA-HCR colorimetric aptamer sensor.

[0031] Figure 2 This section describes the characterization of magnetic microparticles. (A) Schematic diagram of magnetic microparticle synthesis and subsequent amplification; (B) SEM image of Fe3O4 microparticles; (C) SEM image of Fe3O4@Au microparticles; (D) TEM image of Fe3O4 microparticles; (E) TEM image of Fe3O4@Au microparticles; (F) HR-TEM image of Fe3O4@Au microparticles; (GI) EDS elemental distribution map of Fe3O4@Au microparticles; (J) EDS energy spectrum of Fe3O4@Au microparticles; (K) Zeta potential diagram.

[0032] Figure 3 This is a feasibility verification of the sensor.

[0033] Figure 4 The performance verification of the sensor includes: (A) the absorbance spectra corresponding to different GT concentrations; (B) the linear relationship between the logarithm of absorbance and the logarithm of concentration; (C) the comparison of absorbance of GT, OTA, AFB1, DON, OA, and HA; and (D) the linear relationship between the logarithm of absorbance and the logarithm of concentration in bronchoalveolar lavage fluid. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: like Figure 1The diagram illustrates the principle of an RCA-HCR-based colorimetric aptamer sensor provided by this invention. Gold nanoparticles are deposited on the surface of Fe3O4 magnetic microparticles and modified with carboxyl groups. The aptamer is coupled to the magnetic microparticles via hybridization with amino-cDNA. When a target is present in the system, the aptamer preferentially binds to the target and is released from the surface of the magnetic microparticles, exposing the cDNA. After being added to the amplification system, the circular template CT in the system binds to the cDNA, and rolling circle amplification is performed under the catalysis of phi29 DNase. The CT is pre-encoded with a sequence complementary to the HCR primer fragment, so the synthesized RCA product carries a large number of primer fragments that can initiate HCR. After binding with H1 and H2 in the system, HCR occurs, further amplifying the signal. Since the ends of H1 and H2 are modified with biotin, the amplification product contains many sites that can bind to streptavidin. After the addition of streptavidin-HRP, it is coupled to the magnetic particles and can catalyze the substrate TMB to produce a visible colored product.

[0035] Example 1: A method for detecting mycotoxins based on an RCA-HCR colorimetric aptamer sensor, comprising the following steps: 1. Preparation of Fe3O4@Au magnetic microparticles Figure 2 A is a schematic diagram of the synthesis. Take a 1 mg / mL Fe3O4 solution and add a 50 mM gold trichloride (AuCl3) solution, stirring for 10 min to ensure thorough mixing. After boiling the mixture, slowly add a 1% (w / w) sodium citrate solution to the Au... 3+ Gold nanoparticles were reduced to Fe3O4 and deposited in situ on the Fe3O4 surface. The solution was kept boiling and stirred continuously for 30 min to ensure uniform dispersion of the gold nanoparticles. The solution was then slowly cooled to room temperature. The product was magnetically separated and washed three times with ultrapure water to obtain the preliminary Fe3O4@Au nanomaterials.

[0036] The HS-PEG-COOH solution was slowly added dropwise to 100 μL of Fe3O4@Au dispersion to make the final concentration of the HS-PEG-COOH solution 5 mM. The mixture was incubated at room temperature for 2-4 hours. After the reaction, the mixed solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 3000 g for 20 min, and the supernatant was discarded after centrifugation. The supernatant was then resuspended in ultrapure water. The "centrifugation-discarding supernatant-resuspending" steps were repeated 3 times. 1 mL of deionized water was added to prepare Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL. The Fe3O4@Au dispersion was prepared by diluting the prepared Fe3O4@Au nanomaterials with deionized water to 10 mg / mL.

[0037] like Figure 2As shown, to verify the successful preparation of Fe3O4@Au magnetic microparticles, their morphology was characterized using scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM). Figure 2 As shown in (BE), the synthesized Fe3O4@Au magnetic microparticles are regular spherical with a particle size of about 300 nm, have good dispersibility, and the gold nanoparticles are uniformly deposited on the Fe3O4 surface.

[0038] Further analysis of its elemental composition and distribution was conducted using energy dispersive X-ray spectroscopy (EDS). Figure 2 (GI) indicates that Fe, O, and Au elements are uniformly distributed within the particles. Meanwhile, EDS energy dispersive spectroscopy (EDS) Figure 2 The relatively weak Au peak intensity in J) indicates that Au is mainly supported on the iron oxide surface in the form of nanoparticles or thin layers. Combined with the morphological and elemental analysis results, the successful synthesis of Fe3O4@Au magnetic microparticles can be confirmed.

[0039] Simultaneously, the zeta potential of the relevant products was measured. Au exhibits a relatively high surface potential due to surface ligand modification, ion adsorption, or reductive deposition. Figure 2 K shows that the Zeta potential of Fe3O4@Au magnetic particles is higher than that of Fe3O4, which further indicates that Au has been deposited in situ on the Fe3O4 surface.

[0040] 2. Preparation of the ring template 100 μL of ligation primer and 100 μL of 5'-phosphorylated padlock probe were mixed and heat-treated at 95 °C for 5 min, then slowly cooled to room temperature. 100 U of T4 DNA ligase and 1×T4 DNA ligase buffer were added, and the mixture was incubated overnight at 25 °C to promote ligation. After ligation, 100 U of exonuclease III (ExoIII) and 50 U of exonuclease I (ExoI) were added to digest the remaining single-stranded DNA and other byproducts. The reaction was carried out at 37 °C for 2 h. The ligase activity was terminated by heating the reaction system to 80 °C for 10 min, yielding a circular DNA product, which is the circular template (CT). An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added, the mixture was vortexed for 20 s, and centrifuged at 12000 ×g for 5 min. Take the upper aqueous phase, add 0.1 volume of 3M NaAc (pH = 4), and let stand at -20℃ for 3-8 h. Centrifuge at 12000 ×g for 20 min and discard the supernatant. Wash the precipitate twice with 70% ethanol solution, air dry, and redissolve in 50-100 μL PBS.

[0041] 3. Preparation of Fe3O4@Au@cDNA@APT magnetic probe Take 1 mL of Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL and activate them with equal volumes of EDC and NHS. After activation, wash twice, add amino-modified cDNA, incubate at room temperature for 60 min, and then wash three times with magnetic separation. Subsequently, add 50 μL of aptamer (diluted to 10 μM with PBS) and hybridize with cDNA for 60 min. After washing and resuspending, obtain the Fe3O4@Au@cDNA@APT magnetic probe.

[0042] 4. Incubation of Fe3O4@Au@cDNA@APT magnetic probe with mycotoxin 10 μL of Fe3O4@Au@cDNA@APT magnetic probes were mixed with GT solution (mycotoxin solution) and PBS buffer, respectively, and incubated at room temperature for 30 min. After incubation, the supernatant was removed by magnetic separation, retaining the magnetic particles, and the mixture was washed three times.

[0043] 5. RCA Amplification: Add the RCA amplification system to the product obtained above and react at 30℃ for 40 min; wherein, the RCA amplification system includes phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer; after adding phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer to 20 μL of deionized water, the concentration of phi29 DNA polymerase in the RCA amplification system is 4 U, the concentration of dNTPs is 10 mM, and the concentration of circular template is 7.25 nM; 6. Triggers HCR: After washing the RCA amplification products obtained above, hairpin probes H1 and H2, both of which are modified with biotin at their ends, were added, and the mixture was reacted at 30°C for 90 min.

[0044] 7. After the reaction in step 6 was completed, the system was washed twice on a magnetic rack, and streptavidin-HRP was added and incubated for 10 min. After washing away unbound HRP, TMB and H2O2 were added, and the system was incubated at room temperature in the dark for 10 min. Finally, dilute hydrochloric acid was added to terminate the reaction, and the absorbance at 450 nm was measured using a microplate reader. The experimental results showed that ( Figure 3 Without GT, the system produced only a weak colorimetric signal; with the addition of GT, the aptamer preferentially binds to GT, exposing the cDNA and binding to CT, thereby initiating RCA and HCR, significantly enhancing signal accumulation and substrate colorimetric response on the surface of the magnetic particles, producing a visible yellow color in the solution, indicating the feasibility of the constructed sensor. Simultaneously, as negatively charged nucleic acid molecules gradually couple to the surface of the magnetic particles and undergo cascade amplification, the negative value of the zeta potential of the magnetic particles gradually increases (…). Figure 2 K), which also proves that the amplification process is realized on the surface of magnetic particles.

[0045] The relevant nucleic acid sequences used in Example 1 are shown in Table 1: Table 1 DNA Sequence

[0046] Example 2: Investigation of the Sensor's Detection Range A series of GT standard concentration gradients (0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100 ng / mL) were set up and added to the detection system (i.e., a series of GT standards were incubated with Fe3O4@Au@cDNA@APT magnetic probes, followed by steps 5-7 of Example 1). After the reaction, the absorbance value at 450 nm was measured, and the measured absorbance value was imported into Origin software to fit with the GT concentration. The results showed that as the GT concentration increased, the absorbance value of the system gradually increased ( Figure 4A). Further linear fitting analysis was performed on the detection results, and the results are as follows: Figure 4 As shown in Figure B, within the range of 0.1–500 ng / mL, the logarithm of the absorbance value (y) showed a good linear correlation with the logarithm of the GT concentration (x), and the linear regression equation was lg(y) = -0.8216 + 0.3196 lg(x), R0. 2 = 0.9830, with a detection limit as low as 0.02 ng / mL, the constructed sensor has high sensitivity.

[0047] Example 3: Investigation of Sensor Specificity A range of common toxins and a component of bronchoalveolar lavage fluid, as well as mixtures thereof, were selected as potential interfering substances, including ochratoxin A (OTA), aflatoxin B1 (AFB1), deoxynivalenol (DON), okadaic acid (OA), and hyaluronic acid (HA). Each component was detected using a sensor (i.e., after incubation with the Fe3O4@Au@cDNA@APT magnetic probe, followed by steps 5-7 of Example 1), and the resulting absorbance values ​​were compared. Experimental results showed that ( Figure 4 (c) When only GT and mixtures containing GT are present in the system, the RCA-HCR cascade amplification reaction can be effectively triggered, producing significant signals. However, for other potentially present non-specific substances (OTA, AFB1, DON, OA, HA), the absorbance signals are lower. This indicates that the constructed RCA-HCR colorimetric sensor has good specific recognition ability for GT and no obvious cross-reaction with other potentially present non-target substances.

[0048] Example 4: Sensor Validation in Real Samples Bronchoalveolar lavage fluid was collected from three patients with non-fungal infections. After centrifugation at 1000 ×g for 10 min at 4°C, the supernatant was collected, mixed into a tube, and diluted with PBS buffer as the detection matrix. A series of gradient concentrations of GT standard solutions (0, 0.5, 1.5, 15, 20, 40, 100 ng / mL) were prepared in the diluted bronchoalveolar lavage fluid. The method established in this invention was used for detection. The measured absorbance values ​​were fitted to the GT concentrations using Origin software to establish a standard curve. Furthermore, low, medium, and high concentrations of GT standard solutions were added to the bronchoalveolar lavage fluid to achieve final concentrations of 1, 10, and 100 ng / mL, respectively, and the concentration and recovery rate were calculated using the method established in this invention. Each experiment was repeated three times. All clinical samples involved were approved by the Biomedical Ethics Committee of the 900th Hospital of the Joint Logistics Support Force (Ethics Number: 2025-025). The results showed ( Figure 4 (D) Within the concentration range of 0.5-100 ng / mL, the logarithm of the absorbance value (y) shows a good linear relationship with the logarithm of the GT concentration (x), and the regression equation is lg(y) = -0.7780 + 0.2982 lg(x), R 2 = 0.9865. This indicates that the sensor has good stability in complex biological samples and can be used for quantitative analysis of real samples.

[0049] In the spiked recovery experiments (as shown in Table 2), the sensor's recovery rate of the target ranged from 96.96% to 105.30%, with relative standard deviations all less than 3.66%. These results indicate that the sensor (i.e., the method established in this invention) possesses good accuracy and has certain application potential in clinical testing.

[0050] Table 2 Results of Spiked Recovery Experiment

[0051] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting mycotoxins based on an RCA-HCR colorimetric aptamer sensor, characterized in that: It includes the following steps: (1) Preparation of Fe3O4@Au magnetic microparticles: Gold nanoparticles were deposited on the surface of Fe3O4 magnetic microparticles and modified with carboxyl groups to obtain Fe3O4@Au magnetic microparticles; (2) Preparation of Fe3O4@Au@cDNA@APT magnetic probe: Fe3O4@Au magnetic microparticles were activated with EDC and NHS, incubated with amino-modified cDNA, and then aptamers were added to allow them to hybridize with cDNA to obtain the Fe3O4@Au@cDNA@APT magnetic probe. (3) Preparation of circular template: The ligation primer and the 5'-phosphorylated padlock probe were mixed and heat-treated, and then T4 DNA ligase and 1×T4 DNA ligase buffer were added and incubated; then exonuclease III and exonuclease I were added to react and obtain the circular DNA product, which is the circular template (CT). (4) The Fe3O4@Au@cDNA@APT magnetic probe is incubated with mycotoxin standard solution or the sample to be tested; (5) RCA amplification: RCA amplification is performed by adding the product obtained in step (4) to the RCA amplification system to obtain the RCA amplification product; wherein, the RCA amplification system includes phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer; after adding phi29 DNA polymerase, dNTPs, circular template, and 1×phi29 buffer to deionized water to 20 μL, the concentration of phi29 DNA polymerase in the RCA amplification system is 4 U, the concentration of dNTPs is 10 mM, and the concentration of circular template is 7.25 nM; (6) Initiating HCR: Add hairpin probes H1 and H2, both of which are modified with biotin at the ends, to the RCA amplification product obtained in step (5) for reaction; (7) Add streptavidin-horseradish peroxidase to the product obtained in step (6), incubate, wash magnetically, add H2O2 and TMB, react at room temperature in the dark, and then detect the absorbance value on an instrument. The sequences of the aptamer, cDNA, ligation primer, padlock probe, and hairpin probes H1 and H2 are as follows: Aptamer: 5'-TCCAGGGGAGGTACGACTGCGTAC-3' cDNA: 5'-GTACGCAGTC-3', Ligation primer: 5'-GACTGGGCCTCAGTCCGTTTCTCATGGTTC-3' Padlock probe: 5'-GACTGAGGCCCAGTCCTTGAATTTGACGACTGCGTACGAACCATGAGAAACG-3' H1: 5'-TGAATTTGACGACTGCGTACGAGTGTGTTCGTACGCAGTCGTC-3' H2: 5'-TCGTACGCAGTCGTCAAATTCAGACGACTGCGTACGAACACAC-3'.

2. The method according to claim 1, characterized in that: The specific method for step (1) is as follows: Take a 1 mg / mL Fe3O4 solution and add 50 mM gold trichloride (AuCl3) solution, stirring for 10 min to ensure thorough mixing. After boiling the mixture, slowly add a 1% (w / w) sodium citrate solution to the Au... 3+ The gold nanoparticles were reduced to gold nanoparticles and deposited in situ on the surface of Fe3O4. The solution was kept boiling and stirred continuously for 30 min to ensure uniform dispersion of the gold nanoparticles. The solution was then slowly cooled to room temperature. The product was magnetically separated and washed three times with ultrapure water. After drying in a vacuum drying oven, Fe3O4@Au nanomaterials were initially obtained. The HS-PEG-COOH solution was slowly added dropwise to 100 μL of Fe3O4@Au dispersion to make the final concentration of the HS-PEG-COOH solution 5 mM. The mixture was incubated at room temperature for 2-4 hours. After the reaction, the mixed solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 3000 g for 20 min, and the supernatant was discarded after centrifugation. The supernatant was then resuspended in ultrapure water. The "centrifugation-discarding supernatant-resuspending" steps were repeated 3 times. 1 mL of deionized water was added to prepare Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL. The Fe3O4@Au dispersion was prepared by diluting the prepared Fe3O4@Au nanomaterials with deionized water to 10 mg / mL.

3. The method according to claim 1, characterized in that: The specific method for step (2) is as follows: take 1 mL of Fe3O4@Au magnetic microparticles with a concentration of 1 mg / mL and activate them with an equal volume of EDC and NHS; After activation and washing twice, amino-modified cDNA was added, and the mixture was incubated at room temperature for 60 min. Then, it was magnetically separated and washed three times. Subsequently, 50 μL of aptamer diluted to 10 μM with PBS was added, and the mixture was hybridized with cDNA for 60 min. After washing and resuspending, Fe3O4@Au@cDNA@APT magnetic probe was obtained.

4. The method according to claim 1, characterized in that: The specific method of step (3) is as follows: Mix 100 μL of ligation primer and 100 μL of 5'-phosphorylated padlock probe and heat-treat at 95℃ for 5 min, then slowly cool to room temperature; add T4 DNA ligase (100 U) and 1×T4 DNA ligase buffer, and incubate at 25℃ overnight to promote ligation; after ligation, add exonuclease III (Exo III) (100 U) and exonuclease I (Exo I) (50 U) to digest the remaining single-stranded DNA and other byproducts; react at 37℃ for 2 h, and terminate the ligase activity by heating the reaction system to 80℃ for 10 min to obtain the circular DNA product, which is the circular template (CT).

5. The method according to claim 1, characterized in that: The specific method of step (4) is as follows: Mix 10 μL of Fe3O4@Au@cDNA@APT magnetic probe with mycotoxin standard solution or sample to be tested, and incubate at room temperature for 30 min; after incubation, remove the supernatant by magnetic separation, retain the magnetic particles, and wash 3 times.

6. The method according to claim 1, characterized in that: The specific method for step (5) is as follows: add the RCA amplification system to the product obtained in step (4) and react at 30℃ for 40 min.

7. The method according to claim 1, characterized in that: The specific method of step (6) is as follows: add hairpin probes H1 and H2, both of which are modified with biotin at the ends, to the RCA amplification product obtained in step (5), and react at 30℃ for 90 min.

8. The method according to claim 1, characterized in that: The specific method of step (7) is as follows: After the reaction in step (6) is completed, the system is placed on a magnetic rack and washed twice. Streptavidin-HRP is added and incubated for 10 min. After washing away the unbound HRP, TMB and H2O2 are added and incubated at room temperature in the dark for 10 min. Finally, dilute hydrochloric acid is added to terminate the reaction, and the absorbance at 450 nm is measured using an enzyme-linked immunosorbent assay (ELISA) reader.

9. The method according to claim 1, characterized in that: The mycotoxin standard solution was obtained by mixing mycotoxin standard with PBS buffer; the concentrations of the mycotoxin standard solution were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 ng / mL; after the Fe3O4@Au@cDNA@APT magnetic probe was incubated with the mycotoxin standard solution, it was followed by steps (5) to (7) in sequence. After the reaction was completed, the absorbance value at 450 nm was detected, and the measured absorbance value and the concentration of the mycotoxin standard solution were imported into Origin software for fitting. It was found that in the range of 0.1-500 ng / mL, the logarithm of the absorbance value (y) and the logarithm of the mycotoxin concentration (x) showed a good linear correlation, and the linear regression equation was lg(y) = -0.8216 + 0.3196 lg(x), R 2 = 0.9830.

10. The method according to claim 9, characterized in that: After the Fe3O4@Au@cDNA@APT magnetic probe is incubated with the sample to be tested, it is then processed sequentially through steps (5) to (7). After the reaction is completed, the absorbance value at 450 nm is detected. The obtained absorbance value is then substituted into the linear regression equation to obtain the concentration of mycotoxin, thus realizing the quantitative detection of mycotoxin in the sample to be tested.

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