An allosteric ratiometric fluorescence sensor co-regulated by aptamers and thiols

Abstract

The present invention belongs to the field of biotechnology, and in particular, relates to an allosteric ratiometric fluorescent sensor co-regulated by an aptamer and a thiol, which rapidly detects aflatoxin through triple cascade amplification. The sensor achieves precise quantification and high sensitivity through target cyclic deconstruction, rolling circle transcription, and ratio fluorescence excitation. The present invention rationally designs a rolling circle transcription allosteric switch based on a three-way connection, and the aflatoxin double-stranded aptamer acts as an activator to achieve spatial conformational change of the allosteric switch after responding to the target. The activated allosteric switch continuously amplifies the fluorescent receptor, achieving precise quantification of aflatoxin within 1 hour. Due to the programmability of the RNA sensor, the method can be applied to a wider range of target analytes.

Description

Technical Field

[0001] The invention belongs to the field of biosensing, and particularly relates to an allosteric ratio fluorescence sensor co-regulated by an aptamer and a thiol. Background Art

[0002] The inherent "plug-and-play" nature of luminescent RNA sensors plays a key role in revealing the complexity of various biological processes, ranging from transcriptional reporter gene analysis, RNA pull-down, and in situ monitoring. Compared with fluorescent protein-based luminescent sensors, luminescent RNA sensors show higher molar fluorescence intensity and a wider color range. Currently, more than 70 in vitro evolved fluorescent RNA aptamer tools are available. The first generation of malachite green RNA aptamers (MGA) discovered for the first time demonstrated their ability to directly track exogenous non-fluorescent malachite green (MG) dye within the binding pocket. This discovery provides a direct and cost-effective solution for the next generation of RNA sensing systems without the need for chemical modification.

[0003] The fundamental challenges faced in building a robust biosensor include overcoming the effects of non-responsive errors in complex biological matrices, namely false positive / false negative phenomena. However, ratiometric detection has greatly solved this problem by adopting a dual-channel signal design compared to traditional single fluorescence strategies. It essentially provides a correction mechanism to minimize interference and improve the accuracy of signal output. Usually, the dual-emission design based on fluorescence is mainly to integrate additional signal-related components into the array. There are two feasible simple strategies reported so far. The first is to functionalize nucleic acids with metal nanoclusters with high light emission or two different molecular beacons, such as upconversion nanoparticle-DNA nanocomplexes or pH-sensitive oligonucleotide conjugates labeled with fluorescein and anthocyanin dyes. The second method uses ligand-competitive dual-fluorescent RNA aptamers to exploit multicolor orthogonality for sequential fluorescence activation.

[0004] However, although the first scheme is the current mainstream design, this method is prone to increase molecular complexity, and non-specific cleavage of labeled primers may lead to undesirable background. The second scheme requires an additional set of genes to be encoded, including fluorescent RNA aptamers or fluorescent proteins, which not only increases the difficulty of RNA synthesis, but also relies on the competition method of ligands to easily cause hybridization perturbations and increase cross-reactivity. These problems often lead to erroneous results and quantification difficulties. In addition, high-performance luminescent clusters or fluorescent dyes are currently being synthesized in laboratory environments and have not yet been fully commercialized, which poses a challenge to their short-term popularization. Therefore, it is urgent to develop a ratiometric luminescence technology that does not require modification, labeling, quantifiable, robust and versatile to meet the needs of multiple target detection. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to propose an allosteric ratiometric fluorescence sensor co-regulated by an aptamer and a thiol.

[0006] To achieve the above object, on the one hand, the present invention provides an allosteric ratio-type aflatoxin fluorescent sensor co-regulated by an aptamer and thiol, characterized in that the sensor is composed of a three-way junction (3WJ) OFF template, a target double-stranded aptamer, a T4 ligase system, a rolling circle transcription amplification system, and a dye molecule;

[0007] The 3WJ OFF template is composed of complementary hybridization of SEQ ID NO: 1 and SEQ ID NO: 10;

[0008] The target double-stranded adapter is composed of complementary hybridization of SEQ ID NO: 12 and SEQ ID NO: 13.

[0009] The T4 ligase system includes 50 mM Tris-HCl, 10 mM magnesium chloride, 1 mM adenosine triphosphate, 10 mM dithiothreitol, and 15% polyethylene glycol 6000, and the pH value of the system is 7.5.

[0010] The rolling circle transcription system comprises 40 mM Tris-HCl, 6 mM magnesium chloride, 10 mM sodium chloride, 10 mM dithiothreitol and 6 mM spermidine, and the pH value of the system is 7.5.

[0011] The dye molecules are malachite green and its reduced product leuco malachite green.

[0012] On the other hand, the present invention provides a method for using the above sensor to detect aflatoxin:

[0013] First, the linear double-circular template (SEQ ID NO: 1) and the circular end-8 (SEQ ID NO: 10) were annealed and incubated at 95°C for 8 minutes, and then the temperature was lowered by 1°C every 15 seconds until 25°C to form a three-way connection OFF template; then, a target double-stranded adapter consisting of complementary input strands and staple strands, a T4 ligase reaction system, and a T7 RNA transcription system were added to the system; after adding aflatoxin targets and malachite green dye, the one-pot system was incubated at 30°C for strand displacement and enzyme ligation reactions; then the system temperature was increased to 37°C, and a rolling circle transcription reaction was performed for 30 minutes; finally, the reaction was terminated at 75°C for 10 minutes, and the malachite green and leucomalachite green fluorescence intensities of the solution system at 655 and 360 nm were measured by a fluorescence spectrophotometer.

[0014] The strand displacement and enzyme ligation reaction time is 10 to 120 minutes.

[0015] In the rolling circle transcription amplification system, the final concentration of the 3WJ OFF template is 0.05-1.5 μM, the final concentration of the T7 RNA polymerase is 0.1-3 U / μL, and the final concentration of the ribonucleoside triphosphate is 0.1-5 mM.

[0016] The final concentration of the dye molecule is 0.01-20 mM.

[0017] On the other hand, the present invention provides the use of the above-mentioned sensor in the development of aflatoxin detection methods and aflatoxin food safety detection kits.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This method introduces MG dye in a common enzymatic environment, and uses dithiothreitol (DTT) in the buffer to simply and quickly synthesize the deconjugated triphenylmethane molecule leucomalachite green (LMG), proving that it has cluster luminescence properties based on spatial conjugation, and uses a single MG dye to achieve dual emission, namely red fluorescence from the combination of MGA and MG, and ultraviolet luminescence from reduced LMG. The ratiometric fluorescence design improves the robustness of traditional RNA sensors.

[0020] (2) The present invention establishes a target aptamer comprising an aptamer chain and a complementary staple chain to respond to aflatoxin, triggers the deconstruction of the target aptamer through aflatoxin, and completes the conformational change of the 3WJ-based rolling circle transcription switch model (TRSM). This design not only incubates the high affinity and selectivity of the sensor to the target, but also triggers the efficient synthesis of the MGA receptor with the help of TRSM, which is conducive to the establishment of an integrated platform for RNA sensors.

[0021] (3) Based on TRSM, the present invention constructs a ratiometric RNA sensor that can achieve accurate quantification of toxins within 1 hour. The entire process is completed in one pot, with a constant temperature and visualized results, which fully meets the needs of on-site detection.

[0022] (4) The sensing strategy of the present invention includes a triple cascade amplification: target cyclic deconstruction, rolling circle transcription (RCT) amplification and ratio fluorescence activation, which provides a double guarantee for sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The conversion mechanism of the color-changing fluorophore and the design principle of the TRSM-based ratiometric fluorescent RNA sensor.

[0024] Figure 2 For identification of the product after MG dye reduction. Where a is the 1H-NMR spectrum of the material before and after reduction; b1) is the color change and physical texture change of MG in aqueous solution and transcription buffer containing DTT, b2) corresponds to the full wavelength UV absorption spectrum in b1), and the maximum absorption positions of MG and LMG are marked, and b3) is the UV absorption spectrum of LMG at different concentrations. c is the LC-MS qualitative analysis of the reduction product, and the internal standard is deuterated LMG.

[0025] Figure 3 Luminescence performance of fluorophores with different photophysical properties in actual transcription. a is the excitation and emission peaks of LMG and MGA-MG; b is the change in the ratio of the two during the transcription process.

[0026] Figure 4 The feasibility of RCT is verified and the transcription efficiency and luminescence ability are evaluated. a is the denaturing gel analysis of the products of the unconverted 3WJ OFF template, circular template and linear template after transcription; b is the yield comparison of RCT and linear transcription; c is the luminescence ability comparison of RCT and linear transcription after combining with MG.

[0027] Figure 5The effect of input chains with different toehold lengths on the amplification efficiency of TRSM. a is real-time monitoring of fluorescence at 655 nm; b quantifies and compares the fluorescence intensity of input chains with different toehold lengths.

[0028] Figure 6 The strand displacement, enzyme ligation and transcription verification of different length toehold input strands in TRSM in denaturing polyacrylamide gel electrophoresis. a is input strand-0, b is input strand-2, c is input strand-4, d is input strand-8, and e is input strand-14.

[0029] Figure 7 This is the gain effect of 15% PEG6000 on the integrated system.

[0030] Figure 8 Optimization of 3WJ OFF template, T7 RNA polymerase and rNTPs. a is the fluorescence spectrum of the transcription products of different concentrations of transcription templates after binding with MG dye; b is the quantified fluorescence intensity; c is the transcription efficiency mediated by different concentrations of T7 RNA polymerase; d is the effect of different concentrations of rNTPs on transcription.

[0031] Fig. 9 Concentration optimization of MG dye in the integrated system.

[0032] Fig.10 Performance test of the ratiometric fluorescence RNA sensor based on TRSM. a1) is the ratiometric fluorescence value of the sensor in response to different concentrations of AFB1; a2) is the corresponding standard curve of AFB1 concentration dependence; b1) is the ratiometric fluorescence spectrum of the sensor in response to other analytes; b2) is the quantitative results of the corresponding specificity and selectivity test. DETAILED DESCRIPTION

[0033] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0034] Example 1: Discovery of color-changing fluorophores and verification of dual-emission fluorescence

[0035] First, 10 μL of MG aqueous solution with different concentrations was added to the transcription buffer containing DTT to make the final concentration 0-20 mM. Then the absorption peak between 200 and 800 nm was measured in the UV-visible spectrophotometer, and the observed lipid flocs were then ultra-low temperature freeze-dried in a freeze vacuum dryer to collect 5 mg of the product. 5 mg of the product and 5 mg of MG powder were dissolved in 500 μL of DMSO-d6, respectively, and their chemical shifts (ppm) were measured at 25 ° C using a nuclear magnetic resonance instrument and quantitative analysis was performed.

[0036] Inspired by the unique luminescent physical properties of three separated benzene rings and the thiol-sensitive MG, the present invention uses a self-contained DTT enzymatic system to reduce the highly conjugated MG dye to non-conjugated LMG ( Figure 1 ). First, it was directly observed from a colorimetric perspective that a 200 μM MG concentration in a transcription buffer containing DTT rapidly changed from blue-green to colorless, and at a higher concentration (MG=5 mM), dispersed lipid flocs were observed with the naked eye. Figure 2 a shows the 1H-NMR spectra of MG dye and lipid floccules after freeze-vacuum drying. Both show signals in the aromatic region (δ6.5-8.0 ppm). Compared with the spectrum of MG dye, the characteristic non-overlapping triplet signal of lipid floccules at 7.77 ppm disappears, the proton chemical shift shifts to higher fields, and the proton signal at position 1 appears ( Figure 2 a). These changes indicate the potential elimination of the electron-withdrawing quaternary ammonium salt substituent (position 2) of the MG dye itself and the overall electron-withdrawing conjugated structure. Based on this, the present invention preliminarily provides evidence that MG is reduced to a fat-soluble LMG containing only three isolated benzene ring structures.

[0037] The present invention continues to analyze this potential color-changing fluorophore from a spectral perspective. First, 10 μL of MG aqueous solution of different concentrations was added to 90 μL of enzyme-free water and transcription buffer at the same time, and the color difference between the two was observed ( Figure 2 b, 1). Specifically, it was observed that the molar extinction coefficient at 617 nm (λmax of MG) was increased from 4.6*10 4 M -1 cm -1 Reduced to 4.3*10 3 M -1 cm -1 At the same time, the characteristic peak bandwidth of the reduction product becomes wider, the maximum absorption red-shifts, and the maximum absorption peak at the same concentration weakens ( Figure 2b, 2). This indicates that the conjugated chromophore structure of the MG dye is broken and the unsaturation of the reduction product is reduced. Although the maximum absorption wavelength of the reduction product highly overlaps with the LMG standard, due to the abnormal peak shape and lack of fine structure, LC-MS was subsequently performed on the MG co-transcription product and the lipid floccules after acetonitrile resuspension to confirm the target molecule ( Figure 2 b, 3). MG-D5 / LMG-D6 were selected as internal standards for multiple reaction monitoring. The elution times (±5%) of LMG and MG analytes and their stable isotope-labeled counterparts were almost identical and their MS spectra were similar, thus enabling the unambiguous identification of the transcriptional environment containing DTT that reduces the MG dye to LMG ( Figure 2 c).

[0038] For preliminary transcription analysis, 1 μL of 10 μM DNA circular template was added, which was composed of 3WJ OFF template and input strand-14 (SEQ ID NO: 6) after allosteric and enzymatic ligation (3WJ OFF template was obtained by complementation of SEQ ID NO: 1 and SEQ ID NO: 11). In the transcription buffer, 2.5 μL of 12 mM MG dye was added at the same time, and the reaction was carried out at 37°C for 1 hour to obtain the tandem MG RNA aptamer. The reaction was then terminated at 75°C for 10 minutes and the fluorescence of MG RNA aptamer-MG (ex = 617 nm, em = 655 nm) / LMG (ex = 260 nm, em = 360 nm) under each excitation was immediately tested by a fluorescence spectrophotometer.

[0039] Then the present invention evaluated the luminescence properties of MGA-MG and the reduction product LMG after co-transcription of MG dye. Figure 3 a shows the excitation spectra and emission light of the two chromophores. LMG has obvious cluster emission characteristics, that is, non-overlapping excitation and emission peaks. To ensure the feasibility of the dual-color fluorophore in the ratiometric RNA sensor, the fluorescence at 655nm / 360nm was monitored for a long time during RCT using a fluorescence spectrophotometer. The results showed that the F655 / 360 ratio reached a peak at 20 minutes and remained stable after transcription termination ( Figure 3 b).

[0040] Example 2: Construction principle of ratiometric RNA sensor based on color-changing fluorophore and TRSM

[0041] A design principle of a ratiometric RNA sensor mediated by dual-color fluorophores and TRSM Figure 1As shown: (1) In the presence of AFB1 target, the target double-stranded aptamer changes from the initial long-range state to the short-range state. Only in this state can it effectively trigger the toehold site (green) on the 3WJ OFF template, and then the chain displacement reaction occurs. Since the reaction occurs in a one-pot system, AFB1 can cyclically expose the toehold binding site of the double-stranded aptamer, thereby realizing the cyclic amplification of the allosteric event. In the absence of AFB1, the target aptamer still remains in the long-range state and cannot provide a binding site for downstream chain displacement. (2) Since the 3WJ OFF template is designed with a weak binding ability cleavage promoter, it cannot bind to T7 RNA polymerase smoothly, and thus cannot function normally. Only when the target triggers the chain displacement reaction, because the target aptamer that restores the short-range contains part of the promoter, the promoter on the transcription template restores the complete full-length sequence. At this time, the ON template uses T4 DNA rapid ligase to synthesize a new phosphodiester bond to fill the gap after the allosteric reaction and form a complete DNA circular template. T7 RNA polymerase then reacts with RCT on the circular template to synthesize a large number of long single-stranded MGAs in series. (3) MGA can quickly bind to MG with high specificity, activating the red fluorescence of the MGA-MG fluorophore. Since it is a one-pot system, MG and RCT coexist, and the reduced LMG is used as a reference signal in this sensor, and the experimental results can ultimately be evaluated using the ratio signal F655 / 360. The nucleic acid sequences used in the experiment are shown in Table 1.

[0042] Table 1. Nucleic acid sequences related to the experiment

[0043]

[0044]

[0045] Note: [5'P], phosphorylation site; the toehold complementary base between the linear double circle template and the input strand is single underlined. The complementary base between the linear double circle template and the circular terminus is double underlined.

[0046] Example 3: Feasibility verification of TRSM-based ratiometric fluorescent RNA sensor

[0047] Before optimization, the probability of TRSM gene leakage was initially evaluated using input strand-14 (SEQ ID NO: 6) as an activator. First, the 3WJ OFF template was prepared by pre-annealing the linear double ring template (SEQ ID NO: 1) and the circular end (SEQ ID NO: 11) at a final concentration of 10 μM. The strand displacement and enzyme ligation reactions verified separately are shown in Table 2 , and the rolling circle transcription system involving MG dye is shown in Table 3 .

[0048] Table 2 Strand displacement reaction system and T4 DNA ligase reaction system

[0049]

[0050]

[0051] Table 3 RCT reaction system of MG dye co-transcription

[0052]

[0053] The strand displacement and enzyme ligation processes were verified by gel electrophoresis. Figure 4 In a, lanes 1 and 3 directly indicate that the 3WJOFF template (complemented by SEQ ID NO: 1 and SEQ ID NO: 11) effectively prevents accidental leakage and no transcription occurs in the absence of the input strand. The stable 3WJ structure protects the cleaved promoter that is weakly contacted and enclosed in the loop. In addition, the RNA yield of RCT initiated by the circular DNA template is significantly higher than that of the classical linear transcription ( Figure 4 b). At the same dye concentration, the tandem MGA generated by RCT showed a fluorescence intensity about 10 times higher than the short single-stranded MGA generated by classical linear transcription ( Figure 4 c) This is mainly attributed to the steric effect and aggregated charge distribution caused by the engineered multivalency in the long tandem. The charge effect and high affinity geometry increase the ligand-receptor interaction and inhibit the intramolecular charge transfer and rotation of MG.

[0054] Example 4: Sequence Optimization of TRSM and Condition Optimization of One-Pot Transcription Reaction

[0055] To ensure optimal efficiency, the binding free energy between the input strand and the toehold site in the allosteric event was predicted and verified. The toehold-mediated strand displacement reaction and the enzyme ligation system reacted together, with a reaction time of 0 to 60 minutes, a T4 DNA ligase concentration of about 40 U / μL, and a T7 RNA polymerase reaction concentration of 1 U / μL. The concentration of the MG dye was optimized in the range of 0 to 400 μM, and the fluorescence of MGA-MG could be detected immediately after the reaction.

[0056] 1. Optimization of the length of the toehold site in the input chain

[0057] The input chain was designed to optimize the thermodynamics and kinetics of the 3WJ OFF template conformational change. The toehold region on the template was a variable sequence, so the input chain was designed with five pairs of 0, 2, 4, 8 and 14 nt (as shown in SEQ ID NO: 2-6) according to the length of the toehold region. The real-time thermodynamic drive of different input chains on the 3WJ OFF template (complemented by SEQ ID NO: 1 and SEQ ID NO: 11) was monitored using the MG reporter gene. Within 40 minutes, including 20 minutes of chain displacement, it was obvious that the reaction kinetics of input chain-8 (SEQ ID NO: 5) was not inferior to that of input chain-14 (SEQ ID NO: 6) ( Figure 5 a and b). Both denaturing-PAGE electrophoresis and MG signals indicated that the displacement and transcription of input strand-0 (SEQ ID NO: 2) and input strand-2 (SEQ ID NO: 3) were ineffective ( Figure 6 ). Therefore, the transcription state is combined with the fluorescence response to test whether TRSM needs to form more base pairs (longer toehold length) to provide driving force. The results show that input strand-8 (SEQ ID NO: 5) has achieved the ideal toehold-mediated strand displacement reaction efficiency.

[0058] 2. Effect of molecular crowding agents on chain displacement reaction and reaction time optimization

[0059] The preparation of the 3WJ OFF template and chain displacement in the early stage were the same as the above system and operation. When T4 DNA ligase was used to synthesize the phosphodiester bond at the nick site, the initial reaction system was 10 μL, which contained 3WJ OFF template (10 pmol), input chain (10 pmol) T4 DNA ligase (480U), 1 μL 10X T4 DNA ligase buffer and RNase-free water. After thorough mixing, 15% PEG6000 was added, and the enzyme ligation process at 30°C was set to different time spans, while the transcription time was uniformly set to 37°C, 30 min. The results showed that the molecular crowding effect caused by 15% PEG6000 significantly shortened the chain displacement and enzyme ligation time, and the improvement in the cyclization efficiency thereby improved the RCT efficiency ( Figure 7 ). Therefore, 30 min was selected as the optimal strand displacement and enzyme ligation reaction time in the one-pot system.

[0060] 3. RCT system optimization

[0061] The DNA circular template was prepared by using the previously optimized 15% PEG6000 group. First, the concentration of the circular template was optimized, and the final concentration was set to 0-1.5 μM, while the other components remained unchanged. Figure 8Results a and b show that when the DNA circular template reaches 0.6 μM, the fluorescence value of MGA-MG reaches saturation, so the optimal template concentration is 0.6 μM.

[0062] At the optimal DNA circular template concentration (0.6 μM), T7 RNA polymerase was subsequently optimized, and the final concentration gradient was set to 0-3 U / μL, and the rest of the system remained the same as before. Figure 8 c The results showed that the optimal T7 RNA polymerase concentration was 1 U / μL.

[0063] Finally, rNTPs were optimized and the final concentration was set to 0-5 mM, and the rest of the system remained the same as before. Figure 8 d The results showed that when the rNTPs concentration was too high, the fluorescence value of MGA-MG and the corresponding RNA concentration tended to decrease. This may be because when the rNTPs concentration was too high in a small volume reaction, it inhibited the activation effect of T7 RNA polymerase on the transcription template. Therefore, the optimal rNTPs concentration was optimized to 1 mM.

[0064] 4. Optimization of MG dye

[0065] The MG dye concentration was optimized in the optimal RCT system, and the final concentration of MG dye was set to 0.01-20 mM. The fluorescence curves of LMG and MGA-MG were scanned immediately after transcription termination. In order to ensure efficient fluorescence emission and reduce the transparency of the system after LMG was reduced, the MG concentration was finally optimized to 200 μM ( Fig. 9 ).

[0066] Example 5: Response verification of the TRSM-based ratiometric fluorescent RNA sensor for detecting AFB1 target

[0067] The present invention selects the AFB1 aptamer as a specific recognition element, and further synthesizes the input chain-8 sequence for opening the 3WJ OFF template (obtained by complementation of SEQ ID NO:1 and SEQ ID NO:10) at both ends of the aptamer. Only in the presence of the target, the action chains at both ends can be transformed from the initial long-range state to the short-range state, thereby exposing the allosteric site. In order to reduce the high background caused by the spontaneous bending of the flexible single chain. We designed the corresponding nail chain (SEQ ID NO:13) to improve the thermodynamic stability required for the long range through a semi-stable complementary state. At the same time, the target binding site (GTC and GTG in the binding pocket of the AFB1 aptamer) is also retained on the nail chain to ensure that when the target is present, the target aptamer (obtained by complementation of SEQ ID NO:12 and SEQ ID NO:13) can restore the single-chain flexibility. It is particularly noted here that the target aptamer consists of the AFB1 target aptamer embedded in the input chain-8 and the corresponding nail chain.

[0068] Subsequently, a series of AFB1 standards with different concentrations were added to the optimized system for ratiometric detection. It can be clearly observed that in the absence of AFB1, the red fluorescence of MGA-MG at 655 nm is extremely weak. After AFB1 exposure, the emission peak at 655 nm increased significantly, while the LMG fluorescence remained relatively consistent ( Fig.10 a1). Therefore, by recording the spectra at different AFB1 concentrations ( Fig.10 a2), the logarithmic concentration of AFB1 and the F655 / 360 ratio showed a good linear dependence, the detection range was 10-300 pM, and the detection limit was about 2.5 pmoles. The actual AFB1 content in water and ultra-high temperature sterilized milk was detected by the spike recovery method. The measurement results in Table 4 showed that the average recovery rate of this method was between 98.78% and 106.0%, and the relative standard deviation (RSD) was between 1.42% and 5.42%. Results The ratiometric RNA sensor based on TRSM showed reasonable accuracy and applicability in actual detection.

[0069] Table 4 Average recovery and relative standard deviation of ratiometric RNA sensors based on TRSM a

[0070]

[0071]

[0072] a: (n=3).

[0073] In addition, the sensitivity of this method may increase with the extension of transcription time, and the discrimination of extremely small target amounts is more significant. However, for practical monitoring purposes, the present invention limits the RCT process to 30 minutes. The dual enzymatic platform effectively converts and amplifies the target signal within 1 hour. The preassembled 3WJ OFF template and long-range double-stranded aptamer allow one-pot monitoring. In addition, the aptamer-mediated label-free strategy enhances the versatility of TRSM, enabling its universal application to a wide range of targets.

[0074] Example 6: Specificity of TRSM-based ratiometric fluorescent RNA sensor

[0075] To evaluate the specificity of the proposed method, a panel of other toxins infecting crops was selected (ochratoxin AOTA, zearalenone ZEA, patulin PAT, T-2 toxin; deoxynivalenol DON, fumonisin FB1). These non-target toxins were tested together with AFB1 under the same experimental conditions. The sensor accurately distinguished AFB1 from non-target toxins. In non-specific samples, LMG fluorescence increased slightly, which was comparable to blank samples ( Fig.10b1). In addition, the mixed sample containing AFB1 showed a significantly higher ratio signal ( Fig.10 b2). This result indicates that the design of the double-stranded aptamer gives the present invention good selectivity and specificity.

[0076] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. An allosteric ratiometric aflatoxin fluorescent sensor co-regulated by an aptamer and a thiol, characterized in that: The sensor is composed of a three-way linked OFF template, a target double-stranded adapter, a T4 ligase system, a rolling circle transcription amplification system, and a dye molecule; The three-way junction OFF template is composed of complementary hybridization of SEQ ID NO: 1 and SEQ ID NO: 10; The target double-stranded adapter is composed of complementary hybridization of SEQ ID NO: 12 and SEQ ID NO: 13; The dye molecules are malachite green and its reduced product leuco malachite green.

2. The sensor according to claim 1, characterized in that The T4 ligase system includes 50 mM Tris-HCl, 10 mM magnesium chloride, 1 mM adenosine triphosphate, 10 mM dithiothreitol, and 15% polyethylene glycol 6000, and the pH value of the system is 7.

5.

3. A method for detecting aflatoxin co-regulated by aptamers and thiol, characterized in that: The detection method is: First, the linear double-circular template (SEQ ID NO: 1) and the circular end-8 (SEQ ID NO: 10) were annealed and incubated at 95°C for 8 minutes, and then the temperature was lowered by 1°C every 15 seconds until 25°C to form a three-way connection OFF template; then, a target double-stranded adapter composed of complementary input strands and staple strands, a T4 ligase reaction system and a T7 RNA transcription system were added; after that, aflatoxin targets and malachite green dye were added to form a one-pot system, which was incubated at 30°C for strand displacement and enzyme ligation reactions; then the temperature was raised to 37°C, and a rolling circle transcription reaction was performed for 30 minutes; finally, the reaction was terminated at 75°C for 10 minutes, and the malachite green and leucomalachite green fluorescence intensities of the solution system at 655 and 360 nm were measured by a fluorescence spectrophotometer.

4. The method according to claim 3, characterized in that The strand displacement and enzyme ligation reaction time is 10 to 120 minutes.

5. The method according to claim 3, characterized in that: The final concentration of the malachite green is 0.01-20 mM.

6. Use of the sensor according to any one of claims 1 to 2 in the development of aflatoxin detection methods and aflatoxin food safety detection kits.

7. Use of the detection method according to any one of claims 3 to 5 in a food safety detection kit for aflatoxin.

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