A smart chemiluminescence sensing system, a method for determining aflatoxin B1, and its application in traditional Chinese medicine.

Abstract

This invention discloses an intelligent chemiluminescence sensing system, a method for determining aflatoxin B1, and its application in traditional Chinese medicine. The system comprises: a complex formed by the binding of the aflatoxin B1 aptamer Apt and a partially complementary cDNA sequence of Apt, which releases the cDNA in the presence of aflatoxin B1; a complex composed of hairpin DNA that can be opened by the released cDNA and two hemin molecules that bind to both ends of the hairpin DNA and quench each other; and an endonuclease whose recognition site is located on the double-stranded DNA formed by the binding of cDNA and hairpin DNA, capable of cleaving the complex B into two hemin-labeled sequences and releasing the cDNA. This system can detect aflatoxin B1 with high sensitivity, accuracy, and speed without washing and drying, and is effective and accurate in the detection of aflatoxin B1 in traditional Chinese medicine, representing a new technology and method for effectively serving the quality control of traditional Chinese medicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, specifically relating to an intelligent chemiluminescence sensing system, a method for determining aflatoxin B1, and its application in traditional Chinese medicine. Background Technology

[0002] In recent years, mycotoxin contamination has led to frequent safety incidents involving traditional Chinese medicine (TCM), posing a significant threat to human health and hindering the competitiveness of TCM in the international market. Among more than 400 types of mycotoxins, aflatoxin B1 (AFB1) is considered the most common and harmful, as it contaminates various crops, including grains, oilseeds, and nuts, and exhibits strong mutagenic, teratogenic, and carcinogenic properties (AFB1 is 68 times and 10 times more toxic than arsenic and potassium cyanide, respectively, and 75 times and 900 times more carcinogenic than dimethylnitrosamine and dimethylaniline, respectively). AFB1 contamination has attracted increasing attention from governments worldwide, and its maximum limit is typically set at the ppb level. For example, the 2023 edition of the British Pharmacopoeia and the 2020 edition of the United States Pharmacopoeia set the limit for AFB1 at 2 μg / kg. -1 And 5 ppb, the 2020 edition of the Chinese Pharmacopoeia stipulates that the content of AFB1 in consumer products shall not exceed 5 μg / kg. -1 Therefore, the accurate determination of trace amounts of AFB1 is of great significance.

[0003] Currently, the most commonly used methods for detecting mycotoxins include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). However, ELISA requires cumbersome washing and drying steps and often suffers from insufficient sensitivity. While HPLC and LC-MS offer high sensitivity, they involve complex sample pretreatment, expensive equipment, and large quantities of organic solutions, requiring rigorous operator training. Therefore, developing simple, rapid, accurate, and sensitive novel detection methods for AFB1 is crucial.

[0004] Chemiluminescence (CL) has excellent application prospects in food safety detection due to its advantages such as not requiring an external excitation light source, high sensitivity, and simple operation. A series of chemiluminescence immunosensing methods have been developed for the detection of trace amounts of AFB1. However, these methods still cannot avoid the cumbersome washing and drying steps, which introduces significant errors to the detection results and wastes time. To address this problem, this invention application is hereby filed. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an intelligent chemiluminescence sensing system, a method for determining aflatoxin B1, and its application in traditional Chinese medicine.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A smart chemiluminescence sensing system, comprising:

[0008] Complex A: A complex formed by the binding of the aflatoxin B1 aptamer Apt and a partially complementary sequence of cDNA to Apt, which releases the cDNA in the presence of aflatoxin B1;

[0009] Complex B: A complex consisting of hairpin DNA that can be released by cDNA binding and two hemins that bind to both ends of the hairpin DNA and quench each other.

[0010] Restriction endonucleases are enzymes that recognize cDNA and hairpin DNA on the double-stranded DNA, and can cleave complex B into two hemin-labeled sequences and release cDNA.

[0011] Preferably, the nucleotide sequence of the aflatoxin B1 aptamer Apt is shown in sequence No. 1.

[0012] More preferably, the nucleotide sequence of the cDNA is shown in sequence No. 2.

[0013] More preferably, the nucleotide sequence of the hairpin DNA is shown in sequence No. 3.

[0014] More preferably, the restriction endonuclease is the restriction endonuclease Nt.BbvCI.

[0015] A method for determining aflatoxin B1 based on the above-mentioned intelligent chemiluminescence sensing system includes the following steps:

[0016] Step S1: Dissolve Apt, cDNA, and complex B powder separately in TE buffer to prepare stock solutions, then use rCutsmart + The buffer solution was diluted to a diluent.

[0017] Step S2: Mix equal amounts of Apt and cDNA dilution solution and react at room temperature to generate Apt-cDNA;

[0018] Step S3: Take an appropriate amount of Apt-cDNA, complex B dilution buffer, restriction endonuclease and an appropriate amount of sample solution containing aflatoxin B1, mix them at 37°C, and after reacting for a certain time, add an appropriate amount of chemiluminescent substrate that can be catalyzed by hemin and immediately measure the chemiluminescence intensity.

[0019] The application of the above-mentioned intelligent chemiluminescence sensing system in the determination of aflatoxin B1 in traditional Chinese medicine.

[0020] The above method is used to determine aflatoxin B1 in traditional Chinese medicine.

[0021] Beneficial effects:

[0022] The intelligent chemiluminescence sensing system provided by this invention enables simple and rapid detection of AFB1 using three DNA sequences and restriction enzymes. The three DNA sequences include a chemiluminescence switch and a homogeneous aptamer sensor, exhibiting stability and extremely low biotoxicity. AFB1 recognition, hemin dimer depolymerization, and hemin peroxidase activity can be achieved in a single reaction. Signal amplification is generated with the help of the restriction enzyme, resulting in a wide linear range for chemiluminescence analysis of AFB1. This system requires no washing or separation, is easy to operate, inexpensive, efficient, and environmentally friendly, and requires no specialized training for operators. Attached Figure Description

[0023] Figure 1 : A schematic diagram illustrating the basic principle of the method of this invention;

[0024] Figure 2 (A) Base pair optimization of HP and cDNA'; (B) Base pair optimization of Apt and cDNA'; (C) Validation of the signal switching capability of the final selected cDNA using UV-vis visible spectroscopy;

[0025] Figure 3 : Feasibility verification of AFB1 hijacking Apt; (A) Fluorescence quenching and recovery strategy; (B) Fluorescence spectrum of Cy5-labeled Apt at λex=648nm, the solution was reacted with (1) TE buffer, (2) BHQ2-labeled cDNA, (3) (2) + 100ng mL - 1 AFB1 mixture, incubated; (C) CL kinetic curve at 600V and gain 2, solutions were (1) HP, (2) (1) + cDNA, (3) (1) + Apt-cDNA, (4) (3) + 10ng mL -1 AFB1;

[0026] Figure 4 Verification of the cyclic amplification signal of Nt.BbvCI; (A) Chemiluminescence kinetics at 800V and gain 2; (B) Intensity of chemiluminescence signal at 100s; The solutions were: (1) Apt-cDNA and HP, (2) Apt-cDNA, HP and Nt.BbvCI, (3) Apt-cDNA, HP and 10ng mL -1 AFB1, (4)Apt-cDNA, HP, 10ng mL -1AFB1 and Nt.BbvCI;

[0027] Figure 5 Optimization of experimental conditions at 800V voltage and gain of 2; (A) optimization of HP concentration, (B) optimization of Apt-cDNA concentration, (C) optimization of reaction time; AFB1 concentration in group S is 10 ng / mL. -1 Group N has no AFB1;

[0028] Figure 6 pH optimization of the reaction system at 800V and gain of 2; AFB1 concentration in group S was 10 ng / mL. -1 Group N has no AFB1;

[0029] Figure 7 Optimization of incubation temperature at 800V and gain of 2; AFB1 concentration in group S was 10 ng / mL. -1 Group N has no AFB1;

[0030] Figure 8 The prehybridization reaction time of Apt with cDNA was optimized at a voltage of 600V and a gain of 2 without Nt.BbvCI; the AFB1 concentration in group S was 10 ng / mL. -1 Group N has no AFB1;

[0031] Figure 9 (A) CL kinetic curves of AFB1 at different concentrations under optimal conditions; (B) Standard curves of AFB1 detected by intelligent CL sensing system with and without Nt.BbvCI.

[0032] Figure 10 Specificity assessment of the AFB1 intelligent chemiluminescence sensing system was conducted, with all mycotoxins at a concentration of 10 ng / mL. -1 ;

[0033] Figure 11 Detection performance of the CL sensor system after 0, 7, 14, and 21 days of storage, with an AFB1 concentration of 10 ng / mL. -1 The measurement conditions were 800V and a gain of 2.

[0034] Figure 12 Intra-batch and inter-batch studies of the CL sensor system (n=5), with an AFB1 concentration of 10 ng / mL. -1 The measurement conditions were 800V and a gain of 2.

[0035] Figure 13 : AFB1 ELISA results used for comparison; from left to right: 0, 3.0 × 10⁻⁶ -2 9.0×10 -2 2.7×10 -18.1×10 -1 ng mL -1 (A) ELISA result image and (B) standard curve (excluding 0 ng / mL) of AFB1 -1 ). Detailed Implementation

[0036] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0037] I. Experimental Materials

[0038] Restriction endonuclease Nt.BbvCI; DNA sequence (as shown in Table 1-3); TE buffer; methanol; PBS buffer; AFB1 standard; chemiluminescent substrates (luminol and hydrogen peroxide), etc.

[0039] IFFM-E flow injection chemiluminescence analyzer; FLS1000 transient and steady-state fluorescence spectrometer; Mithras 2 LB 943 multi-functional microplate reader; Nano-100 micro-volume UV spectrophotometer; high-speed low-temperature centrifuge; analytical balance; rotary mixer; magnetic stirrer, etc.

[0040] Table 1. DNA Sequences

[0041]

[0042] Table 2. Sequence optimization of cDNA and HP complementary base pairs

[0043]

[0044] Table 3. Sequence optimization for cDNA and Apt complementary base pairs

[0045]

[0046] II. Experimental Methods

[0047] 1. Construction of Intelligent Chemiluminescence Sensing System

[0048] The aptamer (Apt) and its partially complementary sequence (cDNA) and hairpin DNA-dual hemin (HP) powder were dissolved in 1×TE buffer to prepare stock solutions with a final concentration of 100 μM. The solutions were then processed using rCutsmart. + The buffer was used to dilute Apt, cDNA, and HP to 0.8 μM, 0.8 μM, and 2 μM, respectively.

[0049] First, 100 μL of 0.8 μM Apt and 100 μL of 0.8 μM cDNA were mixed and reacted at room temperature for 2 hours to generate Apt-cDNA. Then, 5 μL of 2 μM HP, 5 μL of 0.4 μM Apt-cDNA, 10 μL of different concentrations of AFB1 and 1 U of Nt.BbvCI were mixed at 37 °C and reacted for 30 min. Finally, 20 μL of chemiluminescent substrate was added and the assay was performed immediately.

[0050] 2. Preparation of Chinese herbal medicine samples to be tested

[0051] Three batches of coix seed from Fujian, Hubei, and Hebei, and a slice of medicinal herbs from Guizhou were ground into powder and passed through a No. 2 sieve. 2.5g of the sample was accurately weighed and dispersed in 10mL of 70% methanol solution. 0.5g of sodium chloride was added, and the mixture was extracted by shaking at 37℃ for 30min. The mixture was then centrifuged at 5000rpm for 30min. The supernatant was diluted 5 times with PBS buffer and filtered through a 0.22μm filter membrane to obtain the test solution.

[0052] 3. ELISA Operation Procedures (Existing Technology)

[0053] First, dilute the sample extract 4-fold with diluent and filter through a 0.22 μm filter membrane. Add 50 μL of standard or sample, 50 μL of HRP-labeled secondary antibody, and 50 μL of LAFB1 antibody to the corresponding wells, gently vortex to mix, cover with a cover film, and incubate at 37°C for 20 minutes. Carefully remove the cover film, shake off the liquid in the wells, add 250 μL of washing working solution, and wash three times, patting dry with absorbent paper for the last wash. Add 50 μL / well of substrate solution A and 50 μL / well of substrate solution B, gently vortex to mix, cover with a cover film, and incubate at 37°C in the dark for 10 minutes. Add 50 μL / well of stop solution and immediately measure using a microplate reader at 450 nm (630 nm as the reference wavelength).

[0054] 4. Statistical methods

[0055] Data are expressed as mean±SEM(x±s).

[0056] III. Experimental Results and Discussion

[0057] 1. Measurement Principle

[0058] The principle is as follows Figure 1As shown, a mixed solution of Apt-cDNA complex, HP, and Nt.BbvCI was reacted at 37°C. When AFB1 was absent, the cDNA was bound by Apt and therefore could not open HP. HP maintained its hairpin structure, and the two hemins quenched each other, resulting in only weak catalytic activity; at this point, the "signal was off." The endonuclease recognition site in HP, located within the circular single strand of the hairpin, could not be recognized by the endonuclease Nt.BbvCI, and no signal amplification reaction occurred.

[0059] When AFB1 is present, Apt precisely recognizes and binds to it, releasing cDNA and activating hemin. The two hemin molecules will move away from each other as the hemin is activated, restoring their catalytic activity and catalyzing the luminol-hydrogen peroxide reaction to produce a strong chemiluminescent signal; this is known as "signal activation." The more AFB1 present in the system, the stronger the chemiluminescent signal. Simultaneously, the endonuclease recognition site in the hemin is located within the DNA double strand, allowing Nt.BbvCI to cleave the hemin into two hemin-labeled short DNA strands. The released cDNA continues to activate other hemin molecules. Ultimately, one molecule of AFB1 can release many quenched hemin molecules, resulting in a stronger chemiluminescent signal under the same conditions.

[0060] 2. Base pair optimization

[0061] The number of complementary base pairs determines the binding affinity of oligonucleotide sequences; therefore, UV absorption spectroscopy was used to optimize the number of complementary base pairs between HP and cDNA. A series of cDNAs with 13, 14, 15, 16, and 17 complementary base pairs to HP were designed, such as... Figure 2 As shown in Figure A, when the number of complementary base pairs between HP and cDNA' is greater than 15, the absorption wavelength of the HP and cDNA' mixture undergoes a significant red shift (from 370 nm to 400 nm), indicating that the hemin dimer in HP disappears. This means that cDNA' with more than 15 complementary base pairs to HP can hybridize with HP and open a hairpin structure. Given that more complementary base pairs between HP and cDNA' may affect the efficiency of endonuclease cleavage, 15 was chosen as the number of complementary base pairs between HP and cDNA.

[0062] To ensure that Apt can block cDNA from opening HP in the absence of AFB1, UV absorption spectroscopy was used to optimize the complementary base number between cDNA and Apt. A series of cDNAs with 12, 13, and 14 complementary base pairs to Apt were designed, such as… Figure 2As shown in Figure B, when the number of complementary base pairs between cDNA and Apt is greater than 13, the absorption wavelength of the mixture of Apt, HP, and cDNA undergoes a blue shift (from 400 nm to 370 nm). This demonstrates that the hemin dimer in HP remains unchanged, meaning that cDNA with more than 13 complementary base pairs to Apt can be captured by Apt without activating HP. Given that a greater number of complementary base pairs between cDNA and Apt would affect the competition of AFB1 for Apt, 13 was chosen as the number of complementary base pairs between Apt and cDNA.

[0063] UV absorption spectroscopy was also used to verify that the selected cDNA could cause the retention / disappearance of hemin dimers in HP when Apt protection was present or absent. Figure 2 C). This result also demonstrates that the three DNA sequences can enable the signal to be switched on / off by the AFB1 homogeneous sensor.

[0064] 3. Feasibility Verification

[0065] To verify the feasibility of the aptamer sensor, Cy5 and its quencher BHQ2 were used for FL experiments. Cy5 and BHQ2 were modified at specific sites on Apt and cDNA to ensure that Cy5 and BHQ2 could approach each other sufficiently after cDNA and Apt hybridization. The experimental principle is as follows: Figure 3 As shown in Figure A.

[0066] Experimental results are as follows Figure 3 As shown in line B, Cy5-modified Apt emits strong fluorescence upon excitation at 648 nm (line 1). When incubated with BHQ2-modified cDNA, the fluorescence intensity significantly decreases (line 2), indicating that successful hybridization of cDNA and Apt leads to the quenching of Cy5 by BHQ2. Further addition of 100 ng / mL... -1 After AFB1, the FL intensity recovered (line 3), indicating that competition from AFB1 can cause cDNA and Apt to separate, which in turn leads to the separation of Cy5 and BHQ2.

[0067] After demonstrating the varying binding strengths of Apt, cDNA, and AFB1, chemiluminescence was also used to verify the feasibility of using the designed HP, Apt, and cDNA as AFB1 sensors. Figure 3 As shown in line C, when only HP is present, the chemiluminescence intensity is very weak (line 1), indicating that the two hemins approach each other and form a dimer; the chemiluminescence intensity of the HP and cDNA mixture is the highest (line 2), because free cDNA can open a large amount of HP; the chemiluminescence of the HP and Apt-cDNA system (line 3) is much lower than that of HP and cDNA alone, which is due to the restriction effect of Apt on cDNA; after adding 10 ng / mL -1In the case of AFB1, chemiluminescence returned to a higher value (line 4), which further proves that AFB1 has a higher affinity for Apt than cDNA, releasing a large amount of cDNA to open HP.

[0068] 4. Validation by cyclic amplification of endonuclease

[0069] Chemiluminescence spectroscopy was used to verify the ability of Nt.BbvCI to cycle and amplify signals. For example... Figure 4 As shown, the Apt-cDNA and HP (group 1) signals were lowest due to the quenching effect of dual hemin. The chemiluminescence signal of the mixed solution of Apt-cDNA, HP, and Nt.BbvCI was slightly increased (group 2), possibly due to a side reaction caused by trace amounts of free cDNA. Apt-cDNA, HP, and 10 ng / mL -1 The significantly increased chemiluminescent signal of AFB1 (group 3) is due to AFB1 aptamer competition, releasing cDNA to open hemoglobin (HP), eliminating the hemin dimer, and restoring its ability to catalyze luminol peroxide. The strongest CL signal was observed at Apt-cDNA, HP, Nt.BbvCI, and 10 ng / mL. -1 In the AFB1 mixed solution (group 4), it is shown that Nt.BbvCI drives in-situ signal amplification.

[0070] 5. Chemiluminescence kinetics

[0071] From the performance of all the dynamic curves, it can be seen that ( Figure 3 (C and 4A) The chemiluminescent reaction occurs rapidly after the addition of the substrate and the emission signal tends to stabilize at about 100 s. Therefore, the emission value at 100 s is used for reading.

[0072] 6. Optimization of experimental conditions

[0073] To achieve optimal experimental results with the chemiluminescence system, experimental conditions, including HP concentration, Apt-cDNA concentration, and incubation time, need to be optimized. (0 ng / mL) -1 Chemiluminescence of AFB1 (14% methanol) as noise, 10 ng / mL -1 AFB1 uses chemiluminescence as a signal.

[0074] HP, acting as a key factor in the chemiluminescence signal switching and endonuclease signal amplification strategy, is the core of the entire CL system. The CL signal-to-noise ratio (S / N) is used as an indicator to optimize HP concentration. Figure 5As shown in Figure A, the signal-to-noise ratio was highest at an HP concentration of 0.5 μM. This is because at low concentrations, it cannot provide sufficient probe for cleavage by Nt.BbvCI, while at high concentrations, it introduces a high background signal. Ultimately, 0.5 μM was chosen for subsequent experiments.

[0075] Apt-cDNA is also important as an aptamer sensor, such as Figure 5 As shown in Figure B, both CL signal and noise increased to varying degrees with increasing Apt-cDNA concentration, with the optimal S / N ratio occurring at an Apt-cDNA concentration of 0.1 μM. When the Apt-cDNA concentration was too low, the amount of cDNA provided was insufficient to activate HP. When the Apt-cDNA concentration was too high, excessive cDNA led to an increase in background noise. The results indicate that the optimal concentration of Apt-cDNA is 0.1 μM.

[0076] Incubation time also plays a crucial role in the competitive system of AFB1, Apt-cDNA, and HP, as well as in the amplification of endonuclease signals. Figure 5 As C knows, during the process of increasing the reaction time from 10 min to 40 min, the chemiluminescence signal has the highest S / N ratio at 30 min, indicating that at this time point, the competitive reaction and cycle amplification are sufficient, so 30 min is chosen as the incubation time.

[0077] The effects of pH and temperature on this system are multifaceted, as they directly influence the luminol-hydrogen peroxide reaction, as well as the stability of the DNA double helix and enzyme activity, thus affecting the efficiency of signal cycling amplification. Figure 6 and Figure 7 As shown, the optimal signal-to-noise ratio is achieved at pH 7.9 and an incubation temperature of 37°C.

[0078] In addition, the prehybridization time of Apt and cDNA affects the competitive ability of AFB1 and the number of non-specifically activated HPs. For example... Figure 8 The results show that shorter pre-hybridization times lead to high noise, but excessively long pre-hybridization times reduce the signal. Therefore, 2 hours was chosen as the optimal pre-hybridization time.

[0079] 7. Wash-free, high-sensitivity AFB1 sensor

[0080] Under optimal experimental conditions, the performance of the intelligent CL sensing system for detecting AFB1 was investigated. Following one-step incubation and catalytic chemiluminescence, the chemiluminescence signal of the homogeneous system could be read out immediately. Figure 9 As shown in Figure A, the CL intensity increases with increasing AFB1 concentration. According to... Figure 9 The red line for B is at 1.0 × 10 -2 ~100ng mL -1Within this range, the chemiluminescence intensity is directly proportional to the logarithm of the AFB1 concentration. The standard curve for AFB1 is Y = 2793.56952logX + 10751.1111, with a correlation coefficient of 0.9951, where Y and X represent the chemiluminescence intensity and the AFB1 concentration, respectively. The limit of detection was calculated to be 1.5 pg / mL using a standard deviation of 3. -1 .also, Figure 9 The black line in B is the AFB1 standard curve without endonuclease involvement in signal cyclic amplification. In contrast, the chemiluminescence system with endonuclease involvement exhibits a slope (sensitivity) that is twice as high and a wider linear range.

[0081] 8. Specificity assessment

[0082] The established CL system was incubated with AFB1, ochratoxin (OTA), zearalenone (ZEN), and mixtures thereof, all at a concentration of 10 ng / mL. -1 .like Figure 10 As shown, the CL system exhibits a significant response to analytes containing AFB1, while the response to other mycotoxins is negligible, indicating that the system has good specificity for AFB1.

[0083] 9. Stability, intra-batch precision, and inter-batch precision testing

[0084] 100 μM Apt, cDNA, and HP were diluted to 0.8 μM, 0.8 μM, and 2 μM, and stored at 4°C for later use. At 7, 14, and 21 days, they were mixed with Nt.BbvCI and subjected to AFB1 CL assays. Figure 11 As shown, after 21 days of storage, the CL system can still retain 91% of its original strength.

[0085] The analytical performance of the CL system prepared in single batches and in five batches was investigated: 10 ng / mL... -1 The CL response of AFB1 (n=5) yielded a similar chemiluminescent signal, such as Figure 12 As shown, the intra-batch precision is RSD = 2.7%, and the inter-batch precision is RSD = 3.1%.

[0086] The above results indicate that the CL system has good stability and repeatability.

[0087] 10. Detection of AFB1 in actual samples

[0088] To verify the potential of the proposed CL system in practical sample detection, the content of AFB1 in the traditional Chinese medicine Coix seed and its processed slices was determined. Compared with the detection results of commercial ELISA kits, the chemiluminescence results of the four batches of samples showed good consistency, such as... Figure 13 As shown in Table 4.

[0089] Recovery experiments were conducted by adding 1 ng / mL to each of the four samples. -1 The AFB1 standard was used. The recovery rates ranged from 85.53% to 116.91%, as shown in Table 5, demonstrating the accuracy of the CL system.

[0090] Table 4. Comparison of results of traditional Chinese medicine samples detected by CL system and ELISA kit (n=3)

[0091]

[0092] a Before the assay, the sample was diluted 5 times with 0.01M PBS (pH 7.4).

[0093] b Samples were prepared according to the instructions of the ELISA kit.

[0094] Table 5. CL system used for AFB1 spike recovery experiments in traditional Chinese medicine samples.

[0095]

[0096] The above results demonstrate that the highly sensitive wash-free chemiluminescence system provided by this invention can easily and rapidly detect AFB1 using three DNA sequences and an endonuclease. The three DNA sequences include a chemiluminescence switch and a homogeneous aptamer sensor, exhibiting stability and extremely low biotoxicity. AFB1 recognition, hemin dimer depolymerization, and hemin peroxidase activity activation can be achieved in a single reaction. Signal amplification is generated with the aid of the restriction endonuclease Nt.BbvCI, resulting in a wide linear range for the chemiluminescent analysis of AFB1, with a detection limit as low as 1.5 pg / mL. -1 This intelligent detection system requires no washing or separation, is easy to operate, inexpensive, efficient, and environmentally friendly. It can be extended to the real-time detection of other mycotoxins through sequence design, without requiring specialized operator training. It also holds promise for rapid detection of AFB1 in large numbers of real samples, providing new insights into food and drug quality control.

[0097] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. An intelligent chemiluminescence sensing system, characterized in that, include: Complex A: A complex formed by the binding of the aflatoxin B1 aptamer Apt and a partially complementary sequence of cDNA to Apt, which releases the cDNA in the presence of aflatoxin B1; Complex B: A complex consisting of hairpin DNA that can be released by cDNA binding and two hemins that bind to both ends of the hairpin DNA and quench each other. Restriction endonucleases: Recognition sites located on the double-stranded DNA formed by the binding of cDNA and hairpin DNA, which can cleave complex B into two hemin-labeled sequences and release cDNA. in: The nucleotide sequence of the aflatoxin B1 aptamer Apt is shown in sequence No. 1; The nucleotide sequence of the cDNA is shown in sequence No.

2.

2. The intelligent chemiluminescence sensing system according to claim 1, characterized in that: The nucleotide sequence of the hairpin DNA is shown in sequence No.

3.

3. The intelligent chemiluminescence sensing system according to claim 2, characterized in that: The restriction endonuclease is Nt.BbvCI.

4. A method for determining aflatoxin B1 based on the intelligent chemiluminescence sensing system according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step S1: Dissolve Apt, cDNA, and complex B powder separately in TE buffer to prepare stock solutions, then use rCutsmart + The buffer solution was diluted to a diluent. Step S2: Mix equal amounts of Apt and cDNA dilution solution and react at room temperature to generate Apt-cDNA; Step S3: Take an appropriate amount of Apt-cDNA, complex B dilution buffer, restriction endonuclease and an appropriate amount of sample solution containing aflatoxin B1, mix them at 37 °C, and after reacting for a certain time, add an appropriate amount of chemiluminescent substrate that can be catalyzed by hemin and immediately measure the chemiluminescence intensity.

5. The application of the intelligent chemiluminescence sensing system according to any one of claims 1 to 3 in the determination of aflatoxin B1 in traditional Chinese medicine.

6. The application of the method of claim 4 in the determination of aflatoxin B1 in traditional Chinese medicine.

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