A rapid aflatoxin b1 fluorescence detection method and kit

Abstract

This invention proposes a method and kit for detecting aflatoxin B1 (AFB1) using a multivalent probe fluorescence sensing based on functionalized magnetic beads, belonging to the field of food safety testing. FSNC magnetic beads are prepared, and fluorescently labeled aptamer-initiator chain complexes are immobilized to construct a sensing platform. AFB1 in the sample can specifically bind to the aptamer, releasing the initiator chain, triggering a catalytic hairpin assembly reaction, forming a DNAzyme, and cleaving the substrate to generate a P-chain. The P-chain competitively binds to achieve cascade signal amplification, ionizing the fluorescently labeled aptamer. After magnetic separation, the fluorescence intensity is detected at 519 nm, achieving quantitative detection of AFB1. This method has a linear range of 200 fM–10 nM, a detection limit of 92.67 fM, and a reaction time of 50 min, exhibiting advantages such as high sensitivity, strong specificity, good anti-interference, and fast speed. Combined with the separation and enrichment characteristics of magnetic beads, it can be adapted to complex food matrices such as beer and wine, and is suitable for rapid detection of AFB1, demonstrating good application value in on-site food safety screening.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to an analytical method and kit for detecting aflatoxin B1. Background Technology

[0002] Aflatoxin B1 (AFB1) is a class of highly carcinogenic secondary metabolites produced by fungi, widely found in grains, edible oils, alcoholic beverages and their products, posing a serious threat to food safety and human health. Therefore, establishing rapid and reliable detection methods is of great significance for AFB1 monitoring. In recent years, fluorescence detection methods based on nucleic acid aptamers have attracted widespread attention due to their good specificity and designability. Catalytic hairpin assembly (CHA), as an enzyme-free isothermal nucleic acid amplification strategy, has advantages such as mild reaction and high speed; multivalent Mg... 2+ Multivalent DNAzymes exhibit synergistic catalytic effects, resulting in significantly higher cleavage efficiency compared to monovalent systems. However, existing methods still face the challenge of long reaction times in complex sample systems, making it difficult to meet the demand for rapid detection of trace AFB1. Therefore, it is necessary to construct a rapid-response AFB1 detection method combining multivalent catalysis and CHA amplification to improve the reliability and speed of the detection system and meet the needs of practical sample analysis.

[0003] This invention constructs a method for detecting AFB1 using a multivalent probe fluorescence sensing strategy based on functionalized magnetic beads. First, Fe3O4@SiO2@NH2@CM-β-CD (FSNC) magnetic beads are synthesized. Utilizing the host-guest recognition mechanism of CM-β-CD, a complex (Apt-T) of a ferrocene-modified initiating chain T and the nucleic acid aptamer Apt is co-immobilized on the surface of the FSNC magnetic beads, resulting in functionalized FSNC-T. In the presence of the target analyte AFB1, the initiating chain T is induced to expose and further react with hairpins H1 and H2 in the solution system, initiating a CHA cycle and generating a detectable fluorescence signal. Compared with commonly used nucleic acid aptamer fluorescence sensing methods, this invention not only achieves highly sensitive detection of AFB1 but also possesses advantages such as ultra-high efficiency and ultra-low background, showing promising application prospects in the field of food safety detection. Summary of the Invention

[0004] This invention aims to provide a highly sensitive and rapid method for detecting AFB1. To this end, a multivalent probe fluorescence sensing strategy based on functionalized magnetic beads is constructed to achieve the detection of trace amounts of AFB1. For example... Figure 6As shown, when the target compound AFB1 is absent, the nucleic acid aptamer Apt and the initiator chain T complex are tightly bound to the FSNC magnetic beads. Hairpins H1 and H2 in the system cannot undergo self-hybridization due to complementary pairing at their intrachain stems, thus failing to trigger the CHA reaction. Simultaneously, the double hairpin CP complex remains stable due to tight intrachain binding, preventing the entire system from undergoing a large-scale reaction. After magnetic separation, the supernatant was tested, and the system only showed a low background signal. When the target compound AFB1 is present, the FAM-modified Apt on FSNC-A specifically binds to AFB1 and is released into the system. This process not only generates an initial weak fluorescence signal but also exposes the initiator chain T, providing triggering conditions for the subsequent CHA reaction. The T chain can open hairpin H1 in solution to form T-H1; subsequently, the opened hairpin H1 and H2 have highly complementary sequences, further hybridizing to form the H1-H2 complex, which displaces the T chain, allowing it to re-participate in the cyclic reaction. Simultaneously, the hybridization process of H1 and H2 allows the previously blocked Mg... 2+ The dependent DNAzyme and its linker arms and enzyme strand structures are exposed, forming catalytically active bivalent Mg2+ at both ends. 2+ These are DNAzymes that cyclically cleave double hairpin CPs with rA-modified sites. The resulting P-chain competes with the Apt-T and Apt-AFB1 complexes to generate a more stable Apt-P complex, prompting the release of AFB1 and its continued specific binding to Apt. Simultaneously, the released T-chain participates in the CHA cycle, further generating divalent Mg. 2+ The DNAzyme-dependent reaction accelerates the reaction process and continuously improves signal amplification efficiency, leading to the formation and release of more nucleic acid aptamers (Apt) and initiation chain T complexes into the system. Finally, the supernatant is magnetically separated, and its fluorescence intensity is measured at 519 nm, achieving highly sensitive, low-background, and rapid detection of AFB1.

[0005] The FSNC magnetic beads are dissolved in ultrapure water. The nucleic acid aptamer Apt, the initiator chain T complex, hairpin H1, hairpin H2, double hairpin CP, and E chain are all dissolved in 20 mM Tris-HCl buffer solution (containing 20 mM tris(hydroxymethyl)aminomethane, 120 mM NaCl, 20 mM KCl, 10 mM MgCl2, pH=7.4).

[0006] The 5' end of the nucleic acid aptamer Apt is modified with a fluorescent (FAM) group, the 5' end of the initiating chain T is modified with a ferrocene group, and the C chain is modified with a ribonuclease (rA) cleavage site; the sequence of the nucleic acid aptamer Apt is the sequence shown in SEQ ID No. 1, the sequence of the initiating chain T is the sequence shown in SEQ ID No. 2, the sequence of the hairpin H1 is the sequence shown in SEQ ID No. 3, the sequence of the hairpin H2 is the sequence shown in SEQ ID No. 4, the C sequence is SEQ ID No. 5, the P sequence is SEQ ID No. 6, and the E sequence is SEQ ID No. 7.

[0007] In the fluorescence detection method described above, preferably, the concentration of H1 in the hair clip is 40, 50, 60, 70, or 80 nM; more preferably, it is 60 nM.

[0008] In the fluorescence detection method described above, preferably, the H2 concentration of the hairpin is 40, 50, 60, 70, or 80 nM; more preferably, it is 60 nM.

[0009] In the fluorescence detection method, preferably, the C-chain concentration is 40, 50, 60, 70, or 80 nM; more preferably, it is 60 nM.

[0010] In the fluorescence detection method described above, preferably, the concentration of the P chain is 10, 20, 30, 40, or 50 nM; more preferably, it is 30 nM.

[0011] In the fluorescence detection method, preferably, the Mg²⁺ concentration is 5, 10, 15, 20, or 25 mM; more preferably, it is 10 mM.

[0012] In the fluorescence detection method described above, preferably, the concentration of the FSNC magnetic beads is 6, 8, 10, 12, or 14 mg / mL; more preferably, it is 10 mg / mL.

[0013] In the fluorescence detection method, preferably, the reaction temperature is 4, 15, 25, 37, or 45 °C; more preferably, it is 25 °C.

[0014] In the fluorescence detection method, preferably, the reaction time is 10, 30, 50, or 70 min; more preferably, it is 50 min.

[0015] As part of the same technical concept, the present invention also provides an AFB1 detection kit. The kit includes the nucleic acid sequence and reagents described in the detection method.

[0016] The kit includes a first container containing the FSNC magnetic beads, which are prepared with ultrapure water to a concentration of 10 mg / ml before use.

[0017] The kit includes a second container containing the nucleic acid aptamer Apt and the initiation chain T complex, which is diluted with Tris-HCl buffer to a concentration of 60 nM before use.

[0018] The kit includes a third container containing the hairpin H1, which is diluted with Tris-HCl buffer to a concentration of 60 nM before use.

[0019] The kit includes a fourth container containing the hairpin H2, which is diluted with Tris-HCl buffer to a concentration of 60 nM before use.

[0020] The kit includes a fifth container containing the CP complex, which is diluted to a concentration of 30 nM with Tris-HCl buffer before use.

[0021] The kit includes a sixth container containing the 20 mM Tris-HCl buffer solution (20 mM tris(hydroxymethyl)aminomethane, 120 mM NaCl, 20 mM KCl, 10 mM MgCl2, pH=7.4).

[0022] As part of the same technical concept, the present invention also provides a method for using an AFB1 detection kit.

[0023] The kit is used as follows: the FSNC magnetic beads in the first container are prepared into a solution with a mass concentration of 10 mg / mL using ultrapure water before use; the second container contains the nucleic acid aptamer Apt and the initiator chain T complex, and is diluted with Tris-HCl buffer to a concentration of 60 nM before use; the third container contains hairpin H1, and is diluted with Tris-HCl buffer to a concentration of 1 μM before use; the fourth container contains hairpin H2, and is diluted with Tris-HCl buffer to a concentration of 1 μM before use; the fifth container contains the CP complex, and is diluted with Tris-HCl buffer to a concentration of 1 μM before use.

[0024] The above-described assay kit is used as follows: Take 200 μL of solution in the first container, remove the supernatant using magnetic separation, and wash three times with Tris-HCl buffer. Add 300 μL of solution to the second container and react at 25°C for 2 h. After the reaction, remove the supernatant using magnetic separation and wash three times repeatedly with Tris-HCl buffer. Take 12 μL of solution from the third and fourth containers, and 6 μL of solution from the fifth container. Add Tris-HCl buffer from the sixth container to bring the volume to 200 μL and react at 25°C for 50 min. Measure the fluorescence intensity at 519 nm using a fluorescence spectrophotometer.

[0025] This invention has prominent substantive features and significant technological advancements, as detailed below:

[0026] (1) Using divalent Mg 2+ The DNAzyme-dependent cleavage strategy effectively enhanced signal output and shortened reaction time. Under the same conditions, comparative analysis verified that divalent Mg... 2+ The fluorescence signal generated by DNAzyme is about 1.5 times that of the monovalent system. (2) Apt with fluorescent group FAM is modified onto FSNC magnetic beads and combined with CHA strategy and bivalent substrate cleavage to achieve continuous accumulation and amplification of signal, which can further improve the sensitivity and accuracy of AFB1 detection, while reducing background signal interference, thereby achieving high sensitivity detection of AFB1. Instruction manual illustrations

[0027] Figure 1 This is a schematic diagram illustrating the fabrication process of FSNC magnetic beads.

[0028] Figure 2 This is a scanning electron microscope (SEM) characterization image of FSNC magnetic beads.

[0029] Figure 3 This is an X-ray diffraction characterization pattern of FSNC magnetic beads.

[0030] Figure 4 These are Fourier transform infrared spectral characterizations of Fe3O4, FSN, CM-β-CD, and FSNC magnetic beads.

[0031] Figure 5 This is a characterization diagram of the hysteresis loops of Fe3O4, FSN, and FSNC magnetic beads.

[0032] Figure 6 This is a schematic diagram of the AFB1 detection principle.

[0033] Figure 7 It is a characteristic graph of a nanoparticle size potentiometric analyzer.

[0034] Figure 8This is a polyacrylamide gel electrophoresis characterization image of a multivalent probe fluorescence sensing strategy based on functionalized magnetic beads.

[0035] Figure 9 This is a feasibility analysis diagram for fluorescence sensing methods.

[0036] Figure 10 This is a graph showing the optimized concentration of hairpin substrate H1.

[0037] Figure 11 This is a graph showing the optimized concentration of H2 in the hairpin substrate.

[0038] Figure 12 This is a graph showing the optimization of C-chain concentration.

[0039] Figure 13 This is a graph showing the optimal concentration of the P-chain.

[0040] Figure 14 It is Mg 2+ Concentration optimization diagram.

[0041] Figure 15 This is a graph showing the optimized concentration of FSNC magnetic beads.

[0042] Figure 16 This is a diagram showing the optimized reaction time of the detection system.

[0043] Figure 17 This is a diagram showing the optimized reaction temperature of the detection system.

[0044] Figure 18 It is both divalent and monovalent Mg 2+ PAGE results of comparative validation of DNAzyme dependence.

[0045] Figure 19 It is both divalent and monovalent Mg 2+ Comparative validation graph of DNAzyme dependence.

[0046] Figure 20 This is a graph showing the sensitivity and linear range of this method at different concentrations of AFB1.

[0047] Figure 21 It is a selective analysis chart of blank, T2, DON, FB, OTA, OTB, ZEN, AFG1 and AFB2 in the detection system. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments.

[0049] Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonia (NH3·H2O), and anhydrous ethanol (C2H6O) were purchased from Sinopharm Chemical Reagent Co., Ltd.; tetraethyl orthosilicate (TEOS) and aminopropyltriethoxysilane (APTES) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; carboxymethyl-β-cyclodextrin (CM-β-CD) was purchased from Shanghai E. En Chemical Technology Co., Ltd.; N-hydroxysuccinimide (NHS) and 1-ethyl-carbodiimide (EDC) were purchased from Aladdin Reagent (Shanghai). The following reagents were purchased from Sigma-Aldrich: nitrogen (N2) from Changsha Gaoke Gas Co., Ltd.; disodium ethylenediaminetetraacetate (EDTA-2Na) from Maclean's; potassium chloride (KCl), sodium chloride (NaCl), and magnesium chloride (MgCl2) from Xilong Scientific; AFB1, aflatoxin G1 (AFG1), ochratoxin B (OTB), ochratoxin A (OTA), aflatoxin B2 (AFB2), T2 toxin (T2), vomitoxin (DON), zearalenone (ZEN), and fumonisin B (FB); ammonium persulfate (APs), tris(hydroxymethyl)aminomethane (Tris), N,N,N,N-tetramethylethylenediamine (TEMED), and disodium ethylenediaminetetraacetate (EDTA-2Na) from Maclean's; potassium chloride (KCl), sodium chloride (NaCl), and magnesium chloride (MgCl2) from Xilong Scientific; and all other reagents were of analytical grade. All water used in this experiment was ultrapure water (≥18.2 MΩ∙cm), and all DNA purified by HPLC in this experiment was obtained from Sangon Biotech (Shanghai) Co., Ltd.

[0050] Table 1 shows the sequence information of the nucleic acid aptamer Apt, priming chain T, hairpin H1, hairpin substrate H2, C chain, P chain, and E chain.

[0051] Example 1: First, FSNC magnetic beads were prepared to provide a reliable foundation for subsequent functionalization of magnetic beads and the construction of sensing methods. The preparation process of FSNC magnetic beads was as follows: First, Fe3O4 magnetic nanoparticles were prepared using a co-precipitation method. Then, Fe3O4@SiO2@NH2 and FSNC magnetic beads were prepared sequentially using a solution blending method. The preparation flow chart is shown below. Figure 1 As shown. (1) Preparation of Fe3O4: Weigh 1 g FeCl2·4H2O and 2.79 g FeCl3·6H2O (molar ratio approximately Fe 2+ :Fe 3+=1:2) was placed in a 500 mL three-necked flask, and 200 mL of ultrapure water (≥ 18.2 MΩ∙cm) was added to completely dissolve the two. The three-necked flask was fixed in a water bath. Nitrogen gas was continuously introduced to remove dissolved oxygen, and mechanical stirring was performed at 1500 r / min. When the water bath temperature rose to 90℃, the stirring rate was increased to 1800 r / min, and 8 mL of 25% ammonia water was quickly added. The reaction was carried out at 90℃ for 1 h. After the reaction was completed, the system was cooled to room temperature, and magnetic separation was performed using an external magnetic field. The system was washed 4-5 times alternately with anhydrous ethanol and ultrapure water to remove residual ammonia water, and finally naked Fe3O4 magnetic nanoparticles were obtained. (2) Preparation of Fe3O4@SiO2@NH2 (FSN): The above Fe3O4 nanoparticles were dispersed in 20 mL of ultrapure water and 80 mL of anhydrous ethanol, placed in a 250 mL round-bottom flask, and ultrasonically dispersed in an ultrasonic cleaner for 15 min. Subsequently, 5 mL of 25% concentrated ammonia and 8 mL of TEOS were added, and the mixture was mechanically stirred at 1500 r / min for 12 h at room temperature. After the reaction was completed, the product was collected by magnetic separation, and washed 4-5 times alternately with anhydrous ethanol and ultrapure water to remove residual TEOS. The product was then washed with water until pH=7 to obtain Fe3O4@SiO2, which was dispersed in ultrapure water for later use. 1 g of Fe3O4@SiO2 after magnetic separation was taken and dispersed in 24 mL of anhydrous ethanol and 6 mL of ultrapure water in a 250 mL round-bottom flask. The mixture was ultrasonically dispersed in an ultrasonic cleaner for 15 min, and 8 mL of APTES was added. The mixture was mechanically stirred at 1500 r / min for 12 h at room temperature. After repeated washing with anhydrous ethanol and ultrapure water for 4-5 times to remove excess APTES, FSN magnetic nanomaterials were obtained and dispersed in ultrapure water for later use. (3) Preparation of FSNC magnetic beads: 1.193 g of CM-β-CD, 0.5 g of EDC, and 0.25 g of NHS were weighed and added to a 50 mL round-bottom flask. 20 mL of ultrapure water was added to dissolve them. The mixture was then mechanically stirred at 1500 r / min for 1 h at room temperature to activate the carboxyl groups of CM-β-CD. Subsequently, 800 mg of the magnetically separated wet weight of FSN was added to the 50 mL round-bottom flask. The mixture was mechanically stirred at 1500 r / min for 24 h at room temperature. After magnetic separation, the obtained product was washed repeatedly with anhydrous ethanol and ultrapure water 4-5 times to remove excess CM-β-CD, thus obtaining FSNC magnetic beads. The obtained FSNC magnetic beads were dispersed in ultrapure water for storage. A portion of the sample was freeze-dried under vacuum to prepare a FSNC magnetic bead solution with a mass concentration of 10 mg / mL. To demonstrate the successful preparation of FSNC magnetic beads, their synthesis was characterized using various methods including scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and magnetic hysteresis loop (VSM). SEM was used to characterize the surface structure and microtube morphology of the FSNC magnetic beads. The results are as follows: Figure 2 As shown in (a), (b), and (c), the FSNC magnetic beads exhibit a near-spherical morphology with relatively uniform particle size. Due to the magnetic properties of the material, there is a certain degree of aggregation among the particles. To further analyze the elemental composition of the FSNC magnetic beads, energy-dispersive X-ray spectroscopy (EDS) was used for characterization, as shown in... Figure 2 (d) is the EDS energy spectrum of FSNC magnetic beads. As can be seen from the figure, FSNC magnetic beads are mainly composed of Fe, Si, O, N and C elements. Figure 2 (f), (g), (h), (i), and (j) are the distribution diagrams of Fe, Si, O, N, and C elements in FSNC magnetic beads, respectively. It can be seen from the diagrams that the elements are evenly distributed, with O being the most abundant, followed by C, Si, Fe, and N. The above results indicate that SiO2 and CM-β-CD have been successfully modified on the surface of Fe3O4 magnetic nanoparticles, thus verifying the successful synthesis of FSNC magnetic beads. The crystal structure characteristics of FSNC magnetic beads were characterized using XRD. The results were presented by... Figure 3 As shown, the FSNC magnetic beads exhibited characteristic diffraction peaks corresponding to Fe3O4 magnetic nanoparticles at 2θ of 30.2º, 35.6º, 43.3º, 53.6º, 57.3º, and 62.8º, proving the existence of diffraction peaks on the (220), (311), (400), (422), (511), and (440) crystal planes, thus indicating that the FSNC nanomaterial has a cubic spinel structure. A broad peak related to SiO2 appeared at 2θ of 20-25º, indicating that SiO2 successfully modified the Fe3O4 magnetic nanoparticles. No peaks related to CM-β-CD appeared in the figure, indicating that they were uniformly distributed in the FSN and did not form crystalline aggregates. XRD characterization results show that the crystal phase of Fe3O4 magnetic nanoparticles did not change significantly during SiO2 coating and CM-β-CD surface functionalization, indicating that the surface modification did not destroy its crystal structure. This further shows that FSNC magnetic beads were successfully synthesized and have good crystal integrity and structural stability. The functional group characteristics of Fe3O4, FSN, CM-β-CD, and FSNC magnetic beads were analyzed by FT-IR. The results are as follows: Figure 4 As shown, in the FT-IR spectrum, Fe3O4 appears at approximately 590 cm⁻¹. -1The presence of a characteristic Fe-O absorption peak at approximately 1050 cm⁻¹ indicates the successful preparation of Fe₃O₄ magnetic nanoparticles. In the FSN spectrum, these peaks are observed at approximately 1050 cm⁻¹. -1 and 1620 cm -1 The observation of asymmetric stretching vibration peaks of Si-O-Si and bending vibration peaks of NH at approximately 3450 cm⁻¹ indicates that SiO₂ coating and amination modification have been successfully achieved. These characteristic peaks prove the successful synthesis of FSN. For CM-β-CD and FSNC magnetic beads, a peak at approximately 3450 cm⁻¹ is observed. -1 Broad characteristic absorption peaks appear at all locations, corresponding to the stretching vibration of -OH; in addition, FSNC magnetic beads show a peak at 2923 cm⁻¹. -1 A new absorption peak was observed in FSNC magnetic beads at 500-600 cm⁻¹, corresponding to the asymmetric stretching vibration of the aliphatic -CH bond in CM-β-CD. Meanwhile, Fe₃O₄, FSN, and FSNC magnetic beads showed absorption peaks at 500-600 cm⁻¹. -1 The presence of distinct Fe-O characteristic absorption peaks within the range indicates that the crystal structure of Fe3O4 was not altered during the modification process. Furthermore, the FSNC magnetic beads exhibit absorption peaks at approximately 1025 cm⁻¹. -1 and 1150 cm -1 The absorption peak at this point can be attributed to the stretching vibrations of the trans-glycoside COC and the coupled CC / CO, proving that CM-β-CD is successfully linked to FSN. In summary, the presence of the characteristic functional groups in the spectrum strongly indicates that Fe3O4, FSN, and FSNC magnetic beads have been successfully prepared. The magnetic properties of Fe3O4, FSN, and FSNC magnetic beads were tested using a VSM. The results are as follows: Figure 5 As shown, the saturation magnetization (Ms) of Fe3O4, FSN, and FSNC magnetic beads are 70.11 emu·g. -1 59.91 emu·g -1 49.95 emu·g -1 The magnetic response gradually decreases. This is mainly attributed to the SiO2 shell coating and the further functionalization modification of CM-β-CD on the surface of Fe3O4 magnetic nanoparticles, which leads to a decrease in the proportion of magnetic components in the material, thus weakening the magnetic response. Nevertheless, FSNC magnetic beads still retain a certain degree of magnetism and can achieve effective magnetic separation and reuse. In addition, FSNC magnetic beads have low coercivity and remanence, indicating that they have good superparamagnetic characteristics. In summary, after coating and surface modification, FSNC magnetic beads still have good magnetic response performance, which can meet the requirements of subsequent magnetic separation operations.

[0052] Example 2: An AFB1 detection method of the present invention, wherein 200 μL of solution is taken in the first container, and the supernatant is removed by magnetic separation; 300 μL of solution is taken in the second container and added to the test solution, and reacted at room temperature for 2 h; after the reaction, the supernatant is removed by magnetic separation, and the solution is washed three times with Tris-HCl buffer solution; 300 μL of solution is taken in the third container and added to the test solution, and reacted at room temperature for 2 h; after the reaction, the supernatant is removed by magnetic separation, and the solution is washed three times with Tris-HCl buffer solution; 4 μL of solution is taken in the fourth and fifth containers respectively and added to the test solution, and reacted at room temperature for 90 min; after the reaction, the supernatant is removed by magnetic separation, and the fluorescence intensity at 519 nm is measured using a fluorescence spectrophotometer.

[0053] Example 3: To verify the successful construction of FSNC-A and facilitate the design and detection of subsequent fluorescence methods, DLS was used to determine the hydration particle size and surface potential of Fe3O4, FSN, FSNC magnetic beads, and FSNC-A. The results are as follows: Figure 7 As shown in (A), the average particle size of Fe3O4 is approximately 639 nm. After aminosilanization modification, the average particle size of FSN increases to 1004 nm. Further, by chemically bonding CM-β-CD onto FSN, the average particle size of the resulting FSNC magnetic beads increases to 1474 nm. Based on this, the introduction of the acid aptamer Apt and the initiator chain T complex yields FSNC-A, whose average particle size further increases to 2298 nm. The results of the gradual increase in particle size with each modification step indicate that the Apt-T complex was successfully modified onto the surface of the FSNC magnetic beads. Figure 7 (B) shows the surface Zeta potentials of Fe3O4, FSN, FSNC magnetic beads, and FSNC-A. The results indicate that the surface potential of Fe3O4 is -9.13 mV, while that of FSN after aminosilanization modification is 11.56 mV. After modifying the FSN surface with CM-β-CD, its potential decreases to -22.80 mV. Further modification with Apt-T further reduces the potential of FSNC-A to -41.33 mV. The combined results of hydration diameter and surface potential demonstrate the successful construction of FSNC-A.

[0054] Example 4: The nucleic acid aptamer Apt and the initiator chain T complex, and the double hairpin CP complex. The nucleic acid aptamer Apt and the initiator chain T complex were prepared by mixing equal amounts of Apt and T. The CP complex was prepared by mixing twice the amount of C and one the amount of P, heating at 95°C for 5 min, and then slowly cooling to room temperature.

[0055] The FSNC magnetic beads, nucleic acid aptamer Apt and priming chain T complex, hairpin H1, hairpin H2 and hairpin CP complex were all dissolved in Tris-HCl buffer.

[0056] Example 5: Feasibility Analysis of AFB1 Detection

[0057] To verify the feasibility of constructing a multivalent probe fluorescence sensing strategy based on functionalized magnetic beads, polyacrylamide gel electrophoresis was first used to validate the feasibility of this method. The results are as follows: Figure 8 As shown in (A), lanes 1-5 are T, H1, H2, C, and P, respectively; lane 6 is a double hairpin CP; and lane 7 is a mixture of H1 and H2. Since H1 and H2 have the same molecular weight, their bands only overlap and no new bands are generated. This indicates that in the absence of the initiating chain T, H1 and H2 can exist stably and cannot complement each other to form bivalent Mg with active structures at both ends. 2+ Dependent DNAzyme; Lane 8 is the T-H1 complex; Lane 9 is a mixture of T-H1 and H2. After the reaction of T-H1 and H2, a band with a slower migration rate appeared, along with a band with the same migration rate as the T chain, indicating that after T hybridizes with H1, an H1-H2 complex can be formed in the presence of H2, releasing the T chain; Lane 10 is a mixture of T-H1, H2, and double hairpin CP. Three new bands appeared in this system, indicating that when divalent Mg is formed... 2+ Following DNAzyme dependence, in Mg 2+ The presence of this element will cut the double hairpin CP, resulting in three single chains. Referring to swimlane 5, one of these single chains may be P. For example... Figure 8 As shown in (B), lane 1 is Apt; lane 2 is the complex of nucleic acid aptamer Apt and initiator chain T; lane 3 shows that after the addition of AFB1, a new band appears, indicating that Apt and AFB1 specifically bind to form Apt-AFB1, leading to the release of initiator chain T; lane 4 is the T-H1 complex; lane 5 is a mixed system of T-H1 and H2. Lane 6 represents a mixture of T-H1, H2, and double hairpin CP; lane 7 represents a mixture of nucleic acid aptamer Apt and the initiator chain T complex, H1, and H2 in the absence of AFB1. Each band in these lanes remained stable, indicating that Apt-T, H1, and H2 do not undergo self-hybridization. Lane 8 represents a mixture of nucleic acid aptamer Apt and the initiator chain T complex, H1, H2, and AFB1. Upon the addition of AFB1, two bands with the same migration rate as in lane 5 appeared, indicating that the presence of AFB1 leads to the release of the initiator chain T, which hybridizes with H1 to form T-H1, and then reacts with H2 to ultimately generate divalent Mg. 2+DNAzyme-dependent reaction; Lane 9 is a mixture of nucleic acid aptamer Apt and initiator chain T complex, H1, H2, and CP. Compared with other lanes, their bands are stable and show no significant changes, indicating that no reaction occurs in the absence of AFB1; Lane 10 is a mixture of Apt-T, H1, H2, CP, and AFB1. It shows a band with the same migration rate as in lane 1, indicating that the addition of AFB1 leads to the release of the T chain, which opens H1 and then reacts with H2 to form divalent Mg. 2+ The DNAzyme-dependent cleavage of the double hairpin CP chain results in the formation of a P chain, which competes with the aptamer Apt, the initiator chain T complex, and Apt-AFB1 to form Apt-P, leading to the release of more T chains, which then participate in subsequent cyclic amplification. These results demonstrate that the established analytical method is feasible in principle.

[0058] The emission spectra of the supernatant obtained by magnetic separation under different conditions were further verified using a fluorescence spectrophotometer. The results are as follows: Figure 9 As shown, without the addition of AFB1, the fluorescence signal of the system is weak, with only a background signal in the solution. When 10 nM of the target compound AFB1 is added, the fluorescence intensity signal at 519 nm increases slightly when only FSNC-A is present. This is because AFB1 specifically binds to Apt on FSNC-A to form Apt-AFB1, thereby promoting the release of a small amount of Apt with fluorescent groups into the solution. When FSNC-A, H1, and H2 are present in the system, the fluorescence intensity is approximately the same as when only FSNC-A is present. This is because the T released from FSNC-A can react sequentially with H1 and H2 to form divalent Mg. 2+ The DNAzyme-dependent reaction initiates the first cycle, but due to the lack of the substrate double hairpin CP, subsequent cleavage reactions cannot be triggered. Therefore, there is no significant difference in fluorescence intensity measured from the supernatant after magnetic separation. When FSNC-A, H1, H2, and double hairpin CP are present in the system, the T chain is released first, then reacts with H1 to generate T-H1. T-H1 continues to react with H2 to replace the T chain while generating divalent Mg. 2+ DNAzyme-dependent. In Mg 2+ In the presence of the CP chain, the hairpin is cleaved to generate a single-chain P. The P chain competes with Apt-T in FSNC-A and Apt in Apt-AFB1 in the system, reacting to generate Apt-P to initiate cycle II. Consequently, a large amount of fluorescent Apt and Apt-P are released into the solution in soluble form. After magnetic separation, the supernatant was collected, and its fluorescence intensity at 519 nm was measured, showing a significant signal enhancement. This indicates that the designed detection strategy can effectively identify AFB1 while simultaneously amplifying the signal.

[0059] Example 6: Optimization of H1 Concentration in Hairpins

[0060] To optimize the detection method, the effect of different H1 concentrations on the fluorescence intensity ratio ΔF / F0 was investigated. ΔF is the difference between the fluorescence intensity F of the reaction system at 519 nm in the presence of 10 nM AFB1 and the fluorescence intensity F0 of the system at the same wavelength in the presence of 0 nM AFB1 (ΔF = F – F0), where F0 is the fluorescence intensity of the system at the same wavelength in the presence of 0 nM AFB1. Following the steps described in Experimental Example 2, and keeping other conditions constant, the H1 concentration was optimized. The results are as follows... Figure 10 As shown, the ΔF / F0 value is maximized when the H1 concentration is 60 nM. Therefore, an H1 concentration of 60 nM is chosen as the optimal condition.

[0061] Example 7: Optimization of H2 concentration in substrate hairpins

[0062] The concentration of the substrate hairpin H2 is an important parameter affecting the fluorescence signal of the system. Reaction concentrations of 40, 50, 60, 70, and 80 nM were set, and the concentration of the substrate hairpin H2 was optimized by keeping other conditions constant, following the steps in Example 2. The results are as follows... Figure 11 As shown, the ΔF / F0 value is optimal when the substrate H2 concentration is 60 nM. Therefore, a substrate H2 concentration of 60 nM is chosen as the optimal condition.

[0063] Example 8: C-chain concentration optimization

[0064] The structure of the double-hairpin CP has a significant impact on the fluorescence response of the amplified system. The C chain is a crucial reactant for generating the double-hairpin CP. Concentrations of 40, 50, 60, 70, and 80 nM were set, and the C chain concentration was optimized by keeping other conditions constant, following the steps in Example 2. The results are as follows... Figure 12 As shown, the ΔF / F0 value is optimal when the C-chain concentration is 60 nM. Therefore, a C-chain concentration of 60 nM is chosen as the optimal condition.

[0065] Example 9: Optimization of P-chain concentration

[0066] The concentration of the P chain is an important parameter affecting the fluorescence signal of the system. Following the steps of Example 3, with other conditions unchanged, the concentrations of the P chain were optimized by setting concentrations of 10, 20, 30, 40, and 50 nM. The results are as follows... Figure 13 As shown, the ΔF / F0 value is optimal when the concentration of the P chain is 30 nM. Therefore, a P chain concentration of 30 nM is chosen as the optimal condition.

[0067] Example 10: Mg 2+ Concentration optimization

[0068] Mg2+ DNAzyme dependent on Mg during catalysis 2+ As a cofactor, Mg2+ cleaves the substrate chain by hydrolyzing the phosphodiester bonds of nucleotides in the double hairpin CP, and its concentration directly affects the catalytic efficiency of the DNAzyme and the signal output of the system. Mg2+ concentrations of 5, 10, 15, 20, and 25 mM were used. 2+ Concentration, following the steps of Example 2, while keeping other conditions unchanged, for Mg 2+ The concentration was optimized. The results are as follows: Figure 14 As shown, when Mg 2+ The ΔF / F0 value was optimal at a concentration of 10 nM. Therefore, Mg was chosen. 2+ The optimal concentration was 10 nM.

[0069] Example 11: Optimization of FSNC magnetic bead concentration

[0070] FSNC magnetic beads are closely related to the formation of fluorescence signal intensity. Therefore, FSNC magnetic bead concentrations of 6, 8, 10, 12, and 14 mg / mL were set, and the concentrations were optimized following the steps in Example 2 while keeping other conditions constant. The results are as follows: Figure 15 As shown, the ΔF / F0 value is optimal when the concentration of FSNC magnetic beads is 10 mg / mL. Therefore, 10 mg / mL is selected as the optimal concentration for use of FSNC magnetic beads.

[0071] Example 12: Optimization of Scale-up Reaction Time

[0072] The detection signal of the system increases with increasing reaction time until equilibrium is reached. Reaction times of 10, 30, 50, and 70 min were set, and the reaction time of the system was optimized by keeping other conditions constant, following the steps of Example 2. The results are as follows... Figure 16 As shown, the ΔF / F0 value reaches its maximum value when the amplification reaction time is 50 min, and 90 min is selected as the optimal amplification reaction time.

[0073] Example 13: Optimization of reaction temperature in the detection system

[0074] Reaction temperature is another key factor for signal recognition and output in this system. Reaction temperatures of 4, 15, 25, 37, and 45 °C were set, and following the steps of Example 2, while keeping other conditions constant, the effect of reaction temperature on the ΔF / F0 ratio was investigated. The results are as follows: Figure 17 As shown, the ratio of ΔF / F0 reaches its maximum at 25 °C. Therefore, 25 °C was chosen as the optimal reaction temperature for the detection system.

[0075] Example 14: Divalent and Monovalent Mg 2+Dependency DNAzyme Comparative Validation

[0076] To verify the difference between divalent and monovalent Mg 2+ The effect of DNAzyme-dependent fluorescence signal output was investigated, and the effects of divalent Mg were verified. 2+ DNA-dependent catalytic enzymes exhibit higher catalytic efficiency. PAGE analysis of the system yielded the following results: Figure 18 As shown. Lanes 1, 2, and 3 correspond to individual T, C, and P chains, respectively; lane 4 is a double hairpin CP; lane 5 is H1 and H2; lane 6 is T-H1; lane 7 is a mixture of T-H1 and H2; and lane 9 is a single E, where E is monovalent Mg. 2+ The DNAzyme-dependent cleavage also cleaves double-hairpin CP; lane 8 is a mixture of T-H1 and H2 with double-hairpin CP, and lane 9 is a mixture of E with double-hairpin CP. Both lanes 8 and 9 show three new bands, but the CP band in lane 9 is brighter than the CP band in lane 8, indicating that under the same experimental conditions, divalent Mg... 2+ DNAzyme-dependent catalytic cleavage is significantly more efficient than monovalent Mg. 2+ The band corresponding to the DNAzyme-dependent DNA. Simultaneously, the brightness of the new bands generated in lanes 8 and 9 indicates the presence of divalent Mg. 2+ The DNA-dependent system exhibits superior catalytic efficiency compared to the monovalent system, demonstrating higher substrate conversion capability. Therefore, the above results fully demonstrate the superiority of divalent Mg... 2+ The design of DNAzyme-dependent cleavage strategies can achieve more efficient cleavage reactions, thereby significantly enhancing the efficiency of fluorescence signal output and improving the sensitivity and response performance of the detection system.

[0077] Furthermore, by comparing divalent and monovalent Mg 2+The cleavage ability of the DNAzyme-dependent DNA cleavage against the double-hairpin CP was further evaluated to assess its impact on fluorescence signal output. The resulting P chain competitively hybridizes with the nucleic acid aptamer Apt and the initiator chain T complex on the surface of FSNC-A, displacing and releasing the T chain to form the Apt-P complex, thereby promoting the release of fluorescently labeled Apt into the solution system. Finally, the cleavage efficiency of the two DNAzymes was evaluated by detecting the fluorescence intensity of the solution. For system A, 200 μL of the modified FSNC-A solution was added to a centrifuge tube, the supernatant was discarded by magnetic separation, and H1-H2 (60 nM) and the CP complex (C: 60 nM, D: 30 nM), which had been annealed at 95 °C for 5 min and formed a stable structure, were added. 20 mM Tris-HCl buffer was added to bring the final volume to 200 μL, and the reaction was carried out at 25 °C for 10–60 min. After the reaction, the supernatant was collected by magnetic separation and transferred to a new tube. The fluorescence intensity of the solution in the new tube was then measured using a fluorescence spectrophotometer. For system B, under the same conditions, H1-H2 are replaced with monovalent Mg. 2+ The DNAzyme-dependent reaction (E strand, concentration 120 nM) was used, with all other reaction conditions remaining consistent. The fluorescence signal intensity results for system A and system B are as follows: Figure 19 As shown. The results indicate that, under the same reaction time conditions, the fluorescence intensity of system A is significantly higher than that of system B. Quantitative analysis reveals that divalent Mg... 2+ The fluorescence signal produced by the DNAzyme-dependent system is approximately 1.5 times that of the monovalent system. This is because, although the total concentration of the E strand is greater than that of H1-H2, H1-H2 contains divalent Mg. 2+ DNAzyme-dependent, its equivalent Mg 2+ The number of DNA-dependent active sites may be comparable to that of system B. Based on this, divalent Mg... 2+ DNA-dependent enzymes may have structural synergistic effects, enabling them to more efficiently recognize and cleave double hairpin CPs, thereby generating more P chains and participating in subsequent cyclic amplification reactions. This, in turn, promotes the continuous accumulation and amplification of fluorescence signals in a shorter time, ultimately resulting in stronger fluorescence signal output.

[0078] Example 15: Sensitivity and linear range of the method at different concentrations of AFB1

[0079] The sensitivity of this method was tested using different concentrations of AFB1 (0 pM, 1 pM, 5 pM, 10 pM, 50 pM, 80 pM, 100 pM, 200 pM, 300 pM, 500 pM, 1 nM, 5 nM, 10 nM) following the steps of Example 3, keeping other conditions constant. The results are as follows: Figure 20As shown in (A), the maximum fluorescence intensity of the reaction system increases with increasing AFB1 concentration; Figure 20 As shown in (B), within the concentration range of 200 fM to 10 nM, the fluorescence intensity of this method exhibits good linearity with the logarithm of AFB1 concentration, and its linear regression equation is: A = 1.8023 + 2.5800·lgC, with a correlation coefficient R. 2 =0.9969; Based on the 3σ / S principle (σ is the standard deviation of the blank sample, and S is the slope of the linear equation), the detection limit is calculated to be 92.67 fM.

[0080] Example 16: Selective analysis of AFB1 detection using the fluorescence analysis method of the present invention

[0081] The experiment used 10 nM T2, DON, FB, OTA, OTB, ZEN, AFG1, and AFB2, and 1 nM AFB1, following the steps of Example 2, keeping other conditions unchanged, to selectively analyze the fluorescence. The results are as follows: Figure 21 As shown, the fluorescence intensity values ​​of other interfering toxins at a concentration 10 times that of AFB1 at 519 nm were similar to those of the blank control group in Trsi-HCl buffer, while the fluorescence intensity response value of adding only 1 nM of AFB1 increased significantly. The results demonstrate that this fluorescence analysis method has good selectivity for AFB1.

[0082] Example 17: Actual Sample Testing

[0083] Beer and wine were selected as actual samples. Different concentrations of AFB1 standard solution were added to the samples, and pretreatment was performed according to the method described in the literature. The detection steps in Example 3 were then followed, and the spiked recoveries of AFB1 in beer and wine samples were calculated. The results showed that the spiked recoveries for both samples were 92.65%-104.43%, with RSDs of 2.05%-7.18%, indicating that the method has good accuracy and reliability. These results demonstrate that this method has good quantitative analytical capabilities in beer and wine samples, showing its promising application prospects in food safety testing.

Claims

1. A detection system for aflatoxin B1 (AFB1), characterized in that... The system includes Fe3O4@SiO2@NH2@CM-β-CD (FSNC) magnetic beads, a complex of a nucleic acid aptamer (Apt) and an initiating chain (T) (Apt-T complex), hairpin H1, hairpin H2, and a complex of nucleic acid chain C and nucleic acid chain P (CP complex).

2. The AFB1 detection system according to claim 1, characterized in that... The sequence of the nucleic acid aptamer Apt is shown in SEQ ID No. 1, the sequence of the initiating chain T is shown in SEQ ID No. 2, the sequence of the hairpin H1 is shown in SEQ ID No. 3, the sequence of the hairpin H2 is shown in SEQ ID No. 4, the sequence of the substrate chain C is shown in SEQ ID No. 5, and the sequence of the product chain P is shown in SEQ ID No.

6.

3. The AFB1 detection system according to claim 1, characterized in that... The Apt-T complex and CP complex are prepared by the following methods: (1) Preparation of nucleic acid aptamer Apt and initiator chain T complex: equal amounts of Apt and T are mixed, heated at 95°C for 5 min, and then naturally cooled to room temperature; (2) Preparation of CP complex: C and P are mixed (the volume ratio of the solution is 2:1), heated at 95°C for 5 min, and then naturally cooled to room temperature.

4. The AFB1 detection system according to claim 1, characterized in that... The FSNC magnetic beads are prepared by the following method: (1) 1 g FeCl2·4H2O and 2.79 g FeCl3·6H2O were dissolved in ultrapure water and placed in a three-necked flask. The mixture was stirred at 1500 r / min and heated to 90 °C under nitrogen protection. Then the stirring rate was increased to 1800 r / min, and ammonia water was quickly added. The mixture was incubated at 90 °C for 1 h. After the reaction was completed, the mixture was cooled to room temperature, and the product was collected by magnetic separation. The product was washed several times with anhydrous ethanol and ultrapure water alternately to obtain Fe3O4 magnetic nanoparticles. (2) Fe3O4 was dispersed in a mixed solution of 20 mL ultrapure water and 80 mL anhydrous ethanol, sonicated for 15 min, and then 5 mL ammonia and 8 mL tetraethyl silicate were added. The mixture was stirred at room temperature for 12 h to obtain Fe3O4@SiO2. After magnetic separation and washing to neutrality, it was dispersed in an ethanol / water mixture, and 3-aminopropyltriethoxysilane was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, after magnetic separation and washing, amino-functionalized Fe3O4@SiO2@NH2 (FSN) magnetic nanomaterials were obtained. (3) Carboxymethyl-β-cyclodextrin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide were dissolved in 20 mL of ultrapure water and stirred at room temperature for 1 h to activate the carboxyl groups. Then, 800 mg of FSN (wet weight) was added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, unreacted substances were removed by magnetic separation and repeated washing to obtain FSNC magnetic beads. Finally, the product was dispersed in ultrapure water for storage and then freeze-dried under vacuum to prepare a 10 mg / mL stock solution for later use.

5. The AFB1 detection system according to claim 1, characterized in that... The 5' end of the nucleic acid aptamer Apt is modified with a fluorescent group.

6. The AFB1 detection system according to claim 1, characterized in that... The 5' end of the initiator chain T is modified with a ferrocene group (Fc).

7. A detection system for AFB1 according to any one of claims 1 to 6, characterized in that... The detection steps are as follows: (1) Take the FSNC magnetic bead solution, magnetically separate and discard the supernatant, add the nucleic acid aptamer Apt and initiate the reaction of the T complex to obtain the functionalized magnetic bead FSNC@Apt-T (FSNC-A). (2) Add the test sample, hairpin H1, hairpin H2 and CP complex to the FSNC-A system, adjust the volume with Tris-HCl buffer, react at room temperature, magnetically separate the supernatant and measure the fluorescence intensity at 519 nm.

8. The AFB1 detection system according to claim 5, characterized in that... The concentration of the FSNC magnetic beads was 10 mg / mL, the concentration of the nucleic acid aptamer Apt and the initiator chain T complex was 60 nM, the concentration of hairpin H1 was 60 nM, the concentration of hairpin H2 was 60 nM, the concentration of the C chain was 60 nM, and the concentration of the P chain was 30 nM.

9. A detection kit comprising the AFB1 detection system according to any one of claims 1 to 6, characterized in that... The kit includes: A first container, the first container containing FSNC magnetic beads; The second container contains the nucleic acid aptamer Apt and the initiation chain T complex; A third container, wherein the third container contains a hair clip H1; A fourth container, wherein the fourth container contains a hairpin H2; The fifth container contains a C-chain and a P-chain; The sixth container contains a 20 mM Tris-HCl buffer solution.

10. A method of using the AFB1 detection kit as described in claim 9, characterized in that... Includes the following steps: (1) Take 200 μL of FSNC magnetic bead solution in the first container, magnetically separate and discard the supernatant, add 300 μL of nucleic acid aptamer Apt and initiator chain T complex solution in the second container, mix and incubate at 25℃ for 2 h, magnetically separate and discard the supernatant, wash 3 times with Tris-HCl buffer to obtain FSNC-A; (2) Add the sample to be tested to the prepared FSNC-A, and add 300 μL of the third container to the test solution. Mix and incubate at 25°C for 2 h. After the mixing and incubation, remove the supernatant by magnetic separation. Wash the solution repeatedly with Tris-HCl buffer solution 3 times. Add 4 μL of the fourth container H1 and the fifth container H2 to the test solution and mix and incubate at room temperature for 90 min. After the mixing and incubation, remove the supernatant by magnetic separation and measure the fluorescence intensity at 519 nm using a fluorescence spectrophotometer.

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