Patent ID: 12209963

DETAILED DESCRIPTION OF EMBODIMENTS

Multiple embodiments of the disclosure are described in detail, the detailed descriptions should not be considered as a limitation of the disclosure, but should be understood as a more detailed description of certain aspects, features, and embodiments of the disclosure.

It should be understood that terms described in the disclosure are only for describing specific embodiments and are not intended to limit the disclosure. Furthermore, for a numerical range in the disclosure, it should be understood that each intermediate value between upper and lower limits of the numerical range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range, as well as any other stated value or intermediate value within the stated range, is also included in the disclosure. The upper and lower limits of these smaller ranges can be independently included or excluded within the numerical range.

Unless otherwise specified, all technical and scientific terms used in this article have same meanings as those commonly understood by those skilled in the art described herein. Although the disclosure only describes some methods and materials, any methods and materials similar or equivalent to those described herein may also be used in an implementation or testing of the disclosure. All literature mentioned in the specification is incorporated by reference to publicly disclose and describe methods and/or materials related to the literature. In case of conflict with any incorporated literature, the content of the specification shall prevail.

It is obvious to those skilled in the art that many improvements and changes can be made to the specific embodiments of the disclosure without departing from a scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from the description of the disclosure. The specification and embodiments of the disclosure are exemplary only.

Terms “including”, “comprising”, “having” and “containing” used in this article are all open terms, which means including but not limited to.

The disclosure is an experimental exploration based on optical sectioning. As shown inFIG.1, a basic principle of the disclosure is that a fluorescence probe aptamer and PC-strand labeled with an optically active group are partially complementary, to form a double-strand (i.e., A-PD inFIG.2), and a quenching group TAMRA is closer to a fluorescent group FAM, resulting in fluorescence quenching. Under a lighting condition, the PC-strand breaks at a position of a photosensitive group (i.e., the PC group), to form two segments. The two segments form double strands with the fluorescent probe aptamer, respectively (i.e., AD and A-nPD inFIG.2). At this time, the aptamer and the PC-strand form two double stranded structures, and a binding force between the PC-strand and the aptamer is weak. When a target molecule AFB1 is introduced, the fluorescent probe aptamer has a specific recognition function on the target molecule AFB1, to form a secondary structure, while the double-stranded structure formed by the PC-strand and the aptamer is disrupted, which causes the quenching group TAMRA to move away from the fluorescent group FAM, resulting in fluorescence restoring. Based on the specific recognition function of the aptamer on the AFB1, the fluorescence is not restored when OTA or ZEN with a similar structure to the AFB1 is added. Through monitoring changes in the fluorescence signal, specific detection of the target molecule AFB1 is achieved, and it is applied to the detection of the target molecule AFB1 in foods (e.g., rice, corn, soybeans).

In the following embodiments, aptamer, PC-strand, none-PC-strand and DNA are synthesized by the Sangon Biotech (Shanghai) Co., Ltd.

Specifically, the nucleotide sequences of the aptamer, the PC-strand, the none-PC-strand and the DNA are as follows:aptamer:5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-FAM-3′ (as shown in SEQ ID NO: 1);PC-strand: S′-TAMRA-GGGCCTAGCG-PC-AAGGGCAC-3′ (as shown in SEQ ID NO: 2);none-PC-strand: 5′-TAMRA-GGGCCTAGCGAAGGGCAC-3′ (as shown in SEQ ID NO: 3); andDNA: 5′-TAMRA-GGGCCTAGCG-3′ (as shown in SEQ ID NO: 4).

A chemical structure of a PC group is as follows:

Embodiment 1

1. Method

1.1 Concentration Optimization of an Aptamer Solution

The aptamer (1 μmol/L) solution with different volumes are added into 7 centrifuge tubes, a Tris (10 millimoles per liter (mM) of Tris, 120 mM of sodium chloride (NaCl), 5 mM of potassium chloride (KCl), and power of hydrogen (pH) is 7.2) buffer solution are respectively added into the 7 centrifuge tubes for stirring evenly to obtain mixed solutions, and a total volume of the mixed solution in each centrifuge tube is 80 microliters (μL). In the 7 centrifuge tubes, final concentrations of the aptamer are respectively as 0.5 μmol/L, 0.25 μmol/L, 0.2 μmol/L, 0.15 μmol/L, 0.1 μmol/L, 0.07 μmol/L and 0.03 μmol/L. After stirring evenly, fluorescence intensity of the aptamer in the 7 centrifuge tubes is detected. Conditions for detecting the fluorescence intensity are as follows: an excitation wavelength is 480 nanometers (nm), a scanning range is 490-700 nm, a voltage is 700 volts (V), and a slit is 5/5.
1.2 Screening of a Reaction Temperature Between the Aptamer and the PC-Strand

4 centrifuge tubes each are added with the aptamer solution, a PC-strand solution and the Tris (10 mM of Tris, 120 mM of NaCl, 5 mM of KCl and pH=7.2) buffer solution for stirring evenly to obtain mixed solutions, and a total volume of the mixed solution in each centrifuge tube is 80 μL. In the 4 centrifuge tubes, a final concentration of the aptamer is 0.15 μmol/L, and a final concentration of the PC-strand is 0.18 μmol/L. The mixed solutions in the 4 centrifuge tubes are respectively reacted at 4° C., 15° C., 25° C. and 37° C. for 1 hour (h), and fluorescence intensity of the aptamer in the 4 centrifuge tubes is detected.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.3 Screening of a Concentration of the PC-Strand

12 centrifuge tubes each are added with the aptamer solution, the PC-strand solution and the Tris (10 mM of Tris, 120 mM of NaCl, 5 mM of KCl and pH=7.2) buffer solution for stirring evenly to obtain mixed solutions, and a total volume of the mixed solution in each centrifuge tube is 80 μL. The mixed solutions in the 12 centrifuge tubes are reacted at 4° C. for 1 h, and then fluorescence intensity of the aptamer in the 12 centrifuge tubes is detected. In the 12 centrifuge tubes, a final concentration of the aptamer is 0.15 μmol/L, and final concentrations of the PC-strand are respectively as 0 μmol/L, 0.03 μmol/L, 0.06 μmol/L, 0.09 μmol/L, 0.12 μmol/L, 0.15 μmol/L, 0.18 μmol/L, 0.21 μmol/L, 0.24 μmol/L, 0.27 μmol/L, 0.3 μmol/L and 0.33 μmol/L.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.4 Base Influence Experiment

As shown in Table 1, a centrifuge tube 1 is added with the aptamer solution, a DNA solution and the Tris (10 mM of Tris, 120 mM of NaCl, 5 mM of KCl and pH=7.2) buffer solution, and a centrifuge tube 2 is added with the aptamer solution, the PC-strand solution and the Tris (10 mM of Tris, 120 mM of NaCl, 5 mM of KCl and pH=7.2) buffer solution for stirring evenly to obtain mixed solutions, and a total volume of the mixed solution in each centrifuge tube is 80 μL. The mixed solutions in the 2 centrifuge tubes are reacted at 4° C. for 1 h, a AFB1 standard solution is added into the 2 centrifuge tubes to react at 37° C. for 1 h, and then fluorescence intensity of the aptamer in the 2 centrifuge tubes is detected. In the 2 centrifuge tubes, a final concentration of the aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, a final concentration of the DNA is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 nanograms per milliliter (ng/ml).

TABLE 1Centrifuge tube numberAptamerDNA/PC-strandAFB 1 standard solution10.15 μmol/LDNA 0.24 μmol/L150 ng/mL20.15 μmol/LPC-strand 0.24 μmol/L150 ng/mL

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.5 PC Group Influence Experiment

As shown in Table 2, 8 centrifuge tubes are taken and numbered as 1-8, and the 8 centrifuge tubes are treated as follows.

The centrifuge tubes 1˜4 each are added with the aptamer solution, the DNA solution and the Tris buffer solution, and the centrifuge tubes 5-8 each are added with the aptamer solution, a none-PC-strand solution and the Tris buffer solution for stirring evenly to obtain mixed solutions. The mixed solutions in the centrifuge tubes 1-8 are reacted at 4° C. for 1 h to obtain reacted solutions. The reacted solutions in the centrifuge tubes 1-2 and 5-6 are not treated with light, the reacted solutions in the centrifuge tubes 2 and 6 are added with the AFB1 standard solution to react at 37° C. for 1 h, and fluorescence intensity of the aptamer in the centrifuge tubes 1-2 and 5-6 is detected, and a total volume of the solution in each centrifuge tube is 80 μL. The centrifuge tubes 1 and 5 are control groups of the centrifuge tubes 2 and 6, and are not added with the AFB1 standard solution. The centrifuge tubes 3-4 and 7-8 are irradiated with ultraviolet light (irradiation intensity is 5 mW/cm2) for 20 min. The centrifuge tubes 4 and 8 are added with the AFB1 standard solution to react at 37° C. for 1 h, and fluorescence intensity of the aptamer in the centrifuge tubes 3-4 and 7-8 is detected, and a total volume of the solution in each centrifuge tube is 80 μL. The centrifuge tubes 3 and 7 are control groups of the centrifuge tubes 4 and 8, and are not added with the AFB1 standard solution.

A final concentration of the aptamer is 0.15 μmol/L, a final concentration of the none-PC-strand is 0.24 μmol/L, a final concentration of the DNA is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 ng/mL.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

TABLE 2CentrifugeAptamerDNANone-PC-strandAFB1 standardTime for Uvtube number(μmol/L)(μmol/L)(μmol/L)solution (ng/ml)treatment (min)10.150.2400020.150.240150030.150.24002040.150.2401502050.1500.240060.1500.24150070.1500.2402080.1500.2415020
1.6 Light Influence Experiment

4 centrifuge tubes are numbered as 1-4, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. The centrifuge tubes 1 and 2 are irradiated with ultraviolet light (irradiation intensity is 5 mW/cm2) for 20 min. The centrifuge tube 2 is added with the AFB1 standard solution to react at 37° C. for 1 h, and fluorescence intensity of the aptamer in the centrifuge tubes 1 and 2 is detected, and a total volume of the solution in each centrifuge tube is 80 μL. The centrifuge tube 1 is a control group of the centrifuge tube 2, and is not added with the AFB1 standard solution. The centrifuge tubes 3 and 4 are not treated with the ultraviolet light. The centrifuge tube 4 is added with the AFB1 standard solution to react at 37° C. for 1 h, and fluorescence intensity of the aptamer in the centrifuge tubes 3 and 4 is detected, and a total volume of the solution in each centrifuge tube is 80 μL. The centrifuge tube 3 is a control group of the centrifuge tube 4, and is not added with the AFB1 standard solution.

A final concentration of the aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 ng/ml.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

30% acrylamide (5 milliliters abbreviated as mL), 5× tris-borate-ethylene diamine tetra acetic acid (TBE) buffer solution (2 mL), formamide (2 mL), urea (4.2 grams abbreviated as g), 10% ammonium persulfate (90 μL) and N,N,N′,N′-tetramethylethylenediamine (90 μL) are mixed evenly, and are placed into a gel electrophoresis plate to be placed at room temperature until the gel sets. DNA samples (10 μL), 6× loading buffer (2 μL) and a GELRED dye are added into each well of the gel electrophoresis plate. An electrophoresis experiment is performed for 1.5 h with a voltage of 90 V, and then gels are taken to observe DNA sample bands under a gel electrophoresis imaging device.

1.7 Lighting Time Influence Experiment

10 centrifuge tubes are numbered as 1-10, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. The centrifuge tubes 1-10 are respectively irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min and 9 min, and are added with the AFB1 standard solution to react at 37° C. for 1 h, and fluorescence intensity of the aptamer in the 10 centrifuge tubes is detected, and a total volume of the solution in each centrifuge tube is 80 μL.

In the 10 centrifuge tubes, a final concentration of the aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 ng/mL.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

4 centrifuge tubes are numbered as 1-4, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. After the centrifuge tube 4 is irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 8 min, the centrifuge tubes 3 and 4 are added with the AFB1 standard solution to react at 37° C. for 1 h, circular dichroisms (CD) of the solutions in the centrifuge tubes 3 and 4 are detected, and a total volume of the solution in each centrifuge tube is 80 μL.

A final concentration of the aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 ng/ml.

Testing conditions of a CD spectrometer are as follows: a spectral scanning range is 200-340 nm, an interval is 0.5 nm, and the Tris buffer solution is used to subtract spectral background.

1.8 Screening of a Reaction Temperature of the AFB1

4 centrifuge tubes are numbered as 1-4, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. The centrifuge tubes 1˜4 are irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 8 min, and are added with the AFB1 standard solution to react at different temperatures (4° C., 15° C., 25° C. and 37° C.) for 1 h, fluorescence intensity of the aptamer in the centrifuge tubes 1˜4 is detected, and a total volume of the solution in each centrifuge tube is 80 μL.

A final concentration of the aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and a final concentration of the AFB1 standard solution is 150 ng/mL.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.9 Sensitivity of AFB1 Detection

13 centrifuge tubes are numbered as 1-13, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. The centrifuge tubes 1-13 are irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 8 min, and are added with the AFB1 standard solution with different concentrations to react at 37° C. for 1 h, the fluorescence intensity is detected, and a total volume of the solution in each centrifuge tube is 80 μL.

A final concentration of the Aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and final concentrations of the AFB1 standard solution are 0 ng/ml, 0.00625 ng/mL, 0.0875 ng/ml, 0.0125 ng/ml, 0.025 ng/ml, 0.0625 ng/ml, 0.625 ng/ml, 6.25 ng/ml, 62.5 ng/ml and 625 ng/mL.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.10 Specificity of the AFB1 Detection

6 centrifuge tubes are numbered as 1-6, and added with the aptamer solution, the PC-strand solution and the tris buffer solution to react at 4° C. for 1 h. The centrifuge tubes 1-6 are irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 8 min. The centrifuge tube 1 is used as a control group, and is not added with mycotoxins. The centrifuge tubes 2-4 are added with mycotoxins: the AFB1 standard solution, an OTA standard solution and a ZEN standard solution, respectively. The centrifuge tube 5 is added with the AFB1 standard solution and the OTA standard solution. The centrifuge tube 6 is added with the AFB1 standard solution and the ZEN standard solution. After the solutions in the centrifuge tubes 1-6 are reacted at 37° C. for 1 h, fluorescence intensity is detected, and a total volume of the solution in each centrifuge tube is 80 μL.

A final concentration of the Aptamer is 0.15 μmol/L, a final concentration of the PC-strand is 0.24 μmol/L, and final concentrations of the AFB1 standard solution, the OTA standard solution and the ZEN standard solution are 150 ng/mL.

The conditions for detecting the fluorescence intensity are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

1.11 Application Experiment of the AFB1 Detection

Extraction of the AFB1 in food samples: the food samples (corn, rice and soybeans) are crushed into powder by using a grinder, and the AFB1 is extracted from the food samples according to Chinese National Food Safety Standards (GB5009. 22-2016). Specifically, 3 g of the food samples are added into 10 mL methanol/water (7:3) solution for oscillation reaction at 30° C. for 1 h to obtain a mixture. The mixture is filtered to obtain filtrate. The filtrate is centrifuged with 12000 revolutions per minute (rpm) at 4° C. for 10 min to obtain supernatant. The supernatant is collected and filtered with a 0.45 microns (μm) filter to obtain filtered supernatant. The filtered supernatant is added with 3 mL Tris solution for stirring evenly to obtain a food sample extraction solution, and the food sample extraction solution is stored at 4° C. for later use.

The AFB1 detection in the food samples includes the following steps.

90 μL food sample (corn, rice and soybeans) extraction solution is added with different volumes of the ABF1 standard solution for stirring evenly to obtained food sample solutions with different ABF1 concentrations (the concentrations of the AFB1 are 0 ng/mL, 0.075 ng/ml, 0.125 ng/mL, 0.75 ng/ml and 1.25 ng/ml), and the food sample solutions are stored at 4° C. for later use.

5 centrifuge tubes are numbered as 1-5, and added with the aptamer solution, the PC-strand solution and the Tris buffer solution to react at 4° C. for 1 h. A final concentration of the aptamer is 0.15 μmol/L, and a final concentration of the PC-strand is 0.24 μmol/L. The centrifuge tubes 1-5 are irradiated with the ultraviolet light (irradiation intensity is 5 mW/cm2) for 8 min, and are respectively added with the above food sample solutions with different ABF1 concentrations to reacted at 37° C. for 1 h, and a total volume of the solution in each centrifuge tube is 80 μL.

Detection conditions in the AFB1 solution are as follows: the excitation wavelength is 480 nm, the scanning range is 490-700 nm, the voltage is 700 V, and the slit is 5/5.

2 Experimental Results

2.1 Screening Result of the Concentration of the Aptamer

The aptamer is a fluorescence probe with good fluorescence characteristic, and the TAMRA can effectively quench FAM fluorescence. Thus, the concentration of the aptamer needs to be screened before using the PC-strand to select a concentration of the aptamer with good luminescence reaction effect, and achieve principles of saving raw materials and economy at the same time. It can be seen fromFIG.3that with the increase of the concentration of the aptamer, the fluorescence intensity gradually increased. When the concentration of the aptamer is 0.15 μmol/L, the fluorescence intensity is 225.78633, and the aptamer solution with the concentration of 0.15 μmol/L can be selected to participate in the follow-up experiments.

2.2 Screening Result of the Reaction Temperature Between the Aptamer and the PC-Strand

Temperature will affect the fluorescence quenching effect to some extent, the reaction temperature needs to be optimized to restore the fluorescence to the optimal effect when the AFB1 is added in the subsequent experiment, to thereby select an optimal temperature for fluorescence quenching. According to the optimized fluorescence curve diagram and histogram of the reaction temperature between the aptamer and the PC-strand made by experiments (as shown inFIGS.4A and4B), it can be seen that with the increase of the reaction temperature, the fluorescence intensity is enhanced, which indicates that low temperature is beneficial to hybridization between the aptamer and the PC-strand, and the fluorescence quenching effect is optimal when the reaction temperature is 4° C. Therefore, the temperature used in the subsequent experiments is 4° C. 2.3 Screening result of the concentration of the PC-strand

It can be seen fromFIGS.5A and5Bthat with the increase of the concentration of the PC-strand, the fluorescence intensity of the aptamer is gradually decreased, and when the concentration of the PC-strand is 0.24 μmol/L (CAptamer:CPC-strand=1:1.6), the fluorescence quenching tends to be stable. Therefore, the concentration of the PC-strand is selected as 0.24 μmol/L in the subsequent experiments.

2.4 Result of the Base Influence Experiment

As shown inFIGS.6A and6B, when the concentration of the AFB1 is 150 ng/ml and without lighting condition, due to a fluorescence resonance energy transfer (FRET) effect, the fluorescence change of A-PD is weak, and the fluorescence signal is enhanced by 2.1 times (as shown inFIG.6B), while the AD without 8 bases and the PC group produces obvious fluorescence signal change, and the fluorescence signal is enhanced by 7.37 times (as shown inFIG.6A). It indicates that increasing the base numbers, the binding force of the aptamer of the AFB1 and the DNA is strong, which reduces the sensing performance.

2.5 Result of the PC Group Influence Experiment

As shown inFIGS.7A and7B, when the concentration of the AFB1 is 150 ng/ml and without the lighting condition, the fluorescence signal of the A-nPD without the PC group increases by 210%, and the fluorescence signal of the AD without the PC group and 8 bases increases by 601%. When the concentration of the AFB1 is 150 ng/mL and with the lighting condition, the fluorescence signal of the A-nPD increases by 223%, and the fluorescence signal of the AD increases by 613%, which indicates that the light has no effect on the fluorescence change of the A-nPD and the AD. Therefore, introducing the PC group into the A-PD is a key to a design of time gating for detecting AFB1.

2.6 Result of the Light Influence Experiment

As shown inFIGS.8A and8B, when the concentration of the AFB1 is 100 ng/ml and with irritation under irritation intensity of 5 mW/cm2, the fluorescence intensity increases by 430%. When the concentration of the AFB1 is 100 ng/ml and without the irritation, the fluorescence intensity increases by 178%. The result shows that light can activate the sensing function and expand the detection range.

It can be seen fromFIG.9that the PC group of the PC-strand is disconnected with the lighting condition.

2.7 Result of the Lighting Time Influence Experiment

As shown inFIGS.10A and10B, when the concentration of the AFB1 is 100 ng/ml and with the irritation under irritation intensity of 5 mW/cm2, the fluorescence intensity of the A-PD increases gradually. Long-term lighting causes more PC groups to be disconnected and more photoactivated probes to bind to the AFB1, resulting in enhanced fluorescence, but after 8 min, the fluorescence gradually stabilized. The result shows that the light can activate the sensing function, and the lighting time is 8 min.

As shown inFIGS.11A and11B, in the CD diagram, a positive peak of CD280 nmis a base complementary pairing peak, and a negative peak of CD240 nmis a DNA spiral structure peak. After the A-PD is irradiated under the irritation intensity of 5 mW/cm2, and when the concentration of the AFB1 is 100 ng/ml, the positive peak of CD280 nmincreases by 39.1%, and the negative peak of CD240 nmdecreases by 565%. When the A-PD is not irradiated with the irritation intensity of 5 mW/cm2, and when the concentration of the AFB1 is 100 ng/ml, the positive peak of CD280 nmdecreases by 20.0%, and the negative peak of CD240 nmdecreases by 163.8%. The result shows that the light is more conducive to the binding of the AFB1 with the adapter, resulting in a change in configuration.

2.8 Result of Influence of the Reaction Temperature on the AFB1 Detection

As shown inFIGS.12A and12B, due to the significant influence of temperature on the specific recognition of target molecules by the adapter, under the irradiation intensity of 5 mW/cm2, and when the concentration of the AFB1 is 100 ng/ml, the change in the reaction temperature has a significant impact on the fluorescence intensity of the reaction system. When the reaction temperature is 37° C., the fluorescence value is the highest. Therefore, 37° C. is selected as the reaction temperature in subsequent reactions.

2.9 Experimental Result of Sensitivity of the AFB1 Detection

As shown inFIGS.13A and13B, the fluorescence value increases with the increase of the concentration of the AFB1 (0-625 ng/ml) after lighting for 8 min. According to the calculation of the fitted line and standard deviation in the line diagram inFIG.13B, a limit of detection (LOD) for detection is 0.07018 ng/ml (LOD=3σ/S).

2.10 Experimental Result of Specificity of the AFB1 Detection

As shown inFIGS.14A and14B, after lighting for 8 min, when the system exists the AFB1 (100 ng/ml), the fluorescence intensity increases by 4.4 times; and when the system exists the OTA or ZEN of the same concentration (100 ng/ml), the fluorescence intensity has hardly changed. When adding the same concentration of the AFB1, the fluorescence intensity significantly increases. The result shows that the system has a good selectivity for the AFB1.

2.11 Experimental Result of Applicability of the AFB1 Detection

As shown inFIGS.15A and15B, after lighting for 8 min, when the system exists the AFB1, the fluorescence intensity enhances with the increase of the concentration of the AFB1. The result shows that the sensor has good applicability to AFB1 in rice systems.

As shown inFIGS.16A and16B, after lighting for 8 min, when the system exists the AFB1, the fluorescence intensity enhances with the increase of the concentration of the AFB1. The result shows that the sensor has good applicability to AFB1 in corn systems.

As shown inFIGS.17A and17B, after lighting for 8 min, when the system exists the AFB1, the fluorescence intensity enhances with the increase of the concentration of the AFB1. The result shows that the sensor has good applicability to AFB1 in soybean systems.

In summary, the disclosure conducts a series of explorations and selects the optimal value from the experimental results. That is, the optimal concentration of the aptamer solution is 0.15 μmol/L, the optimal tris buffer solution includes 10 mM of tris, 120 mM of NaCl, 5 mM of KCl and pH=7.2, which indicates that the fluorescence quenching effect of optical sectioning on the reaction between the aptamer and the PC-strand is better. The optimal reaction temperature between the aptamer and the PC-strand is 4° C., the optimal reaction time is 20 min, and the optimal concentration ratio of the aptamer and the PC-strand is 1:1.2, which proves the feasibility of the experiment on fluorescence recovery by adding AFB1 after optical sectioning, and the optimal time for optical sectioning is 20 min. The optimal reaction temperature for AFB1 is 37° C., the sensitivity of the AFB1 detection is detected, and the sensitivity is calculated as 0.07018 ng/mL. The experimental method is applied to detect the AFB1 content in food samples such as corn, rice, soybeans and the like.

The above embodiments are merely a description of some of the embodiments of the disclosure, and do not limit the scope of the disclosure. Without departing from a design spirit of the disclosure, all variations and improvements made by those skilled in the art to the technical solution of the disclosure should fall within the scope of protection determined by claims of the disclosure.