A cascade fret aptamer sensor and method for detection of ochratoxin a

Abstract

The application discloses a cascade FRET aptamer sensor and method for detecting ochratoxin A (OTA). The sensor comprises a donor nanoparticle and an acceptor nanoparticle; the donor nanoparticle is externally modified with an OTA aptamer (Apt); the acceptor nanoparticle is modified with a complementary strand of the OTA aptamer, and the acceptor nanoparticle can accept the fluorescence emitted by the donor nanoparticle through the hybridization of the OTA aptamer and the complementary strand; the acceptor nanoparticle is externally modified with an acceptor fluorescent group, which can accept the fluorescence emitted by the acceptor nanoparticle and realize the fluorescence emission of the acceptor fluorescent group, thereby forming a FRET sensor similar to cascade transmission; the presence of the OTA makes the hybridization of the aptamer and the complementary strand dissociate, so that the fluorescence of the donor nanoparticle cannot be transmitted to the acceptor nanoparticle, and the acceptor fluorescent group is not excited to emit fluorescence. According to the fluorescence emission intensity of the fluorescent group on the acceptor probe at the excitation wavelength of the donor nanoparticle, the target object can be detected.

Description

Technical Field

[0001] This invention belongs to the field of sensor technology, specifically relating to a cascaded FRET aptamer sensor and method for detecting ochratoxin A (OTA). Background Technology

[0002] Ochratoxins are low-molecular-weight secondary metabolites mainly produced by certain fungi in the genera *Aspergillus* and *Penicillium*. There are four compounds: A, B, C, and D. Among them, ochratoxin A (OTA) is the most toxic, widely distributed, has the highest toxin production, causes the most severe contamination of agricultural products, and is most closely related to human health. The molecular formula of OTA is C0. 20 H 18 ClNO6, with a relative molecular mass of 403.8 and a molar extinction coefficient of 5550, is weakly acidic, and has a pK... a With a molecular weight of 4.4–7.5, it is a stable, colorless crystalline compound, readily soluble in polar solvents and slightly soluble in water. Studies have shown that OTA has a cumulative effect in humans and animals, with the kidneys as its primary target organ. It exhibits strong nephrotoxicity and has potential carcinogenic, teratogenic, and mutagenic effects. OTA's toxicity is second only to aflatoxin, and the International Agency for Research on Cancer (IARC) classifies it as a Group B (II) cancer. B Carcinogens.

[0003] Otolostrum (OTA) is widely found in plant-derived products such as food, feed, and traditional Chinese medicine, including grains, coffee beans, grape juice, beer, chocolate, dairy products, and pork products. It can enter the human and animal body through the food chain. OTA contamination in feed is particularly serious. Recent reports indicate that OTA contamination also exists in traditional Chinese medicine. OTA contamination in traditional Chinese medicine is mainly found in roots and rhizomes, seeds and fruits, and aromatic plants. Astragalus, ginseng, turmeric, forsythia, licorice, lotus seeds, and malt have all been found to contain OTA. Contaminated traditional Chinese medicine not only loses its nutritional and medicinal value but also poses various health risks to humans. Given the widespread distribution and toxicity of OTA, many countries and organizations have established strict limits for OTA in food, feed, and traditional Chinese medicine. my country's national standard GB 2761-2017 stipulates that the limit for OTA in cereals and their products, beans and their products, roasted coffee beans, and ground coffee is 5 μg / kg, the limit for OTA in wine is 2 μg / kg, and the limit for OTA in instant coffee is 10 μg / kg.

[0004] Currently, conventional methods for detecting OTA mainly include thin-layer chromatography (TLC), liquid chromatography (LC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), and immunoassay (IA). While these methods can detect OTA, they have certain drawbacks: TLC has poor sensitivity and specificity; LC and GC methods are expensive and require specialized operators; ELISA is easily affected by environmental and reaction conditions, has poor stability, exhibits non-specific reactions, and is prone to false positives; CE has poor reproducibility. Therefore, it is essential to research and establish rapid, accurate, and sensitive new methods for detecting OTA contamination levels.

[0005] An aptamer is a short, single-stranded oligonucleotide (DNA or RNA) sequence (approximately 25-80 bp in length) that is specifically bound to a target molecule and obtained in vitro from a synthetically produced random oligonucleotide library using the Systematic Evolution of Ligands (SELEX) technique. It exhibits activity similar to monoclonal antibodies, specifically recognizing and binding to biomolecules, and is often referred to as a "chemical antibody." Compared to antibodies, aptamers offer better stability and are simpler to prepare. Their targets can include metal ions, small molecules, proteins, and even whole cells. In recent years, aptamer biosensors have achieved significant results in the detection of mycotoxins in food and traditional Chinese medicine.

[0006] Fluorescence resonance energy transfer (FRET) refers to the phenomenon where, in two different fluorophores, if the emission spectrum of one fluorophore (donor) overlaps to some extent with the absorption spectrum of another fluorophore (acceptor), and the distance between the two fluorophores is appropriate (typically between 5 and 10 nm), the fluorescent donor absorbs energy to reach a high electronic state and then transfers the absorbed energy to the acceptor via a non-radiative energy transition, returning itself to the ground state and exciting the acceptor to emit fluorescence. After energy transfer, the donor fluorescence intensity decreases or is completely quenched, while the acceptor emits a characteristic fluorescence stronger than its own or acts as a quencher to form a non-luminescent ground-state complex with the luminescent molecule. FRET efficiency is related to factors such as the degree of overlap between the donor emission spectrum and the acceptor characteristic absorption spectrum, the spatial distance between the donor and acceptor, and the relative orientation of the transition dipoles of the donor and acceptor.

[0007] Fluorescent polymer nanoparticles (NPs) are novel fluorescent probes obtained by embedding small-molecule organic dyes within a polymer core or adsorbing them onto the surface of the polymer core. However, the confinement of fluorophores within the small space of the polymer core leads to aggregation-induced quenching (ACQ). Utilizing bulky hydrophobic counterions to organically align small-molecule organic dyes facilitates energy cascade transfer between dye molecules, avoids ACQ, and allows for the synthesis of dye-loaded nanoparticles with strong fluorescence emission. Simultaneously, the counterions minimize dye leakage, contributing to the structural stability of the polymer nanoparticles. Compared to inorganic nanoparticles, fluorescent polymer nanoparticles exhibit good biocompatibility, good photostability, ease of preparation and surface functionalization, high loading capacity, and good tunability. Using dye-loaded polymer nanoparticles as energy donors and coupling them with DNA modified by acceptor dyes, excitation can be achieved using an internal dye excitation source. Energy can then be transferred to the acceptor dyes on the nanoparticle surface via the FRET effect, thus constructing FRET-based nanoantennas. By using DNA modified with nanoparticles as signal probes for target molecules, and modulating the fluorescence emission of acceptor dyes by the target molecules, a simple fluorescence sensor can be constructed. For efficient FRET (Fluorescence Emission Transmission), the absorption spectrum of the acceptor dye and the emission spectrum of the donor dye must highly overlap. However, due to the narrow Stokes shift of the dye molecules, the fluorescence emission bands of the donor and acceptor dyes are very close, resulting in very similar fluorescence colors that are difficult to distinguish visually, thus exhibiting a high signal-to-noise ratio in imaging applications. Summary of the Invention

[0008] This invention provides a fluorescent aptamer sensor for OTA detection that utilizes the antenna effect of polymer nanoparticles and FRET cascade transfer. The sensor includes a set of polymer nanoparticles with antenna effect, a nucleic acid aptamer and complementary strand for OTA recognition, a receptor fluorescent group, and related reaction reagents. A group of polymer nanoparticles with an antenna effect comprises two types of polymer nanoparticles, each internally encapsulated with a different fluorescent dye—donor nanoparticles and acceptor nanoparticles. The donor nanoparticles are externally modified with an OTA aptamer (Apt), which hybridizes with the complementary DNA strand externally modified on the acceptor nanoparticles, bringing the two nanoparticles sufficiently close. The donor nanoparticles act as energy donors, and the fluorescence emitted at their excitation wavelength can excite the fluorescence of the acceptor nanoparticles. The acceptor nanoparticles are modified with the complementary strand of the OTA aptamer (i.e., cDNA), and through hybridization between the OTA aptamer and the complementary strand, the acceptor nanoparticles can receive the fluorescence emitted by the donor nanoparticles. Furthermore, the acceptor nanoparticles, externally modified with a acceptor fluorophore, can receive the fluorescence emitted by the acceptor nanoparticles and achieve their own fluorescence emission, forming a cascade-like FRET sensor. The presence of OTA causes dissociation of the hybridization between the aptamer and the complementary strand, preventing the fluorescence of the donor nanoparticles from being transmitted to the acceptor nanoparticles, thus preventing the acceptor fluorophore from being excited and resulting in no fluorescence emission. Target analyte detection can be achieved based on the fluorescence emission intensity of the fluorophore on the acceptor probe at the excitation wavelength of the donor nanoparticles.

[0009] The technical solution adopted in this invention is as follows:

[0010] I. A cascaded FRET aptamer sensor for the detection of ochratoxin A

[0011] The cascaded FRET aptamer sensor is obtained by cross-linking donor polymer nanoparticles and acceptor polymer nanoparticles. The donor polymer nanoparticles are modified with OTA aptamers (Apt), and the acceptor polymer nanoparticles are modified with cDNA and fluorescent acceptor groups.

[0012] II. A method for preparing a cascaded FRET aptamer sensor for ochratoxin A detection

[0013] Includes the following steps:

[0014] Step 1) Synthesize donor polymer nanoparticles and acceptor polymer nanoparticles separately, with different fluorescent dyes loaded on the donor polymer nanoparticles and acceptor polymer nanoparticles;

[0015] Step 2) Modify the donor polymer nanoparticles with OTA aptamers (Apt), and modify the acceptor polymer nanoparticles with the complementary strand of the OTA aptamer (i.e., cDNA) and the acceptor fluorescent group.

[0016] Step 3) The donor polymer nanoparticles and acceptor polymer nanoparticles from Step 2 are cross-linked through DNA hybridization to obtain a cascaded FRET aptamer sensor for the detection of ochratoxin A.

[0017] In step (1):

[0018] Both the donor polymer nanoparticles and the acceptor polymer nanoparticles have shells of poly(methyl methacrylate-co-methacrylic acid) PMMA-AspN3 modified with azide / carboxylic acid bifunctional groups.

[0019] The dye encapsulated inside the donor polymer nanoparticles is a complex of a green cationic dye with an emission wavelength of 500-540 nm and a counterion; the dye encapsulated inside the acceptor polymer nanoparticles is a complex of a yellow cationic dye with an excitation wavelength of 500-540 nm and an emission wavelength of 545-600 nm and a counterion.

[0020] The green cationic dye includes one of the green fluorescent dyes such as Rhodamine 110, Rhodamine Green, and Rhodamine 123; the yellow cationic dye includes one of Rhodamine R18 and TAMRA; the counterion includes tetraphenylborate (TPB) and its fluorinated analogs, such as F5-TPB and F12-TPB.

[0021] The specific method for synthesizing the donor polymer nanoparticles or acceptor polymer nanoparticles is as follows:

[0022] PMMA-AspN3 was dissolved in acetonitrile. A complex salt of cationic dye and counterion was added to the PMMA-AspN3 acetonitrile solution. After shaking at 500-1500 rpm, synthesis buffer was quickly added. Acetonitrile was removed by ultrafiltration. The solution was washed three times with synthesis buffer. Finally, an appropriate amount of synthesis buffer was added to the ultrafiltration tube and sonicated for 1-5 min. The solution was then reconstituted to obtain dye-loaded donor polymer nanoparticles or acceptor polymer nanoparticles.

[0023] The concentration of PMMA-AspN3 dissolved in acetonitrile is 1–5 mg / mL; the concentration of the complex salt dissolved in acetonitrile is 0.5–1.5 μg / μL; the composition of the synthesis buffer includes 10–50 mM PBS or Tris (pH 7.0–8.0), and contains 10–200 mM NaCl, 0–30 mM MgCl2, and 0–30 mM CaCl2.

[0024] The specific method for synthesizing the complex salt of the cationic dye and the counterion is as follows:

[0025] The cationic dye and the counterion were dissolved separately in acetonitrile to obtain two acetonitrile solutions containing the cationic dye and the counterion, respectively. The two acetonitrile solutions were mixed and the acetonitrile was removed by nitrogen blowing to obtain the crude product. The crude product was purified by column chromatography and dried to obtain a complex salt of cationic dye and counterion. The obtained product was dissolved in acetonitrile and stored at 4°C.

[0026] The eluent used in column chromatography purification of the crude product is a mixture of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 98:2; the molar ratio of cationic dye to counterion is 1:20 to 1:5.

[0027] Step (2) specifically involves:

[0028] The method for modifying OTA aptamers on donor polymer nanoparticles is as follows: after mixing ssDNA with donor polymer nanoparticles, react without stirring and in the dark at 25-50℃ for 5-24 hours, and then cool to room temperature;

[0029] The method for modifying cDNA and receptor fluorescent groups on receptor polymer nanoparticles is as follows: after mixing ssDNA, red fluorescent dye and receptor polymer nanoparticles, react without stirring and in the dark at 25-50℃ for 5-24h, and then cool to room temperature.

[0030] The ssDNA modified on the donor polymer nanoparticles includes blocking DNA and OTA aptamers, wherein the ratio of blocking DNA to OTA aptamers is between 120:1 and 60:1; the ssDNA modified on the acceptor polymer nanoparticles includes blocking DNA and cDNA, wherein the ratio of blocking DNA to cDNA is between 60:1 and 20:3.

[0031] The receptor fluorescent groups modified on the receptor polymer nanoparticles are red fluorescent dyes with fluorescence emission >610nm, including Cy5-DBCO, ATTO647N-DBCO, ATTO665-DBCO or Cy5.5-DBCO, and the amount of modified receptor fluorescent groups is 1-60 per receptor polymer nanoparticle.

[0032] The length of the blocked DNA ranges from 10 to 30 bases, and the sequence is composed of any ssDNA that does not react with the OTA aptamer, including polyT or polyA.

[0033] The ssDNA is modified with a DBCO group; the DBCO group is modified at the 5' end of the OTA aptamer, and the OTA aptamer sequence includes: 5'-DBCO-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3', 5'-DBCO-AGCCTCGTCTGTTCTCCCGGCAGTGTGGGCGAATCTATGCGTACCGTTCGATATCGTGGGGAAGACAAGCAGACGT-3' or 5'-DBCO-GGCAGTGTGGGCGAATCTATGCGTACCGTTCGATATCGTG-3';

[0034] The cDNA is a short single-stranded DNA that stably hybridizes with the OTA aptamer. The sequence is a single-stranded nucleotide of 6-16 bases complementary to any position at the 5' end, 3' end, or middle of the OTA aptamer, and the length of the non-complementary region on the side closest to the polymer nanoparticle does not exceed 15 bases. Specifically, it includes: 5'-CACCCACAGGCTAG-DBCO-3', 5'-AGAACAGAGCTCCGA-DBCO-3', or 5'-TTCGCCCACTGACGG-DBCO-3'.

[0035] Polymer nanoparticles are either polymer nanoparticles or acceptor polymer nanoparticles;

[0036] In step (3), the method for crosslinking the acceptor polymer nanoparticles with the donor polymer nanoparticles is as follows:

[0037] The acceptor polymer nanoparticles and donor polymer nanoparticles were mixed in a ratio of 1:1 to 1:3, denatured, slowly cooled to room temperature, and allowed to stand in the dark for 2 hours. After washing five times with nanoantenna washing buffer, the product was dispersed in 1 mL of buffer and stored at 4 °C.

[0038] The denaturation temperature is 70–100°C, and the denaturation time is 3–10 min.

[0039] The nanoantenna washing buffer comprises 10-50 mM PBS or Tris (pH 7.0-9.0) and contains 10-200 mM NaCl, 0-30 mM MgCl2, 0-30 mM CaCl2, and 0-20 mM KCl.

[0040] III. A method for detecting ochratoxin A using a cascaded FRET aptamer sensor.

[0041] Step A: React the cascaded FRET aptamer sensor with the sample solution to be tested;

[0042] Step B: Measure the fluorescence spectrum and, in conjunction with visual detection, determine the results using the following method:

[0043] When the emission peak of the acceptor dye does not change, it indicates that OTA is not present in the sample solution; when the emission peak of the acceptor dye decreases, it indicates that OTA is present in the sample.

[0044] Step A specifically involves:

[0045] The polymer nanoantenna was diluted to a suitable concentration using a binding buffer, added to the sample solution to be tested, and reacted at room temperature in the dark for 5-120 min before measuring the fluorescence spectrum.

[0046] The binding buffer comprises 10-50 mM Tris-HCl buffer (pH 8.5), containing 0-120 mM NaCl, 0-20 mM KCl, and 0-30 mM CaCl2; the diluted concentration of the polymer nanoantenna is 10-10... 4 pM;

[0047] In step B, the fluorescence spectrum measurement conditions are: excitation wavelength 300-480nm, emission wavelength 610-750nm.

[0048] The detection range of the OTA includes, but is not limited to, 0.01-500 ppb.

[0049] The beneficial effects of this invention are:

[0050] 1. High sensitivity

[0051] This invention utilizes polymer nanoparticles encapsulating a large number of fluorescent dyes and using counterions to arrange the fluorescent dyes in a close and orderly manner, thereby achieving extremely rapid energy transfer between the dyes. This allows the energy of a large number of fluorescent dyes to be accepted by a very small number of acceptors on the outside of the polymer nanoparticles. Under the action of low-energy excitation light, the external fluorescent dyes can emit strong fluorescence, thus exhibiting extremely high response sensitivity to the target.

[0052] 2. Results are visible

[0053] By encapsulating different fluorescent dyes within two polymer nanoparticles, fluorescence is excited using the excitation wavelength of the donor fluorescent polymer nanoparticle, and energy is transferred to the acceptor fluorescent polymer nanoparticle, causing its external acceptor dye to emit fluorescence, thus achieving cascaded fluorescence transfer. When no target is present, the fluorescence emitted by the nanoparticles reflects the fluorescence emission of the external fluorescent acceptor; conversely, when a target is present, the fluorescence emitted by the nanoparticles reflects the fluorescence of the donor fluorescent polymer nanoparticle. This avoids the problem of excessive overlap in the emission spectra of the donor and acceptor dyes, resulting in unclear color changes, which is common in traditional FRET sensors, facilitating visual interpretation of the results. Attached Figure Description

[0054] Figure 1 The thin-layer chromatography results correspond to the Rhodamine R18 and counterion F5-TPB complex salt in Example 2. The left side shows Rhodamine R18, and the right side shows Rhodamine R18 / F5-TPB.

[0055] Figure 2 The absorption spectrum of the Rhodamine R18 and counterion F5-TPB composite salt in acetonitrile in Example 2.

[0056] Figure 3 a) Transmission electron microscopy results of dye-loaded polymer nanoparticles in Example 3, with the sample negatively stained with 2% phosphotungstic acid for 10 s; b) Particle size distribution of dye-loaded polymer nanoparticles in Example 3.

[0057] Figure 4 The fluorescence spectra of the nanoantenna aptamer sensor before and after the addition of OTA in Example 3 were obtained. The excitation wavelength Ex was 480 nm, and the emission at wavelengths of 610-700 nm was measured.

[0058] Figure 5 In Example 5, a nano-antenna aptamer sensor was used to detect the fluorescence spectra of OTA at different concentrations (10, 20, 80, 100, 150, 200, 400 ppb), with an excitation wavelength Ex of 480 nm, and the emission at wavelengths of 610-700 nm was measured.

[0059] Figure 6 This is a visual schematic diagram illustrating the detection of ochratoxin A according to the present invention. Detailed Implementation

[0060] The following will describe in more detail through specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0061] Example 1: Preparation of dye-supported donor polymer nanoparticles

[0062] Weigh 10 μmol F5-TPB and dissolve it in 1.6 mL acetonitrile; dissolve 5 μmol Rhodamine 110 in 0.4 mL acetonitrile and mix them rapidly. Dry the acetonitrile under nitrogen. Dissolve the product with an appropriate amount of eluent, purify the crude product by column chromatography (eluent: dichloromethane: methanol = 98:2), dry, and store the product in acetonitrile.

[0063] 400 μg of PMMA-AspN3 was weighed and dissolved in 100 μL of 0.6 μg / μL Rhodamine 110 / F5-TPB acetonitrile solution. The solution was sonicated for 5 min and then rapidly added to 900 μL of synthesis buffer under oscillation at 800 rpm. The acetonitrile was dried by nitrogen blowing to obtain polymer fluorescent nanoparticles (110 / F5-TPB / PMMA-AspN3) loaded with Rhodamine 110 / F5-TPB.

[0064] Example 2: Preparation of dye-loaded acceptor polymer nanoparticles

[0065] Weigh 10 μmol F5-TPB and dissolve it in 1.6 mL acetonitrile; dissolve 5 μmol Rhodamine R18 in 0.4 mL acetonitrile and mix them rapidly. Dry the acetonitrile under nitrogen. Dissolve the product with an appropriate amount of eluent, purify the crude product by column chromatography (eluent: dichloromethane: methanol = 98:2), dry, and store the product in acetonitrile.

[0066] like Figure 1 As shown, Rhodamine R18 did not develop on thin-layer chromatography, while the R18 / F5-TPB composite dye migrated over a longer distance, and its UV absorption spectrum in acetonitrile is as follows. Figure 2 As shown, an absorption peak appears at 555 nm.

[0067] 400 μg of PMMA-AspN3 was weighed and dissolved in 100 μL of 0.6 μg / μL R18 / F5-TPB acetonitrile solution. The solution was sonicated for 5 min and then rapidly added to 900 μL of synthesis buffer under oscillation at 800 rpm. The acetonitrile was dried by nitrogen blowing to obtain polymer fluorescent nanoparticles (R18 / F5-TPB / PMMA-AspN3) loaded with Rhodamine R18 / F5-TPB.

[0068] Example 3: DNA Modification on Polymer Nanoparticles

[0069] Add 60 μM polyA-DBCO and 1 μM Apt-DBCO to 110 / F5-TPB / PMMA-AspN3, mix, and react at 30°C without stirring in the dark. Then cool to room temperature.

[0070] Add 1 μM cDNA-DBCO, 60 μM polyA-DBCO, and 3 μM Cy5-DBCO to R18 / F5-TPB / PMMA-AspN3, mix, and react at 30°C without stirring in the dark. Then allow to cool to room temperature.

[0071] Example 4: Crosslinking of donor nanoparticles and acceptor nanoparticles

[0072] The donor polymer nanoparticles modified with Apt and the acceptor polymer nanoparticles modified with cDNA were mixed, incubated at 95°C for 3 min, cooled to room temperature, and hybridized in the dark. The mixture was then diluted to 500 μL with 30 mM PBS and purified by centrifugation at 3000 rpm for 2 min using an ultrafiltration centrifuge tube (Amicon, 0.5 mL, 100 kDa). This process was repeated 5 times to remove unreacted DNA. The product was dissolved in 1 mL of binding buffer and stored at 4°C.

[0073] Transmission electron microscopy results and particle size distribution of dye-loaded polymer nanoparticles are as follows: Figure 3 As shown, the average particle size is approximately 25 nm. After cross-linking, the nanoparticles successfully constructed a cascaded FRET fluorescent aptamer biosensor, such as... Figure 4 As shown, the emission peak of the receptor dye decreased significantly after the addition of Target.

[0074] Example 5: OTA Detection Based on FRET Fluorescence Changes

[0075] like Figure 6 As shown, it includes the following steps:

[0076] a. Prepare OTA gradient concentration standard solutions with concentrations of 1, 2, 8, 10, 15, 20, and 40 ppm using binding buffer.

[0077] b. Take 10 μL of the nanoantenna from Example 4 into 80 μL of binding buffer, and then add 10 μL of the OTA gradient concentration solution prepared in step a. The final reaction volume is 100 μL. After incubation in the dark at room temperature for 1 h, measure the fluorescence spectrum. Scan the fluorescence spectrum in the wavelength range of 610-750 nm at an excitation wavelength of 480 nm.

[0078] c. Results are shown Figure 5 , Figure 5 As can be seen, the fluorescence emission peak of Cy5 decreases with increasing OTA concentration.

[0079] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All changes, modifications, combinations, substitutions, and simplifications made under the spirit and principle of the present invention should be considered equivalent substitutions and should be covered within the scope of protection of the present invention.

Claims

1. A cascaded FRET aptamer sensor for the detection of ochratoxin A, characterized in that, The cascaded FRET aptamer sensor is obtained by cross-linking donor polymer nanoparticles and acceptor polymer nanoparticles. The donor polymer nanoparticles are modified with OTA aptamers, and the acceptor polymer nanoparticles are modified with cDNA and fluorescent acceptor groups. The fabrication method of the cascaded FRET aptamer sensor includes the following steps: Step 1) Synthesize donor polymer nanoparticles and acceptor polymer nanoparticles separately, with different fluorescent dyes loaded on the donor polymer nanoparticles and acceptor polymer nanoparticles; Step 2) Modify OTA aptamers on donor polymer nanoparticles and modify cDNA and receptor fluorescent groups on acceptor polymer nanoparticles. Step 3) The donor polymer nanoparticles and acceptor polymer nanoparticles from Step 2 are cross-linked through DNA hybridization to obtain a cascaded FRET aptamer sensor for the detection of ochratoxin A. In step (1): Both the donor polymer nanoparticles and the acceptor polymer nanoparticles have shells of PMMA-AspN3 modified with azide / carboxylic acid bifunctional groups. The dye encapsulated inside the donor polymer nanoparticles is a complex of a green cationic dye with an emission wavelength of 500-540 nm and a counterion; the dye encapsulated inside the acceptor polymer nanoparticles is a complex of a yellow cationic dye with an excitation wavelength of 500-540 nm and an emission wavelength of 545-600 nm and a counterion. The green cationic dye includes one of Rhodamine 110, Rhodamine Green, and Rhodamine 123; the yellow cationic dye includes one of Rhodamine R18 and TAMRA; the counterion includes tetraphenylboronic acid ester and its fluorinated analogs; the fluorinated analogs of tetraphenylboronic acid ester include F5-TPB and F12-TPB; Step (2) specifically involves: The method for modifying OTA aptamers on donor polymer nanoparticles is as follows: after mixing ssDNA with donor polymer nanoparticles, react without stirring and in the dark at 25-50℃ for 5-24 hours, and then cool to room temperature; The method for modifying cDNA and receptor fluorescent groups on receptor polymer nanoparticles is as follows: ssDNA modified with DBCO groups, red fluorescent dye and receptor polymer nanoparticles are mixed and reacted without stirring and in the dark at 25-50℃ for 5-24 hours, and then cooled to room temperature. The ssDNA modified on the donor polymer nanoparticles includes blocking DNA and OTA aptamers, wherein the ratio of blocking DNA to OTA aptamers is between 120:1 and 60:

1. The ssDNA modified on the receptor polymer nanoparticles includes blocking DNA and cDNA, wherein the ratio of blocking DNA to cDNA is between 60:1 and 20:

3. The receptor fluorescent groups modified on the receptor polymer nanoparticles are red fluorescent dyes with fluorescence emission >610nm, including Cy5-DBCO, ATTO647N-DBCO, ATTO665-DBCO or Cy5.5-DBCO, and the amount of modified receptor fluorescent groups is 1-60 per receptor polymer nanoparticle.

2. The cascaded FRET aptamer sensor for the detection of ochratoxin A according to claim 1, characterized in that, The specific method for synthesizing the donor polymer nanoparticles or acceptor polymer nanoparticles is as follows: PMMA-AspN3 was dissolved in acetonitrile. A complex salt of cationic dye and counterion was added to the PMMA-AspN3 acetonitrile solution. After shaking at 500-1500 rpm, synthesis buffer was quickly added. Acetonitrile was removed by ultrafiltration. The solution was washed three times with synthesis buffer. Finally, synthesis buffer was added to the ultrafiltration tube and sonicated for 1-5 min. The solution was then reconstituted to obtain dye-loaded donor polymer nanoparticles or acceptor polymer nanoparticles. The concentration of PMMA-AspN3 dissolved in acetonitrile is 1–5 mg / mL; The concentration of the composite salt dissolved in acetonitrile is 0.5–1.5 μg / μL; The synthesis buffer comprises 10-50 mM PBS or Tris and contains 10-200 mM NaCl, 0-30 mM MgCl2, and 0-30 mM CaCl2.

3. The cascaded FRET aptamer sensor for the detection of ochratoxin A according to claim 1, characterized in that, The length of the blocked DNA ranges from 10 to 30 bases, and the sequence is composed of any ssDNA that does not react with the OTA aptamer, including polyT or polyA. The ssDNA is modified with a DBCO group; a DBCO group is modified at the 5' end of the OTA aptamer, and the OTA aptamer sequence includes: 5'-DBCO-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3', 5'-DBCO-AGCCTCGTCTGTTCTCTCCCGGCAGTGTGGGCGAATCTATGCGTAC CGTTCGATATCGTGGGGAAGACAAGCAGACGT-3' or 5'-DBCO-GGCAGTGTGGGCGAATCTATGCGTACCGTTCGATATCGTG-3'; The cDNA is a short single-stranded DNA that stably hybridizes with the OTA aptamer. The sequence is a single-stranded nucleotide of 6-16 bases complementary to any position at the 5' end, 3' end, or middle of the OTA aptamer, and the length of the non-complementary region on the side closest to the polymer nanoparticle does not exceed 15 bases.

4. A cascaded FRET aptamer sensor for the detection of ochratoxin A according to claim 1, characterized in that, In step (3), the method for crosslinking the acceptor polymer nanoparticles with the donor polymer nanoparticles is as follows: The acceptor polymer nanoparticles and donor polymer nanoparticles were mixed in a ratio of 1:1 to 1:3, denatured, slowly cooled to room temperature, and allowed to stand in the dark for 2 hours. After washing five times with nanoantenna washing buffer, the product was dispersed in 1 mL of buffer and stored at 4 °C. The denaturation temperature is 70–100°C, and the denaturation time is 3–10 min. The nanoantenna washing buffer comprises 10-50 mM PBS or Tris and contains 10-200 mM NaCl, 0-30 mM MgCl2, 0-30 mM CaCl2, and 0-20 mM KCl.

5. A method for detecting ochratoxin A using the cascaded FRET aptamer sensor as described in any one of claims 1 to 4, characterized in that, Step A: React the cascaded FRET aptamer sensor with the sample solution to be tested; Step B: Measure the fluorescence spectrum and, in conjunction with visual detection, determine the results using the following method: When the emission peak of the acceptor dye does not change, it indicates that OTA is not present in the sample solution. When the emission peak of the receptor dye decreases, it indicates the presence of OTA in the sample being tested.

6. The method for detecting ochratoxin A according to claim 5, characterized in that, Step A specifically involves: The polymer nanoantenna was diluted to a suitable concentration using a binding buffer, added to the sample solution to be tested, and reacted at room temperature in the dark for 5-120 min before measuring the fluorescence spectrum. The binding buffer consists of 10-50 mM Tris-HCl buffer, which contains 0-120 mM NaCl, 0-20 mM KCl, and 0-30 mM CaCl2. The diluted concentration of the polymer nanoantenna is 10-10. 4 pM.

7. The method for detecting ochratoxin A according to claim 5, characterized in that, In step B, the fluorescence spectrum measurement conditions are: excitation wavelength 300-480nm, emission wavelength 610-750nm.

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