A Detection Method for Sesame Allergen Ses i 2 Based on Perovskite Quantum Dots - Aptamer

Abstract

The present invention relates to a biosensor for detecting sesame protein Ses i 2 and a preparation method thereof. The biosensor is prepared from perovskite quantum dots CsPbBr3@SiO2@mPEG-DSPE, nucleic acid aptamers, and Ti3C2 MXenes, wherein the nucleic acid aptamers are nucleic acid aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO.1 or nucleic acid aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO.2. The biosensor of the present invention is applied to the determination of sesame allergen Ses i 2 protein in food and has great potential in the field of allergen detection.

Description

Technical Field

[0001] The present invention relates to the field of analytical detection, and particularly relates to a biosensor for detecting the sesame allergen Ses i 2 and a detection method thereof. Background Art

[0002] Sesame (Sesamum indicum L.) is an agricultural crop widely cultivated worldwide. It is rich in nutritional value, containing oils, proteins, and various bioactive components. Sesame is not only a delicious food ingredient but also has good medicinal value, capable of regulating the gastrointestinal tract, moisturizing the skin, delaying aging, and enhancing immunity. However, it is also a recognized food allergen subject to international regulation. In 2021, sesame was listed as one of the new eight major food allergens. For people allergic to sesame, after coming into contact with sesame or sesame products, they may experience symptoms such as skin redness, urticaria, respiratory distress, angioedema, and gastrointestinal discomfort. In severe cases, it may even lead to anaphylactic shock, threatening life safety.

[0003] Ses i 2 is a 2S seed storage protein, also known as 2S storage albumin and β-globulin. It belongs to the prolamin superfamily and consists of 148 amino acids with a relative molecular mass of 7×10 3 . This protein is widely present in sesame. Relevant research has confirmed that it can be recognized by the sera of 80% of sesame-allergic patients and is thus considered one of the main allergens of sesame. This protein has strong resistance to digestion by pepsin, trypsin, and chymotrypsin in its natural state, and there are no glycosylation modification sites in its amino acid sequence. These characteristics make Ses i 2 have strong anti-digestibility and also increase the possibility of its clinical cross-allergic reactions, further increasing its sensitization risk.

[0004] Aptamer is a specific structure composed of nucleic acids (DNA or RNA) that can bind to specific target molecules and play a role. Aptamers have the characteristics of high specificity, high affinity, programmability, and easy preparation, which makes them a very important biorecognition element and have been widely used in various fields. Aptamers have extensive applications in biological research (such as gene expression regulation, signal transduction, viral infection, and cancer treatment, etc.). "Magnetic bead-SELEX aptamer screening" is an efficient technique for screening specific target molecules. Magnetic beads are tiny magnetic beads that can bind to target molecules and thus be separated by a magnetic separator. The SELEX technique is a screening method based on exponential enrichment that can quickly find aptamers that bind highly specifically to target molecules. By combining magnetic beads and the SELEX technique, the screening efficiency can be greatly improved, and thus the optimal aptamer can be quickly found.

[0005] A biosensor is composed of a receptor, a signal transducer, and a signal processing device. It can specifically bind various target compounds and convert the binding degree into a measurable signal. Due to the advantages of biosensors in terms of biological receptors, materials, and transduction modes, they are widely used in different fields. Due to the advantages of simple operation, high throughput, high sensitivity, and on-site portability, the biosensing technology detection platform has attracted much attention in the field of rapid detection of food allergens. It can be used for food certification and safety monitoring, including the detection of pathogens, pesticides, additives, and allergens, etc.

[0006] At present, there is no relevant report on the detection of sesame allergens by a biosensor based on perovskite quantum dots and nucleic acid aptamers in the existing technology. Therefore, it is of great significance to screen and obtain Ses i 2 nucleic acid aptamers with high affinity and use them for biosensing detection. Summary of the Invention

[0007] The object of the present invention is to provide a biosensor for detecting Ses i 2 protein of sesame allergens based on perovskite quantum dots - nucleic acid aptamers and its detection method.

[0008] For the above - mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a preparation method of perovskite quantum dots CsPbBr3@SiO2@mPEG - DSPE with excellent water stability and water solubility. The method includes the following steps:

[0010] Step 1: Synthesize SiO2 - coated CsPbBr3 quantum dots by ligand - assisted reprecipitation method;

[0011] Step 2: Use the functional polymer mPEG - DSPE - COOH as a ligand for CsPbBr3@SiO2 to synthesize CsPbBr3@SiO2@mPEG - DSPE.

[0012] Preferably, the step 1 includes the preparation of precursors and the precipitation of quantum dots.

[0013] More preferably, the step 1 includes the following steps:

[0014] (1) Add lead bromide (PbBr2), cesium bromide (CsBr), oleic acid (OA), and oleylamine (OAm) into DMF, stir at high temperature to prepare a precursor solution, and then add ammonia solution;

[0015] (2) Under vigorous stirring, quickly add the precursor solution to toluene containing TMOS, and then stir vigorously to form core - shell CsPbBr3@SiO2 quantum dots, and centrifuge to collect the final product.

[0016] Further preferably, step 1 includes the following steps:

[0017] (1) Add 0.1468 g of PbBr2, 0.0851 g of CsBr, 0.6 mL of oleylamine and 1.8 mL of oleic acid to 10 mL of DMF; then stir at 100 °C for 2 hours to obtain a transparent precursor solution; add 40 μL of an ammonia solution with a concentration of 2.8% to 2 mL of the precursor solution;

[0018] (2) Under stirring at 1500 rpm, quickly add 0.2 mL of the precursor solution to 10 mL of dry toluene containing 5 μL of TMOS, and stir at 30 °C at 1500 rpm; after 10 s, adjust the stirring speed to 150 rpm, and the reaction time is 12 h to form core-shell CsPbBr3@SiO2 quantum dots, and the final product is collected by centrifugation at 9000 rpm for 5 min.

[0019] Preferably, step 2 includes the following steps: dissolve mPEG-DSPE-COOH in ultradry chlorobenzene; then add CsPbBr3@SiO2 quantum dots to the mPEG-DSPE-COOH solution, stir overnight, and completely evaporate the organic solvent; purify CsPbBr3@SiO2@mPEG-DSPE by centrifugation and redisperse it in deionized water.

[0020] More preferably, step 2 includes the following steps: dissolve 20 mg of mPEG-DSPE-COOH in 1 mL of ultradry chlorobenzene; then add 5 mg of CsPbBr3@SiO2 quantum dots to the mPEG-DSPE-COOH solution, stir overnight, and completely evaporate the organic solvent; purify CsPbBr3@SiO2@mPEG-DSPE by centrifugation and redisperse it in deionized water.

[0021] The second aspect of the present invention provides the perovskite quantum dot CsPbBr3@SiO2@mPEG-DSPE prepared by the above method.

[0022] The third aspect of the present invention provides a method for screening nucleic acid aptamers of allergen protein Ses i 2 using a magnetic bead-SELEX exponential enrichment ligand system, and the method includes the following steps:

[0023] Step 1, screening by magnetic bead method: first pre-treat the random library, and then use magnetic bead-SELEX technology for reverse screening and forward screening to screen nucleic acid aptamers of sesame allergen Ses i 2;

[0024] Step 2, Preparation of the secondary library: Using the screened ssDNA as a template, perform PCR amplification, and unwind the amplification product into single strands to prepare the secondary library;

[0025] Step 3, Measurement of fluorescence value: After each round of screening, dilute the secondary library to an appropriate concentration and measure the fluorescence value with a fluorometer;

[0026] Step 4, High-throughput sequencing analysis: Construct a library from the specific amplification product and perform sequencing;

[0027] Step 5, Affinity determination: Perform affinity determination using ELISA experiments to obtain aptamers with affinity.

[0028] Preferably, step 1 includes the following steps:

[0029] (1) Take a random ssDNA library, place it in a 95°C water bath for 5 min, immediately take it out and put it in an ice box for ice bath to allow the ssDNA in the library to fold into a complex tertiary structure;

[0030] (2) Use magnetic bead-SELEX technology to screen for aptamers of sesame allergen Ses i 2, and perform negative and positive screening;

[0031] (3) Negative screening: To enhance the specificity of the screened aptamers, start negative screening from the first round; first couple the activated magnetic beads with the negative target Ses i 1, add the library to the magnetic beads coupled with Ses i 1, incubate with slow shaking at room temperature to immobilize the protein on the magnetic beads; then wash the magnetic beads with PBS, resuspend with PBS, add the initial library, mix well, and incubate with slow shaking at room temperature;

[0032] (4) Positive screening: Add the supernatant in the secondary library to the magnetic beads coupled with Ses i 2, incubate with slow shaking at room temperature, perform magnetic separation, discard the supernatant, resuspend with PBS, heat at 95°C for 5 min, perform magnetic separation, collect the supernatant, and use the supernatant as a template for PCR amplification to prepare the secondary library for subsequent screening.

[0033] More preferably, step 1 includes the following steps:

[0034] (1) Take 20 μL of 100 μmol·L -1 random ssDNA library, add 500 μL of 1×PBS buffer, mix well, then place it in a 95°C water bath for 5 min, immediately take it out and put it in an ice box for ice bath for 5 min, and then let it stand at room temperature for 10 min to allow the ssDNA in the library to fold into a complex tertiary structure;

[0035] (2) Use magnetic bead-SELEX technology to screen aptamers of sesame allergen Ses i 2, perform reverse screening and forward screening, and conduct a total of 10 rounds of screening, among which reverse screening was added in the 1st, 2nd, 3rd, 4th, and 7th rounds;

[0036] (3) Reverse screening: To enhance the specificity of the screened aptamers, reverse screening was added starting from the 1st round; First, couple the activated magnetic beads with the reverse target Ses i 1, add the library to the magnetic beads coupled with Ses i 1, and incubate at room temperature with slow shaking for 30 min; Perform magnetic separation and collect the supernatant;

[0037] (4) Forward screening: Add the supernatant in the secondary library to the magnetic beads coupled with Ses i 2, incubate at room temperature with slow shaking for 30 min, perform magnetic separation, discard the supernatant, add 200 μL of PBS to resuspend, heat at 95 °C for 5 min, perform magnetic separation, collect the supernatant, and measure the DNA concentration in the supernatant with a Qubit fluorometer; Use the supernatant as a template for PCR amplification to prepare a secondary library for subsequent screening.

[0038] Further preferably, the specific methods of steps 1 and (3) are as follows: Dissolve 200 pmol of the initial library in 200 μL of PBS buffer, heat at 95 °C for 5 min, and place it in a refrigerator at 4 °C for 15 min; Take 50 μL of magnetic beads, resuspend them in 200 μL of PBS buffer, wash them 3 times with PBS, add 50 μL, 50 mg·mL -1 of EDC and NHS solution, and slowly shake at room temperature for 30 min to activate the carboxyl groups on the surface of the magnetic beads; Wash the activated magnetic beads 3 times with PBS, resuspend them with 500 μL of CBS buffer, add 6 μL of sesame protein Ses i 1, and slowly shake at room temperature for 2 h to fix the protein on the magnetic beads; Subsequently, wash the magnetic beads 3 times with PBS, add 200 μL of PBS to resuspend, add the initial library, mix well, and incubate at room temperature with slow shaking for 1 h.

[0039] Preferably, step 2 includes the following steps:

[0040] (1) Use the supernatant as a template for PCR amplification to enrich the DNA;

[0041] (2) After the PCR reaction is completed, take streptavidin-coated paramagnetic microspheres, wash them 3 times with PBS, add the PCR product, slowly shake, perform magnetic separation, discard the supernatant, add PBS to resuspend, heat at 95 °C for 10 min to unwind the DNA double strand, the biotin-labeled ssDNA remains fixed on the magnetic microspheres, and the FAM-fluorescent-labeled ssDNA is freed, perform magnetic separation, and collect the supernatant for the next round of screening.

[0042] More preferably, step 2 includes the following steps:

[0043] (1) Using the supernatant as a template, perform PCR amplification to enrich DNA. The PCR system is as follows: 2 μL of supernatant template, 25 μL of PCRmix, 2 μL of each forward and reverse primer, and add water to 50 μL. The PCR program: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 20 s, for 12 cycles, and finally extension at 72 °C for 5 min, and store at 4 °C.

[0044] (2) After the PCR reaction, take 200 μL of streptavidin-coated paramagnetic microspheres, wash them 3 times with PBS, add 500 μL of PCR product, gently shake at 37 °C for 1 h, perform magnetic separation, discard the supernatant, add 200 μL of PBS to resuspend. At this time, the DNA is fixed on the magnetic microspheres. Heat at 95 °C for 10 min to unwind the DNA double strands. The biotin-labeled ssDNA remains fixed on the magnetic microspheres, and the FAM-fluorescently labeled ssDNA is released. Perform magnetic separation and collect the supernatant for the next round of screening.

[0045] Preferably, step 3 includes the following steps: After each round of screening, dilute the PCR product to 0.1 mL with PBS, measure the fluorescence value with a fluorometer, the excitation wavelength is 492 nm, the emission wavelength is 518 nm, and the excitation slit is 10 nm.

[0046] Preferably, step 4 includes the following steps: Hand over the specific amplification product to Sangon Biotech (Shanghai) Co., Ltd. for library construction and sequencing.

[0047] Preferably, step 5 includes the following steps: Dilute sesame protein Ses i 2 with PBS, set 2 replicates for each experimental group, and coat a 96-well plate; Wash the plate with PBST and pat dry the liquid in the plate; Block with 5% BSA solution; Wash the plate with PBST and pat dry the liquid in the plate; Prepare the aptamer to the target concentration, add it to the 96-well plate, and incubate at room temperature for 1 h; Wash the plate with PBST and pat dry the liquid in the plate; Add SA-HRP to each well, incubate at room temperature and then wash the plate; Add the chromogenic solution to each well and incubate at room temperature in the dark; Add H2SO4 solution to each well to terminate the reaction; Gently shake to ensure sufficient reaction; Read with an enzyme-linked immunosorbent assay reader at 450 nm; The results are expressed as OD values (OD value); The data is processed with GraphPad Prism 8 software, and non-linear fitting is performed according to the formula Y = B max X / (K d +X), where X is the aptamer concentration and Y is the OD value.

[0048] More preferably, step 5 includes the following steps:

[0049] Dilute sesame protein Ses i 2 with PBS to 6 μg·mL-1 For each experimental group, set 2 replicates. Add 100 μl to each well and coat the 96-well plate at 37 °C for 1.5 h; Wash the plate 3 times with PBST and pat dry the liquid in the plate; Block with 5% BSA solution for 1 h; Wash the plate 3 times with PBST and pat dry the liquid in the plate; Prepare the aptamer to the target concentrations of 50, 100, 150, 200, 400, 600, 800, and 1000 nM, and add 50 μL to the 96-well plate respectively, and incubate at room temperature for 1 h; Wash the plate 3 times with PBST and pat dry the liquid in the plate; Add 50 μL of SA-HRP diluted 1:200 with buffer to each well, wash the plate after incubating at room temperature for 0.5 h; Add 50 μL of chromogenic solution to each well; Incubate at room temperature for 20 min in the dark; Add 50 μL of 0.5 M H2SO4 solution to each well to terminate the reaction; Gently shake to ensure sufficient reaction; Read with an enzyme-linked immunosorbent assay reader: 450 nm; The results are expressed as OD values (ODvalue); The data is processed using GraphPad Prism 8 software, and according to the formula Y = B max X / (K d +X) for non-linear fitting, where X is the aptamer concentration and Y is the OD value.

[0050] Preferably, the aptamer obtained in step 5 is aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO.1 or aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO.2.

[0051] More preferably, the aptamer obtained in step 5 is aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO.1.

[0052] The fourth aspect of the present invention provides an aptamer for the allergen protein Ses i 2, and the aptamer is aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO.1 or aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO.2.

[0053] Preferably, the aptamer is aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO.1.

[0054] The fifth aspect of the present invention provides a biosensor for detecting sesame protein Ses i 2, and the biosensor is prepared from the above-mentioned perovskite quantum dot CsPbBr3@SiO2@mPEG-DSPE, the above-mentioned aptamer, and Ti3C2 MXenes.

[0055] The sixth aspect of the present invention provides the application of the above biosensor in detecting sesame protein Ses i 2 in food.

[0056] The seventh aspect of the present invention provides a method for preparing a biosensor for detecting sesame protein Ses i 2, the method comprising the following steps:

[0057] Step 1, synthesize Ti3C2 MXenes: Add titanium carbide (Ti3C2T x ) MXenes to DMSO, stir at room temperature, centrifuge, redissolve the obtained precipitate in deionized water, repeat the operation, wash the solution to remove DMSO, and then ultrasonically treat the product;

[0058] Step 2, synthesize the probe CsPbBr3@SiO2@mPEG-DSPE@Apt: Activate the carboxyl groups on the surface of CsPbB r3 @SiO2@mPEG-DSPE using EDC and NHS, centrifuge, redissolve the precipitate in deionized water, add a sterile aqueous solution of the above nucleic acid aptamer to the obtained solution, and bind the nucleic acid aptamer to CsPbBr3@SiO2@mPEG-DSPE;

[0059] Step 3, prepare the CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 biosensor: Mix the aqueous solution of CsPbBr3@SiO2@mPEG-DSPE@Apt and the aqueous solution of Ti3C2 for hybridization; Collect the final CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 by centrifugation, wash with PBS buffer, and resuspend to obtain.

[0060] Preferably, step 1 includes the following steps: Add 0.05 g of Ti3C2T x MXenes to 1 mL of DMSO, stir at room temperature for 24 h; then centrifuge, redissolve the obtained precipitate in deionized water, repeat the operation, wash the solution to remove DMSO; ultrasonically treat the obtained product for 2 h and centrifuge at 3500 rpm for 1 h; The supernatant is the exfoliated Ti3C2 MXenes two-dimensional nanomaterial;

[0061] Preferably, step 2 includes the following steps: Take 5 mg of CsPbBr3@SiO2@mPEG-DSPE, add 1 mL each of EDC and NHS with a concentration of 25 mg·mL -1 , shake for 30 min to activate the carboxyl groups on the surface of CsPbBr3@SiO2@mPEG-DSPE, centrifuge, and redissolve the precipitate in deionized water; Add 500 μL of a sterile aqueous solution of the nucleic acid aptamer S1 with a concentration of 10 μM to the obtained solution; Then incubate the mixture at 25 °C for 12 h to bind the nucleic acid aptamer S1 to the surface of CsPbBr3@SiO2@mPEG-DSPE.

[0062] Preferably, step 3 includes the following steps: Mix 100 μL of CsPbBr3@SiO2@mPEG-DSPE@Apt aqueous solution and 200 μL of Ti3C2 (0.8 mg / mL) aqueous solution for 30 min for hybridization. Collect the final CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 by centrifugation, wash it with PBS buffer, and resuspend it in 150 μL of PBS buffer to obtain the product.

[0063] The beneficial effects of the present invention are as follows:

[0064] 1. The present invention provides a method for synthesizing perovskite CsPbBr3 with excellent water stability and water solubility, which can be directly used as a fluorescent probe in aqueous solution.

[0065] 2. The present invention obtains the allergen protein Ses i 2 nucleic acid aptamer through magnetic bead-SELEX screening. As the number of screening rounds increases, nucleic acid aptamers with high affinity are enriched. After sequencing, the nucleic acid sequence of the nucleic acid aptamer with high affinity and high specificity for the target protein can be obtained, which can be used to detect the allergen protein Ses i 2 and for the prevention of sesame allergy.

[0066] 3. The present invention provides a biosensor for detecting sesame protein Ses i 2 based on perovskite quantum dots and nucleic acid aptamers, which can be used to detect sesame protein Ses i 2 in actual samples. Description of the Drawings

[0067] Figure 1 is the transmission electron microscope image of the perovskite quantum dot CsPbBr3@SiO2@mPEG-DSPE in Example 1 of the present invention;

[0068] Figure 2 is the change in fluorescence quantum yield of CsPbBr3 and CsPbBr3@SiO2@mPEG-DSPE in the aqueous phase within 30 days in Example 1 of the present invention;

[0069] Figure 3 is the detection result of the SELEX screening fluorescence value in Example 2 of the present invention;

[0070] Figure 4 is the determination result of the affinity of the candidate nucleic acid aptamer in Example 2 of the present invention;

[0071] Figure 5The fluorescence intensity (FL) of CsPbBr3@SiO2@mPEG-DSPE@aptamer (red), CsPbBr3@SiO2@mPEG-DSPE@aptamer and Ti3C2 (black), CsPbBr3@SiO2@mPEG-DSPE@aptamer and Ti3C2 and Ses i 2 protein (blue) in Example 3 of the present invention, with the maximum emission wavelength being 519 nm;

[0072] Figure 6 is the calibration graph of the fluorescence intensity varying with the concentration of Ses i 2 in Example 3 of the present invention;

[0073] Figure 7 is the specific result of the biosensor for Ses i 2 protein in Example 3 of the present invention. Detailed implementation manners

[0074] The present invention is further illustrated by way of examples, but the present invention is not limited to the following examples only.

[0075] Example 1: Synthesis of perovskite quantum dots CsPbBr3@SiO2@mPEG-DSPE

[0076] 1. Preparation of precursors and precipitation of quantum dots

[0077] (1) Add 0.1468 g of PbBr2, 0.0851 g of CsBr, 0.6 mL of oleylamine (OAm) and 1.8 mL of oleic acid (OA) to 10 mL of DMF. Then stir at 100 °C for 2 h to obtain a transparent precursor solution. Add ammonia solution (40 μL, 2.8%) to 2 mL of the precursor solution.

[0078] (2) Under vigorous stirring (1500 rpm), quickly add 0.2 mL of the precursor solution to 10 mL of dry toluene containing 5 μL of TMOS, and carry out vigorous stirring (1500 rpm) at 30 °C. After 10 s, adjust the stirring speed to 150 rpm, and the reaction time is 12 h to form core-shell CsPbBr3@SiO2 quantum dots. The final product is collected by centrifugation at 9000 rpm for 5 min.

[0079] 2. Use the functional polymer mPEG-DSPE-COOH as a ligand for CsPbBr3@SiO2 to synthesize CsPbBr3@SiO2@mPEG-DSPE

[0080] Dissolve 20 mg of mPEG-DSPE-COOH in 1 mL of ultradry chlorobenzene. Add 5 mg of CsPbBr3@SiO2 quantum dots to the mPEG-DSPE-COOH solution, stir overnight, and completely evaporate the organic solvent. Purify CsPbBr3@SiO2@mPEG-DSPE by centrifugation and redisperse it in deionized water. The transmission electron microscopy image of the perovskite quantum dots CsPbBr3@SiO2@mPEG-DSPE is as Figure 1 shown.

[0081] The fluorescence intensity of the CsPbBr3@SiO2@mPEG-DSPE composite decreases slowly over time and remains at 75% of the initial fluorescence value after 30 days ( Figure 2 ). These results reveal that the CsPbBr3@SiO2@mPEG-DSPE composite has higher stability in the aqueous phase and shows great potential as a fluorescent labeling material in the aqueous phase system.

[0082] Example 2. Screening of nucleic acid aptamers against sesame allergen protein Ses i 2

[0083] 1. Pretreatment of the random library, negative selection and positive selection

[0084] (1) Take 20 μL of 100 μmol·L -1 (2 nmol) random ssDNA library, add 500 μL of 1×PBS buffer, mix well, place it in a 95°C water bath for 5 min, take it out and immediately place it in an ice bath for 5 min. After the ice bath is completed, let it stand at room temperature for 10 min to allow the ssDNA in the library to fold into a complex tertiary structure.

[0085] (2) Use the magnetic bead-SELEX technique to screen for nucleic acid aptamers against sesame allergen Ses i 2, perform negative selection and positive selection, and perform 10 rounds of screening in total. Negative selection was added in the 1st, 2nd, 3rd, 4th, and 7th rounds.

[0086] (3) Negative selection: To enhance the specificity of the screened nucleic acid aptamers, negative selection was added starting from the 1st round. First, couple the activated magnetic beads with the negative target Ses i 1, add the library to the magnetic beads coupled with Ses i 1, and incubate with slow shaking at room temperature for 30 min. Perform magnetic separation and collect the supernatant.

[0087] The specific method is as follows: Dissolve 200 pmol of the initial library in 200 μL of PBS buffer, heat it at 95°C for 5 min, and let it stand in a 4°C refrigerator for 15 min. Take 50 μL of magnetic beads, resuspend them in 200 μL of PBS buffer, wash them 3 times with PBS, add 50 μL, 50 mg·mL -1The EDC and NHS solutions were slowly shaken at room temperature for 30 min to activate the carboxyl groups on the surface of the magnetic beads. The activated magnetic beads were washed 3 times with PBS, resuspended in 500 μL of CBS buffer, and 6 μL of sesame protein Ses i 1 was added. The mixture was slowly shaken at room temperature for 2 h to immobilize the protein on the magnetic beads. Subsequently, the magnetic beads were washed 3 times with PBS, resuspended in 200 μL of PBS, and the initial library was added. The mixture was mixed well and slowly shaken and incubated at room temperature for 1 h.

[0088] (4) Positive screening: The supernatant in the secondary library was added to the magnetic beads conjugated with Ses i 2, and the mixture was slowly shaken and incubated at room temperature for 30 min. After magnetic separation, the supernatant was discarded, and the magnetic beads were resuspended in 200 μL of PBS. Then, the mixture was heated at 95 °C for 5 min, followed by magnetic separation. The supernatant was collected, and the DNA concentration in the supernatant was measured using a Qubit fluorometer. The supernatant was used as a template for PCR amplification to prepare the secondary library for subsequent screening.

[0089] 2. PCR amplification, dissociation of the amplified product into single strands, and preparation of the secondary library

[0090] (1) Using the supernatant as a template, PCR amplification was performed to enrich the DNA. The PCR system was as follows: 2 μL of supernatant template, 25 μL of PCRmix, 2 μL of each forward and reverse primer, and water was added to make up to 50 μL. The PCR program was: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 20 s, for 12 cycles, and finally extension at 72 °C for 5 min, and stored at 4 °C.

[0091] (2) After the PCR reaction was completed, 200 μL of streptavidin-coated paramagnetic microspheres were taken, washed 3 times with PBS, and 500 μL of the PCR product was added. The mixture was slowly shaken at 37 °C for 1 h, followed by magnetic separation. The supernatant was discarded, and the magnetic beads were resuspended in 200 μL of PBS. At this time, the DNA was immobilized on the magnetic microspheres. The DNA double strands were denatured by heating at 95 °C for 10 min. The biotin-labeled ssDNA remained immobilized on the magnetic microspheres, while the FAM-fluorescent-labeled ssDNA was released. After magnetic separation, the supernatant was collected for the next round of screening.

[0092] 3. Measurement of the fluorescence value of the secondary library using a fluorometer

[0093] After each round of screening, the PCR product was diluted to 0.1 mL with PBS, and the fluorescence value was measured using a fluorometer. The excitation wavelength was 492 nm, the emission wavelength was 518 nm, and the excitation slit was 10 nm. The results are as Figure 3 shown.

[0094] 4. High-throughput sequencing analysis

[0095] The specific amplification products obtained from the last round of screening were sent to Sangon Biotech (Shanghai) Co., Ltd. for library construction and sequencing. The sequencing results of the candidate nucleic acid aptamers of Ses i 2 are shown in Table 1.

[0096] Table 1 Candidate aptamer nucleic acid sequences

[0097]

[0098] 5. Affinity determination by ELISA

[0099] Dilute sesame protein Ses i 2 with PBS to 6 μg·mL -1 , set 2 replicates for each experimental group, add 100 μl to each well, and coat a 96-well plate at 37 °C for 1.5 h. Wash the plate 3 times with PBST and pat dry the liquid in the plate. Block with 5% BSA solution for 1 h. Wash the plate 3 times with PBST and pat dry the liquid in the plate. Prepare the nucleic acid aptamer to the target concentration (50, 100, 150, 200, 400, 600, 800, 1000 nM), add them to the 96-well plate respectively, add 50 μL to each well, and incubate at room temperature for 1 h. Wash the plate 3 times with PBST and pat dry the liquid in the plate. Add 50 μL of SA-HRP diluted 1:200 with buffer to each well, wash the plate after incubating at room temperature for 0.5 h. Add 50 μL of chromogenic solution to each well. Incubate at room temperature for 20 min in the dark. Add 50 μL of 0.5 M H2SO4 solution to each well to terminate the reaction. Gently shake to ensure sufficient reaction. Read with an enzyme-linked immunosorbent assay reader: 450 nm. The results are expressed as OD values. The data was processed using GraphPad Prism 8 software and non-linearly fitted according to the formula Y = B max X / (K d +X) (X is the nucleic acid aptamer concentration, Y is the OD value). The results showed that only nucleic acid aptamers S1 and S2 showed affinity. As Figure 4 shown, the affinity constants kd were 67.02 nM and 97.65 nM respectively. According to the inverse relationship between the Kd value and affinity, aptamer S1 was selected as the aptamer for Ses i 2.

[0100] Example 3. A biosensor for detecting sesame protein Ses i 2 based on perovskite quantum dots and nucleic acid aptamers

[0101] 1. Fabrication of the CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 biosensor

[0102] (1) Synthesis of Ti3C2 MXenes: Add 0.05 g of titanium carbide (Ti3C2T x) MXenes were added to 1 mL of DMSO and stirred at room temperature for 24 h. Then, it was centrifuged, and the obtained precipitate was redissolved in deionized water. The operation was repeated to wash the solution and remove DMSO. Ti3C2T x was sonicated for 2 h and centrifuged at 3500 rpm for 1 h. The supernatant was the exfoliated two-dimensional nanomaterial of Ti3C2 MXenes, dispersed in water.

[0103] (2) Synthesis of the probe CsPbBr3@SiO2@mPEG-DSPE@Apt: Take 5 mg of CsPbBr3@SiO2@mPEG-DSPE, add 1 mL each of EDC and NHS with a concentration of 25 mg·mL -1 and shake for 30 min to activate the carboxyl groups on the surface of CsPbBr3@SiO2@mPEG-DSPE. Then, centrifuge and redissolve the precipitate in deionized water. Add 500 μL of a sterile aqueous solution of the nucleic acid aptamer S1 with a concentration of 10 μM to the obtained solution. Then, incubate the mixture at 25 °C for 12 h to bind the nucleic acid aptamer S1 to the surface of CsPbBr3@SiO2@mPEG-DSPE.

[0104] (3) Preparation of the CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 biosensor: Mix 100 μL of the aqueous solution of CsPbBr3@SiO2@mPEG-DSPE@Apt and 200 μL of the Ti3C2 (0.8 mg·mL -1 ) aqueous solution for 30 min for hybridization. Collect the final CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 by centrifugation, wash it with PBS buffer, and resuspend it in 150 μL of PBS buffer to obtain.

[0105] 2. Feasibility analysis of the biosensor

[0106] Synthesize water-stable CsPbBr3@SiO2@mPEG-DSPE@Apt with a concentration of 2 mg·mL -1 . Then, take 1 mL each of Ti3C2 (1 mg·mL -1 ) and PBS solution and mix them with 1 mL of the perovskite aqueous solution, and shake well for 1 h. Measure the fluorescence spectra of the two tubes of solutions and record the data. Add 5.0 μg·mL -1 of the Ses i 2 protein standard to the CsPbBr3@SiO2@mPEG-DSPE@Apt-Ti3C2 solution, observe the recovery of the fluorescence signal, measure the fluorescence spectrum of the solution, and record the data. As Figure 5 shown, the fluorescence recovered after adding the protein standard.

[0107] 3. Determination of the detection limit and specificity of the biosensor

[0108] (1) Experiments were carried out under optimal conditions (the reaction time for Ti3C2 to quench CsPbBr3@SiO2@mPEG-DSPE-Apt was 30 min, and the concentration of Ti3C2 was 0.002 mg·mL -1 ). After connecting and assembling the perovskite sensor, different concentrations of Ses i 2 protein (0, 0.5, 10, 20, 40, 60, 80, 100, 120 ng·mL -1 ) were added for incubation, the fluorescence intensity in the supernatant was measured, and three parallel experiments were carried out for each sample. The standard curve for Ses i 2 protein detection was made based on the fluorescence values of the supernatants obtained by adding different concentrations of Ses i 2 protein. As Figure 6 shown, the regression equation of the standard curve was (F - F0) / F0 = 0.003925X + 0.1037, the correlation coefficient was R 2 = 0.9926, the linear range of the detection system was 0.5 - 120 ng / mL, the limit of detection (LOD) was 6.96 ng·mL -1 (S / N = 3), and the limit of quantification (LOQ) was 23.18 ng·mL -1 (S / N = 10).

[0109] (2) To evaluate the specificity of this fluorescence detection method, sesame proteins (Ses i 3, Ses i 4, Ses i5, Ses i 6, Ses i 7) and walnut and Brazil nut proteins that cross-react with Ses i 2 were selected for specificity testing. The concentrations of these 7 proteins of Ses i 2 were all adjusted to 0.1 mg·mL -1 , and then they were respectively added to the CsPbBr3@SiO2@mPEG-DSPE-Apt probe solution. After mixing evenly, fluorescence detection was carried out to evaluate the specificity of this method. As Figure 7 shown, this sensor has specificity and affinity for detecting Ses i 2 protein.

[0110] Although the specific embodiments of the present invention have been described, those skilled in the art should recognize that various changes and modifications can be made to the present invention without departing from the scope or spirit of the present invention. Thus, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. An aptamer of the allergen protein Ses i 2, characterized in that, The nucleic acid aptamer is the nucleic acid aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO. 1 or the nucleic acid aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO.

2.

2. A biosensor for detecting sesame protein Ses i 2, characterized in that, The biosensor is prepared from perovskite quantum dots CsPbBr3@SiO2@mPEG-DSPE, nucleic acid aptamers, and Ti3C2 MXenes; The nucleic acid aptamer is the nucleic acid aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO. 1 or the nucleic acid aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO.

2.

3. The biosensor for detecting Ses i 2 of sesame protein according to claim 2, characterized in that, The preparation method of the perovskite quantum dots CsPbBr3@SiO2@mPEG-DSPE includes the following steps: Step 1. Synthesize SiO2-coated CsPbBr3 quantum dots by ligand-assisted reprecipitation method; Step 2. Synthesize CsPbBr3@SiO2@mPEG-DSPE using the functional polymer mPEG-DSPE-COOH as a ligand for perovskite nanocrystals.

4. The biosensor for detecting sesame protein Ses i 2 according to claim 3, characterized in that, The said Step 1 includes the following steps: (1) Add lead bromide, cesium bromide, oleic acid, and oleylamine into DMF, stir at high temperature to prepare a precursor solution, and then add ammonia solution; (2) Under vigorous stirring, quickly add the precursor solution to toluene containing TMOS, and then stir vigorously to form core-shell CsPbBr3@SiO2 quantum dots, and collect the final product by centrifugation.

5. The biosensor for detecting sesame protein Ses i 2 according to claim 4, characterized in that, The said Step 2 includes the following steps: Dissolve mPEG-DSPE-COOH in ultradry chlorobenzene; then add CsPbBr3@SiO2 quantum dots into the mPEG-DSPE-COOH solution, stir overnight, and completely evaporate the organic solvent; purify CsPbBr3@SiO2@mPEG-DSPE by centrifugation and redisperse it in deionized water.

6. Application of the biosensor according to any one of claims 2-5 in detecting Ses i 2 in food.

7. A method for preparing a biosensor for detecting sesame protein Ses i 2, characterized in that, The said method includes the following steps: Step 1. Synthesize Ti3C2 MXenes: Add titanium carbide (Ti3C2T x ) MXenes into DMSO, stir at room temperature, centrifuge, redissolve the obtained precipitate with deionized water, repeat the operation, wash the solution to remove DMSO, and then ultrasonically treat the product; Step 2. Synthesize the probe CsPbBr3@SiO2@mPEG-DSPE@Apt: Activate the carboxyl groups on the surface of CsPbB r3 @SiO2@mPEG-DSPE with EDC and NHS, centrifuge, redissolve the precipitate in deionized water, add a sterile aqueous solution of the aptamer to the resulting solution, and bind the aptamer to CsPbBr3@SiO2@mPEG-DSPE; The nucleic acid aptamer is the nucleic acid aptamer S1 with a nucleic acid sequence as shown in SEQ ID NO. 1 or the nucleic acid aptamer S2 with a nucleic acid sequence as shown in SEQ ID NO. 2; Step 3. Prepare the CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 biosensor: Mix the aqueous solution of CsPbBr3@SiO2@mPEG-DSPE@Apt and the aqueous solution of Ti3C2 for hybridization; collect the final CsPbBr3@SiO2@mPEG-DSPE@Apt@Ti3C2 by centrifugation, wash it with PBS buffer solution, and resuspend it to obtain.

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