A method for preparing an RCA-GO aptamer fluorescent biosensor and its application in the simultaneous fluorescence detection of AFB1 and OTA.

Abstract

The application belongs to the field of biosensors and detection technologies, and relates to a preparation method of an RCA-GO aptamer fluorescent biosensor, which comprises the following steps: first, preparing a quantum dot coupled fluorescent probe; then, preparing a circular DNA template CDT; and finally, preparing a rolling circle amplification product RCAP, which is hybridized with the quantum dot coupled fluorescent probe at a volume ratio of 1:1, and GO solution is added to prepare the RCA-GO aptamer fluorescent biosensor after incubation at 37 DEG C. The prepared RCA-GO aptamer fluorescent biosensor is applied to fluorescent detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). The quantum dots have low preparation cost, high coupling activity, and a mature and stable preparation method. The aptamer that is not combined with the target substance is combined with Padlock to form CDT, and a rolling circle amplification reaction is performed, so that the fluorescent signal is greatly enhanced, the detection of AFB1 and OTA is realized, the theoretical basis is mature, and the reliability is good. The fluorescent probe coupled with the quantum dots has stable signal and high sensitivity, and the double-stranded structure (RCAP / QDs-cDNA) formed has good stability and is not prone to false positive signals.

Description

Technical Field

[0001] This invention belongs to the field of biosensor and detection technology, and relates to fluorescent biosensors, particularly to a method for preparing an RCA-GO aptamer fluorescent biosensor and its application in the simultaneous fluorescence detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). Background Technology

[0002] Mycotoxins are a group of secondary metabolites produced by fungi, contaminating approximately 25% of the world's food products. Aflatoxin B1 (AFB1), a metabolite from secondary fungi, is one of the most widely distributed and most toxic mycotoxins in nature. Ochratoxin A (OTA) is also a mycotoxin, primarily produced by *Aspergillus ochraceus*, *Penicillium verrucosum*, and *Aspergillus carbonate*. Identification of mycotoxins in food samples can provide early warning and monitoring of food contamination. However, in some cases, low concentrations or trace amounts pose a significant challenge to mycotoxin detection. For example, in the pre-contamination stages of grains, mycotoxins may not be completely metabolized, producing metabolites at concentrations too low to be detected. Therefore, these issues have created a need for routine quantitative or qualitative detection of mycotoxins in food crops with ultra-high sensitivity, high versatility, and low background. Currently, commonly used detection methods include high-performance liquid chromatography (HPLC), fluorescence, electrochemical assays, and immunoassays. In addition, many signal amplification strategies have been developed to achieve ultra-high sensitivity, such as polymerase chain reaction (PCR), hybridization chain reaction (HCR), rolling circle amplification (RCA), catalytic hairpin amplification (CHA), and many deoxyribozymes involved in this cycle. Their targets are cyclic, and the number can be amplified by several orders of magnitude, with detection limits (LODs) reaching pM or nM.

[0003] Rounding amplification (RCA) is an isothermal amplification technique that uses single-stranded circular DNA molecules as templates to synthesize DNA, producing repeating sequences complementary to the circular template. The product size ranges from hundreds to tens of thousands of bases. A typical RCA reaction requires four components: (1) a circular DNA template; (2) a DNA / RNA initiating strand partially complementary to the RCA template; (3) a DNA / RNA polymerase with strand displacement activity; and (4) deoxyribonucleotide triphosphates (dNTPs). RCA has significant advantages such as simplicity, high sensitivity, high throughput, and high selectivity. Therefore, this amplification strategy has been widely used in the diagnosis of various targets, including nucleic acids, proteins, and cells.

[0004] Based on circular amplification (RCA) and graphene oxide (GO), this invention designs a fluorescent aptamer sensor for the simultaneous detection of AFB1 and OTA. The aptamer is designed as a primer, and Padlock is used as a template for circular amplification. In the absence of the target aptamer, Padlock hybridizes with the aptamer and is circularized by T4 DNA ligase. Subsequently, Phi29 polymerase initiates RCA using this template. As RCA proceeds, a circular amplification product (RCAP) of single-stranded DNA (ssDNA) containing multiple copies of the target aptamer sequence is generated. QDs-cDNA is then used as a fluorescent signal probe. Because the cDNA (complementary DNA) is partially complementary to the aptamer, the cDNA will be complementary to multiple sites on the RCAP, forming an RCAP / QDs-cDNA double-stranded molecule. This double-stranded probe cannot be quenched by GO, which has a stronger affinity for ssDNA. In the presence of the target, they inhibit the formation of circular DNA template (CDT) and ssDNA because the aptamers have a stronger affinity for the target, preventing hybridization with Padlock. Therefore, RCAP can only be synthesized in the absence of the target. Furthermore, at higher target concentrations, more QDs-cDNA probes are adsorbed onto the GO surface, and more fluorescence quenching is observed. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to disclose a fluorescent biosensor capable of simultaneously detecting two mycotoxins (AFB1 and OTA) in food, namely, a method for preparing the RCA-GO aptamer fluorescent biosensor.

[0006] To solve the problems of the prior art, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing an RCA-GO aptamer fluorescent biosensor includes the following steps:

[0008] (1) Preparation of quantum dot-coupled fluorescent probes

[0009] Prepare a solution containing 20 mg·mL using Tris-HCl buffer as the solvent. -1 EDC and 10 mg·mL -1 A mixed solution of NHS;

[0010] Mix 2 mL of g-CNQDs and 200 μL of the above EDC and NHS mixed solution, incubate at room temperature for 1 h to activate surface carboxyl groups, and then add 100 μL of cDNA. 1 Gently stir at 4℃ for 12 hours, centrifuge at 10000 rpm for 10 minutes, and wash three times to obtain the coupled fluorescent probe g-CNQDs-cDNA. 1 ;

[0011] Following the same steps, the coupled fluorescent probe CdTe QDs-cDNA was prepared. 2 ;

[0012] The quantum dot-coupled fluorescent probe (g-CNQDs-cDNA) was prepared. 1 and CdTe QDs-cDNA 2 Disperse in 2 mL Tris-HCl buffer and store at 4 °C;

[0013] Among them, the cDNA 1 and cDNA 2 The concentration of all components was 10 nM; the concentration of the Tris-HCl buffer solution was 10 mM and the pH was 8.0.

[0014] (2) Preparation of circular DNA template (CDT)

[0015] The adapters Apt (Apt 1 and Apt 2) and the rolling ring templates Padlock (Padlock 1 and Padlock 2) were annealed at 95°C for 10 min, then in a water bath at 65°C for 10 min, and then gradually cooled to room temperature.

[0016] The reaction system comprises aptamer Apt, rolling circle template Padlock, T4 ligase, 10×T4 ligase buffer, and 50% PEG 4000 buffer, mixed in a volume ratio of 5–10 μL:5–10 μL:0.5–1 μL:1–2 μL:1–2 μL, preferably 6 μL:6 μL:0.6 μL:1.2 μL:1.2 μL. The mixture is then diluted with deionized water to a total volume of 20 μL, incubated in a 22°C water bath, and the T4 ligase is inactivated at 65°C to obtain the final mixture.

[0017] Among them, Apt 1 is mixed with Padlock 1, and Apt 2 is mixed with Padlock 2;

[0018] The incubation time in a water bath at 22℃ is 2 hours, and the inactivation time at 65℃ is 10 minutes.

[0019] (3) Preparation of RCAP

[0020] Add 10 μL of the mixture from step (2) to the RCA reactant, which contains Phi29 polymerase, 10×Phi29 buffer and 500 μM dNTPs in a volume ratio of 1:3:4. Add deionized water to make the total volume 20 μL. Incubate at 37°C for 10 h and terminate the reaction at 65°C for 10 min to inactivate Phi29 polymerase. The resulting solution is the product of rolling circle amplification (RCAP).

[0021] (4) Fabrication of RCA-GO aptamer fluorescent biosensor

[0022] The product of rolling amplification (RCAP) was hybridized with the fluorescent probe conjugated in step (1) at a volume ratio of 1:1, and the volume was made up to 200 μL with deionized water. The reaction was carried out in a water bath at 37°C, and 5 μL of 200 mg·mL⁻¹ was added. -1 The RCA-GO aptamer fluorescent biosensor was prepared by mixing the GO solution and incubating at 37°C for 30 min.

[0023] The hybridization reaction time of RCAP with the coupled fluorescent probe was 1 hour; the reaction was carried out in a 37°C water bath for 1 hour.

[0024] sequence list

[0025]

[0026] cDNA involved in this invention 1 cDNA 2 The composition of Apt 1, Padlock 1, Apt 2, and Padlock 2 is shown in the sequence listing above. During the creation of the computer-readable sequence listing file, to ensure successful verification, the 5' phosphate group was deleted from both rolling circle template 1 (Padlock 1) and rolling circle template 2 (Padlock 2); complementary DNA... 1 (cDNA 1 ) and complementary DNA 2 (cDNA 2 Please replace “NH2-” with “nh” in the original text.

[0027] The DNA sequence used in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] The second objective of this invention is to disclose the application of the prepared RCA-GO aptamer fluorescent biosensor, namely, for the simultaneous fluorescence detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA).

[0029] The specific fluorescence detection method includes the following steps:

[0030] (A) Containing 3 μL of 0.05, 0.1, 0.5, 1.0, 10, 25, 50 and 100 ng·mL -1 AFB1 and OTA were mixed with a system containing 3 μL of 10 nM aptamer, 3 μL of 10 nM Padlock, 0.3 μL of T4 ligase, 0.6 μL of 10×T4 ligase buffer and 0.6 μL of 50% PEG 4000, respectively, and the volume was made up to 20 μL with deionized water. The mixture was incubated at 22 °C for 2 h to obtain the reaction solution.

[0031] (B) Take 10 μL of reaction solution and mix it with 0.5 μL of Phi29 polymerase, 1.5 μL of 10×Phi29 buffer and 2 μL of 500 μM dNTPs. Add deionized water to make up the volume to 20 μL. Incubate at 37℃ for 10 h to inactivate Phi29 polymerase and obtain the amplification product.

[0032] (C) Hybridize the amplification product with the coupled fluorescent probe at a volume ratio of 1:1, and add deionized water to a final volume of 200 μL. Incubate at 37°C for 10 h, then add 5 μL of 200 mg / mL solution. -1 The RCA-GO aptamer fluorescent biosensor was prepared by incubating the GO solution at 37℃ for 30 min. The fluorescence emission peak of the solution was detected, and standard curves of fluorescence intensity versus corresponding AFB1 and OTA concentrations were plotted.

[0033] (D) Take 3 μL of the extracted sample and mix it with a system containing 3 μL of 10 nM aptamer, 3 μL of 10 nM Padlock, 0.3 μL of T4 ligase, 0.6 μL of 10×T4 ligase buffer, and 0.6 μL of 50% PEG 4000, and bring the volume to 20 μL. Incubate at 22℃ for 2 h. Take 10 μL of the reaction solution and mix it with 0.5 μL of Phi29 polymerase, 1.5 μL of 10×Phi29 buffer, and 2 μL of 500 μM dNTPs. Add deionized water to make the volume 20 μL, incubate at 37℃ for 10 h, and then inactivate the Phi29 polymerase to obtain the amplification product. Hybridize the amplification product with the conjugated fluorescent probe at a volume ratio of 1:1 and bring the volume to 200 μL with deionized water. React in a 37℃ water bath for 10 h, and add 5 μL of 200 mg·mL⁻¹ solution. -1 Mix the GO solution and incubate at 37°C for 30 min. Measure the fluorescence intensity and input it into the standard curve in step (C) to obtain the concentrations of AFB1 and OTA in the sample.

[0034] In a preferred embodiment of the present invention, in step (C), the excitation wavelength of the fluorescence detector is 330 nm.

[0035] In a preferred embodiment of the present invention, in step (D), the sample to be tested is pretreated as follows: 5g of the sample to be tested is weighed, cultured at 37°C and 50% humidity for 20 days, and an acetonitrile-water (85:15, v / v) mixture is added and centrifuged at 10000 rpm for 5 minutes to fully dissolve it. The supernatant is purified by filtration through a 0.22 μm filter membrane, which contains AFB1. Similarly, a methanol-water (70:30, v / v) mixture is added to the sample cultured for 20 days, and centrifuged and dissolved at the same speed and time. The supernatant is purified by filtration through a 0.22 μm filter membrane, which contains OTA. The supernatant is diluted to different concentrations (25, 50, and 100 times) with a buffer solution and stored at 4°C.

[0036] The detection limit for AFB1 in this invention is 0.21 ng·mL. -1 The detection limit for OTA is 0.19 ng·mL. -1 .

[0037] The g-CNQDs quantum dots and CdTe quantum dots described in this invention are either commercially available or self-made. The following discloses a self-made method.

[0038] (1) Preparation of g-CNQDs quantum dots

[0039] Sodium citrate and urea are mixed and ground into powder, then transferred to a polytetrafluoroethylene-lined stainless steel autoclave and heated at 160–200°C for 50–70 min, preferably at 180°C for 60 min. After natural cooling to room temperature, the black solid is dispersed in 30 mL of deionized water and washed by centrifugation at 10,000 rpm to remove impurities. The upper layer solution is aspirated and dialyzed in a 100 Da dialysis bag for 20–30 h, preferably 24 h, to obtain g-CNQDs quantum dots. The molar ratio of sodium citrate to urea is 1–2:3–8, preferably 1:6.

[0040] (2) Preparation of CdTe QDs quantum dots

[0041] Sodium borohydride and tellurium powder are dissolved in deionized water and nitrogen gas is passed through to remove oxygen. The solution is then allowed to stand at 4°C for 2–8 hours, preferably 4 hours, to obtain a sodium telluride solution. The mass ratio of sodium borohydride to tellurium powder is 0.06–0.09:0.01–0.02, preferably 0.084:0.0125.

[0042] Cadmium chloride and 3-mercaptopropionic acid (MPA) were dissolved in distilled water and stirred rapidly under nitrogen atmosphere. Sodium hydroxide solution was added dropwise to adjust the pH to 9-10. Sodium telluride solution was then rapidly transferred and refluxed in a 100°C water bath for 2.5 hours. The mixture was then washed with acetone, and the product was CdTe quantum dots. The reaction ratio of cadmium chloride, 3-mercaptopropionic acid, distilled water, and sodium telluride solution was 0.05-0.06 g: 30-40 μL: 20-30 mL: 0.8-1.5 mL, preferably 0.057 g: 37 μL: 25 mL: 1 mL, and the concentration of sodium hydroxide was 1 M.

[0043] This invention utilizes graphite-phase carbon nitride quantum dots (g-CNQDs) and CdTe quantum dots (CdTe QDs) as energy donors, conjugating them with complementary DNA (cDNA) as signal probes (A), and graphene oxide (GO) as a quencher for specific target recognition. A rolling circle template (Padlock) binds to an aptamer and triggers a rolling circle amplification reaction (RCA) for signal amplification, achieving highly sensitive and simultaneous detection of the target. In the absence of the target, the rolling circle template (Padlock) binds to an aptamer and triggers an RCA to obtain the amplified product (RCAP). The RCAP forms an RCAP / QDs-cDNA duplex with the signal probe (B). This duplex cannot adsorb onto GO, therefore the fluorescence is not quenched. In the presence of AFB1 and OTA, the aptamer recognizes the target and forms a complex. The aptamer cannot bind to the rolling ring template, so no RCA and double strands are formed. The signal probe is adsorbed by GO, which leads to fluorescence quenching, thus achieving highly sensitive detection.

[0044] When the target compounds AFB1 and OTA are present, aptamers are designed as primers, and Padlock is used as a template for circular amplification. The aptamers have a higher affinity for the target compounds, causing them to specifically and preferentially bind to their corresponding aptamers, preventing hybridization with Padlock and thus inhibiting the formation of circular DNA templates (CDT) and circular amplification products (RCAP). Therefore, RCAP can only be synthesized in the absence of the target compounds. Furthermore, when the target concentration is low, there are more RCAPs, and the QDs-cDNA probe hybridizes with the RCAP to form a double-stranded structure, preventing adsorption onto the GO surface, resulting in unquenched fluorescence and high fluorescence intensity. When the target concentration is high, more QDs-cDNA probes are adsorbed onto GO, causing fluorescence quenching and a decrease in fluorescence intensity. By employing fluorescence resonance energy transfer (FRET) and circular amplification strategies, the simultaneous detection of AFB1 and OTA in food can be achieved.

[0045] Beneficial effects

[0046] The quantum dot material used in this invention has low preparation cost, strong coupling activity, and a mature and stable preparation method. The aptamer not bound to the target analyte binds to Padlock to form a CDT, which undergoes a rolling amplification reaction, greatly enhancing the fluorescence signal and enabling the detection of AFB1 and OTA. The theoretical basis is mature and the reliability is high. The fluorescent probe coupled with quantum dots has a stable signal and high sensitivity. The formed double-stranded structure (RCAP / QDs-cDNA) has good stability and is not prone to false positive signals. This method is relatively simple to operate, highly sensitive, and has a low background signal, making it suitable for highly sensitive detection and analysis, and has the advantage of low cost. The disclosed method can be applied to the determination of AFB1 and OTA in real samples. Attached Figure Description

[0047] Figure 1 Schematic diagrams of the fluorescent sensor structure and detection process are shown, where (A) is a quantum dot-coupled fluorescent probe and its adsorption by GO; (B) is the detection process of the fluorescent biosensor in the absence of the target analyte; and (C) is the detection process of the fluorescent biosensor with the target analyte.

[0048] Figure 2 TEM images of quantum dots: (A) are g-CNQDs quantum dots, and (B) are CdTe quantum dots;

[0049] Figure 3 Zeta potential images before and after quantum dot coupling with DNA;

[0050] Figure 4 Fluorescence and UV-Vis spectra of quantum dots before and after DNA coupling: (A) is the fluorescence spectrum of g-CNQDs quantum dots before and after coupling, (B) is the UV-Vis spectrum of g-CNQDs quantum dots before and after coupling, (C) is the fluorescence spectrum of CdTe quantum dots before and after coupling, and (D) is the UV-Vis spectrum of CdTe quantum dots before and after coupling.

[0051] Figure 5 Fluorescence spectra of the prepared sensor when detecting different concentrations of (A) AFB1 and OTA;

[0052] (B) AFB1 standard curve linear curve and (C) OTA standard curve linear curve. Detailed Implementation

[0053] The present invention will be described in detail below with reference to embodiments, so that those skilled in the art can better understand the present invention, but the present invention is not limited to the following embodiments.

[0054] Example 1

[0055] A method for preparing an RCA-GO aptamer fluorescent biosensor includes the following steps:

[0056] 1. Preparation and activation of dual-emission quantum dot materials

[0057] (1) Preparation of g-CNQDs quantum dots: Weigh 0.486g sodium citrate and 0.606g urea, mix and grind into powder, pour into a 20mL polytetrafluoroethylene-lined stainless steel autoclave, heat at 180℃ for 1h and then cool naturally to room temperature; disperse the obtained black solid product in 20mL deionized water, centrifuge at 10000rpm, wash three times to remove impurities; aspirate the upper layer solution and dialyze in a 100Da dialysis bag for 24h to further remove impurities, and obtain g-CNQDs quantum dots;

[0058] (2) Preparation of CdTe quantum dots: 0.084 g sodium borohydride and 0.0125 g tellurium powder were poured into a 10 mL centrifuge tube, 5 mL of deionized water was added, nitrogen gas was passed through for 15 min to remove oxygen, and the solution was allowed to stand at 4 °C for 4 h to obtain sodium telluride solution; 0.057 g cadmium chloride, MPA and distilled water were mixed and stirred rapidly in nitrogen gas, and sodium hydroxide was added dropwise to adjust the pH to 9-10; the sodium telluride solution was quickly transferred to a solution with nitrogen atmosphere, and then refluxed in a constant temperature water bath at 100 °C and washed with acetone to obtain CdTe quantum dots;

[0059] (3) Preparation of quantum dot-coupled DNA probes: EDC and NHS powders were dissolved in Tris-HCl buffer solution (10 mM, pH = 8.0) to obtain 20 mg / mL of each. -1 and 10 mg·mL -1 The concentration was determined by mixing 2 mL of QDs and 200 μL of (EDC+NHS) and incubating at room temperature for 1 h to activate the surface carboxyl groups. 100 μL of cDNA was added to the solution, and the mixture was gently stirred at 4 °C for 12 h. The solution was then centrifuged at 10,000 rpm for 10 min and washed 3 times. The coupled probe was redispersed in 2 mL of Tris-HCl buffer and stored at 4 °C.

[0060] 2. Preparation of RCA reaction

[0061] (1) Preparation of circular DNA template (CDT): First, the aptamer and Padlock were annealed separately and mixed 1:1 in a 65°C water bath for 10 min, and then gradually cooled to room temperature. The entire reaction system contained 6 μL of aptamer, 6 μL of Padlock, 0.6 μL of T4 ligase, 1.2 μL of 10×T4 ligase buffer and 1.2 μL of 50% PEG 4000 buffer, and the total volume was prepared to 20 μL with deionized water. The above mixture was incubated at 22°C for 2 h and reacted at 65°C for 10 min to inactivate T4 ligase.

[0062] (2) Preparation of RCA reaction: After the ligation reaction, the mixture was added to the RCA reaction mixture, which contained 0.5 μL Phi29 polymerase, 1.5 μL 10×Phi29 buffer and 2 μL dNTPs (500 μM), and deionized water was added to make the total volume of the solution 20 μL. Then, the entire mixture was incubated at 37 °C for 10 h, and the reaction was terminated at 65 °C for 10 min to inactivate Phi29 polymerase. The resulting solution was the product of rolling circle amplification (RCAP).

[0063] 3. Fabrication of RCA-GO aptamer fluorescent biosensor

[0064] The rolling amplification product (RCAP) was hybridized to the conjugated fluorescent probe at a volume ratio of 1:1, and the volume was brought up to 200 μL with deionized water. The reaction was carried out in a water bath at 37 °C, and 5 μL of 200 mg / mL solution was added. -1 The RCA-GO aptamer fluorescent biosensor was prepared by mixing the GO solution and incubating at 37°C for 30 min.

[0065] Example 2

[0066] The method for detecting aflatoxin B1 (AFB1) and ochratoxin A (OTA) in food based on the RCA-GO aptamer fluorescent biosensor prepared in Example 1 comprises the following steps:

[0067] All DNA strands were prepared to the required concentration in PBS buffer, heated at 95°C for 10 min, and then allowed to cool naturally at room temperature for subsequent dilution.

[0068] 3 μL of different concentrations of AFB1 and OTA (concentrations of 0.05, 0.1, 0.5, 1, 10, 25, 50, and 100 ng / mL) were added. -1The amplified product was mixed with 3 μL of aptamer (10 nM), 3 μL of Padlock (10 nM), 0.3 μL of T4 ligase, 0.6 μL of 10×T4 ligase buffer, and 0.6 μL of 50% PEG 4000, and the volume was brought to 20 μL. The mixture was incubated at 22 °C for 2 h. 10 μL of the reaction solution was then mixed with Phi29 polymerase and incubated at 37 °C for 10 h to inactivate the Phi29 polymerase. The amplified product was mixed with the GO / QDs-cDNA probe and incubated at 37 °C for 1 h. The fluorescence emission peaks of the solution were measured using a fluorescence spectrometer, and a standard curve of fluorescence intensity versus corresponding AFB1 and OTA concentrations was plotted.

[0069] To evaluate the effectiveness of the established method on real samples, different natural positive samples (wheat flour, mung bean flour, and chickpeas) were selected, and the contents of AFB1 and OTA were detected.

[0070] Table 1. Measurement of AFB1 and OTA in real samples using aptamer fluorescent biosensors.

[0071]

[0072] Note: Relative recovery rate = detected concentration / original concentration

[0073] The test results are shown in Table 1, and all tests achieved good relative recovery rates.

[0074] Compared with other detection methods, this method uses quantum dot materials with low preparation cost, strong coupling activity, and a mature and stable preparation method. The aptamer that has not bound to the target analyte binds to Padlock to form CDT, which undergoes rolling amplification reaction, greatly enhancing the fluorescence signal and enabling the detection of AFB1 and OTA. The theoretical basis is mature and the reliability is good. The fluorescent probe signal after coupling with quantum dots is stable and highly sensitive. The double-stranded structure (RCAP / QDs-cDNA) formed has good stability and is not prone to false positive signals.

[0075] like Figure 1As shown, this invention designs a fluorescent aptamer sensor for the simultaneous detection of AFB1 and OTA based on RCA and GO. The aptamer is designed to work with a primer to generate a rolling circle amplification template (CDT) in conjunction with Padlock. In the absence of the target analyte, Padlock hybridizes with the aptamer and circularizes via T4 ligase. Subsequently, Phi29 polymerase initiates RCA using this template. As the RCA reaction proceeds, a long single-stranded DNA containing multiple copies of the target aptamer sequence, i.e., the rolling circle amplification product (RCAP), is produced. QDs-cDNA is selected as the fluorescent signal probe. Because the cDNA is partially complementary to the aptamer, it will be complementary to multiple sites on the RCAP, forming an RCAP / QDs-cDNA duplex. GO has a stronger affinity for ssDNA; therefore, this duplex cannot be adsorbed by GO, and the fluorescence cannot be quenched. When the target analyte is present, the formation of the circular DNA template (CDT) and ssDNA is inhibited because the aptamer has a stronger affinity for the target analyte, preventing hybridization with Padlock. Therefore, RCAP can only be synthesized in the absence of the target substance. Furthermore, when the target substance concentration is high, more QDs-cDNA probes are adsorbed onto the GO surface, leading to a decrease in fluorescence intensity.

[0076] like Figure 2 As shown in (A), g-CNQDs are well dispersed with an average particle size of 1.98 nm; Figure 2 As shown in (B), the average particle size of CdTe QDs is 2.43 nm.

[0077] Figure 3 The Zeta potentials of g-CNQDs and CdTe QDs under neutral pH conditions were characterized, at -2.023 and -24.167 mV, respectively, indicating that both surfaces are negatively charged. Simultaneously, DNA is composed of a negatively charged phosphate backbone. Therefore, when cDNA is coupled to QDs, the increased size leads to a decrease in Zeta potential emission, with potentials of -10.413 and -31.677 eV, respectively.

[0078] Figure 4 The fluorescence spectrum of (A) shows that the emission wavelength of g-CNQDs is 456 nm, and after being coupled with DNA, a redshift of about 6 nm occurs, resulting in a final emission wavelength of 462 nm. Figure 4 (B) The UV-Vis absorption spectrum shows that g-CNQDs have a characteristic absorption peak at 332 nm, which is attributed to aromatic sp. 2 The π-π* conjugated structure of hybrid carbon. When g-CNQDs were coupled with cDNA1, a characteristic absorption peak of DNA appeared at 257 nm, which initially proved that g-CNQDs were successfully coupled with cDNA1; Figure 4The fluorescence spectrum of (C) shows that the emission wavelength of CdTe QDs is 643 nm, which is redshifted by about 11 nm after coupling, and the final emission wavelength is 654 nm. Figure 4 (D) The UV-Vis absorption spectrum shows that CdTe QDs have a characteristic absorption peak at 581 nm. When CdTe QDs are coupled with cDNA2, the characteristic absorption peak of DNA appears at 262 nm, which proves that the coupling of CdTe QDs with cDNA2 is successful.

[0079] Figure 5 As shown in Figure A, standard solutions containing different concentrations of AFB1 and OTA were co-incubated with aptamers, Padlock, T4 ligase, Phi29 polymerase, and dNTPs. It can be seen that the fluorescence intensity gradually decreases with increasing target concentration. The response curve of the fabricated sensor to AFB1 is shown in Figure A. Figure 5 As shown in B, the linear regression equation is y = -943.5x + 10137.1, and the linear correlation coefficient R0 is... 2 =0.9921, at AFB1 concentrations of 0.05-100 ng / mL -1 At that time, the detection limit was 19 pg·mL. -1 Similarly, the sensor's response curve to OTA is as follows: Figure 5 As shown in C, the linear regression equation is y = -1019.3x + 11835.3, and the linear correlation coefficient R0 is... 2 =0.9906, at OTA concentrations of 0.05-100 ng / mL -1 At that time, the detection limit was 17 pg·mL. -1 (3σ / k).

[0080] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a RCA-GO aptamer fluorescent biosensor, characterized in that, The method comprises the following steps: (1) Preparation of quantum dot coupled fluorescent probe A mixed solution containing 20 mg-mL -1 of EDC and 10 mg-mL -1 of NHS was prepared with Tris-HCl buffer solution as solvent; Mix 2 mL g-CNQDs, 200 μΐ, of the above mixed solution of EDC and NHS, incubate at room temperature for 1 h to activate the surface carboxyl group, add 100 μΐ, of cDNA 1 , 4°C for 12 h, centrifuge at 10000 rpm for 10 min, wash 3 times, and prepare the coupled fluorescent probe g-CNQDs-cDNA 1 ; The conjugated fluorescent probe CdTe QDs-cDNA was prepared by the same procedure as in the previous step 2 ; The prepared quantum dot coupled fluorescent probe is dispersed in 2 mL of Tris-HCl buffer solution and stored at 4°C; (2) Preparation of circular DNA template CDT The aptamers Apt, i.e., Apt 1 and Apt 2, and the padlock templates Padlock, i.e., Padlock 1 and Padlock 2, are separately annealed at 95°C for 10 min, in a water bath at 65°C for 10 min, and gradually cooled to room temperature; The reaction system comprises the aptamers Apt, the padlock templates Padlock, T4 ligase, 10×T4 ligase buffer and 50% PEG 4000 buffer, and is mixed in a volume ratio of 5-10 μL: 5-10 μL: 0.5-1 μL: 1-2 μL: 1-2 μL, and is diluted with deionized water to a total volume of 20 μL, and is incubated in a water bath at 22°C, and the T4 ligase is inactivated at 65°C, to obtain a mixture; (3) Preparation of RCAP 10 μL of the mixture of step (2) is added to the RCA reaction, which contains Phi29 polymerase, 10×Phi29 buffer and 500 μM of dNTPs in a volume ratio of 1:3:4, and deionized water is added to a total volume of 20 μL, and is incubated at 37°C for 10 h, and the reaction is terminated at 65°C for 10 min to inactivate the Phi29 polymerase, and the obtained solution is the rolling circle amplification product RCAP; (4) Preparation of RCA-GO aptamer fluorescent biosensor The product of rolling circle amplification, RCAP, was hybridized with the coupled fluorescent probe prepared in step (1) at a volume ratio of 1:1 and supplemented with deionized water to a volume of 200 µL, and reacted in a 37 °C water bath. 5 µL of 200 mg·mL -1 of GO solution was added and mixed, and incubated at 37 °C for 30 min to prepare the RCA-GO aptamer fluorescent biosensor.

2. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (1), the cDNA 1 and the cDNA 2 were both at a concentration of 10 nM.

3. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (1), the concentration of the Tris-HCl buffer solution is 10 mM, and the pH is 8.

0.

4. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (2), the aptamers Apt, the padlock templates Padlock, T4 ligase, 10×T4 ligase buffer and 50% PEG 4000 buffer are mixed in a volume ratio of 6 μL: 6 μL: 0.6 μL: 1.2 μL: 1.2 μL.

5. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (2), Apt 1 is mixed with Padlock 1, and Apt 2 is mixed with Padlock 2.

6. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (2), the incubation time in a water bath at 22°C is 2 h.

7. The method for preparing RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (2), the inactivation time at 65°C is 10 min. 8.The method for preparing the RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (4), the hybridization reaction time of RCAP and the coupled fluorescent probe is 1 h. 9.The method for preparing the RCA-GO aptamer fluorescent biosensor according to claim 1, characterized in that: In step (4), the reaction is performed in a water bath at 37°C for 1 h.

10. The use of the RCA-GO aptamer fluorescent biosensor prepared by the method according to any one of claims 1-9, characterized in that: It is used for simultaneously detecting aflatoxin B1 and ochratoxin A by fluorescence.

Images