A polyadenine-coated au np adhesive flare material, and a preparation method and application thereof

Abstract

The application discloses a kind of polyadenine covered AuNPs viscous flare materials and its preparation method and application, belong to the technical field of biosensor.It includes the following steps: (1) polyadenine is mixed with AuNPs solution culture, centrifugal, and polyadenine covered AuNPs are obtained;(2) P1 is mixed with polyadenine covered AuNPs solution, freeze reaction, then F1 is incubated, and the polyadenine covered AuNPs viscous flare material is obtained.The DNA covered AuNPs viscous flare for aflatoxin B1 (AFB1) detection is prepared in the application.The higher hybridization efficiency of poly A covered AuNPs improves the sensitivity of viscous flare for aflatoxin B1 detection, realizes the detection of trace aflatoxin B1 in actual sample, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a polyadenine-coated AuNPs viscous flare material, its preparation method, and its application. Background Technology

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by certain types of Aspergillus flavus and Aspergillus parasiticus fungi. It is widely found in contaminated foods, especially agricultural products such as grains, nuts, seeds, and spices. AFB1 is one of the most potent carcinogens known, with acute toxicity exceeding that of potassium cyanide and arsenic by tens of times. It is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), posing a serious threat to human health. The main danger of AFB1 lies in its severe hepatotoxicity. When a person ingests food containing AFB1, the toxin is absorbed by the intestinal system, particularly preferentially by the duodenum. Unabsorbed portions are excreted through the excretory system. However, even small doses of AFB1 can accumulate in the liver, causing chronic poisoning. Long-term or excessive exposure to AFB1 can trigger a range of health problems. Therefore, the detection of AFB1 in food and feed is crucial. With the development of science and technology, various advanced detection technologies have emerged to improve the accuracy and efficiency of detection.

[0003] Currently, various methods are widely used for the detection of aflatoxin B1 (AFB1), including thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, immunoassay, electrochemical techniques, fluorescence detectors, and colorimetric biosensors. These instrumental analytical methods are known for their high sensitivity, accuracy, and good repeatability. However, these detection devices are typically expensive, resulting in high costs during the detection process, and they also require a high level of technical expertise from operators. These factors significantly limit their application in large-scale sample processing and on-site detection. Therefore, developing a simple, cost-effective, and highly stable AFB1 detection method is of particular importance and practical significance. Summary of the Invention

[0004] The purpose of this invention is to provide a polyadenine-coated AuNPs viscous flare material, its preparation method, and its application, to solve the problems existing in the prior art. This polyadenine-coated AuNPs viscous flare material has many advantages, including low time cost, high specificity, high sensitivity, and the ability to be mass-produced, enabling highly sensitive analysis of AFB1 in the environment or food.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of this invention is a method for preparing a polyadenine-coated AuNPs viscous flare material, comprising the following steps:

[0007] (1) Mix polyadenine with AuNPs solution, culture and centrifuge to obtain polyadenine-coated AuNPs;

[0008] (2) P1 is mixed with a polyadenine-coated AuNPs solution, frozen, and then F1 is added for incubation to obtain the polyadenine-coated AuNPs viscous flare material.

[0009] The second technical solution of the present invention is a polyadenine-coated AuNPs viscous flare material prepared by the preparation method.

[0010] The third technical solution of the present invention is the application of the polyadenine-coated AuNPs viscous flare material in the detection of aflatoxin B1.

[0011] The fourth technical solution of the present invention is a product for detecting aflatoxin B1, comprising the polyadenine-coated AuNPs viscous flare material.

[0012] The fifth technical solution of the present invention is a method for detecting aflatoxin B1, which uses the polyadenine-coated AuNPs adhesive flare material or the product to detect aflatoxin B1.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] (1) This invention synthesizes poly-A-coated AuNPs for subsequent DNA chain functionalization. By pre-coating the surface of AuNPs with poly-A, partial functional chains are blocked, allowing for precise control of the orientation and conformation of surface-modified oligonucleotides and fine adjustment of hybridization ability. At the same time, this method has a relatively short preparation time, stable synthesis, and long shelf life, making it suitable for mass production and application.

[0015] (2) This invention prepares DNA-coated AuNPs sticky flares for the detection of aflatoxin B1 (AFB1). The high hybridization efficiency of polyA-coated AuNPs improves the sensitivity of the sticky flares for the detection of aflatoxin B1, realizing the detection of trace amounts of aflatoxin B1 in actual samples, and has broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the detection principle of aflatoxin B1.

[0018] Figure 2 In the image, (a) is a schematic diagram of P1 functionalization on polyA-covered AuNPs and polyA-uncovered AuNPs. (b) is the surface density of P1 functionalized on polyA-covered and uncovered AuNPs.

[0019] Figure 3 In the diagram, (a) is a schematic diagram of F1 hybridization. (b) shows the hybridization efficiency of F1 on P1-functionalized poly-A covered and uncovered AuNPs. (c) shows the number of P1 connections on poly-A covered AuNPs and the hybridization efficiency of F1.

[0020] Figure 4 In the figures, (a) is a schematic diagram of the functionalization of P1 on the surface of AuNPs covered with polyA at different densities. (b) is the surface density (stripes / AuNP) of P1 and the connection efficiency of P1 on AuNPs covered with A5 at different densities. (c) is the number of P1 connections on AuNPs covered with A5 at different densities and the hybridization efficiency of F1.

[0021] Figure 5 In the diagram, (a) is a schematic diagram of the detection of S1 by a poly-A-covered AuNPs viscous flare. (b) are fluorescence curves of different concentrations of S1 detected by the poly-A-covered AuNPs viscous flare. (c) are the fluorescence intensity curves of the poly-A-covered AuNPs viscous flare versus the S1 concentration. Inset: Linear relationship between fluorescence intensity and S1 concentration.

[0022] Figure 6 Electron micrograph of AuNPs covered by A5. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0029] This invention provides a method for preparing a polyadenine-coated AuNPs viscous flare material, comprising the following steps:

[0030] (1) Mix polyadenine with AuNPs solution, culture and centrifuge to obtain polyadenine-coated AuNPs;

[0031] (2) P1 is mixed with a polyadenine-coated AuNPs solution, frozen, and then F1 is added for incubation to obtain the polyadenine-coated AuNPs viscous flare material.

[0032] In some specific implementations, the AuNPs have a diameter of 13 nm.

[0033] In some specific embodiments, the length of the polyadenine is 5 to 50; the molar ratio of the polyadenine to AuNPs is (100 to 1400):1.

[0034] In some specific embodiments, the polyadenine has a length of 5; the molar ratio of polyadenine to AuNPs is 600:1.

[0035] In some specific implementations, the conditions for the mixed culture include: adding phosphate buffer to a final concentration of 8-12 mM, adding NaCl to a final concentration of 0.1-0.3 M, and incubating overnight at room temperature;

[0036] The centrifugation conditions were: centrifugation at 12000 rpm for 20 min at 4℃.

[0037] In some specific implementations, the nucleotide sequence of P1 is shown in SEQ ID NO.1;

[0038] The nucleotide sequence of F1 is shown in SEQ ID NO.2.

[0039] In some specific implementations, the concentration of P1 is 30 mM, the concentration of F1 is 15 mM, and the concentration of polyadenine-coated AuNPs is 8-12 nM; the volume ratio of P1, F1 to polyadenine-coated AuNPs is 3:0.5:100.

[0040] In some specific implementations, the conditions for the freezing reaction include: freezing at -20°C for 2-4 hours;

[0041] The incubation process specifically involves shaking the incubator at 37°C for 3-5 hours.

[0042] The present invention also provides a polyadenine-coated AuNPs viscous flare material prepared by the preparation method described above.

[0043] This invention also provides the application of the polyadenine-coated AuNPs viscous flare material in the detection of aflatoxin B1.

[0044] This invention also provides a product for detecting aflatoxin B1, comprising the polyadenine-coated AuNPs viscous flare material.

[0045] This invention also provides a method for detecting aflatoxin B1, which uses the polyadenine-coated AuNPs adhesive flare material or the product to detect aflatoxin B1.

[0046] Apt-AFB1 (the aptamer strand of AFB1) and S1 can hybridize to form a hybrid duplex. When the sample to be tested contains AFB1, Apt-AFB1 specifically interacts with AFB1 and then releases S1. Therefore, this invention uses the detection of S1 as a target to determine the content of aflatoxin B1. Thus, the aflatoxin B1 detection system provided by this invention requires a duplex (the aptamer strand of AFB1 and its complementary strand).

[0047] Example 1

[0048] Preparation of polyadenine (polyA)-coated AuNPs

[0049] 50 mL of a 1 mM HAuCl4 solution was heated. The solution was brought to a boil with vigorous stirring, and then 5 mL of a 38.8 mM sodium citrate solution was rapidly added. Heating continued for 20 minutes to obtain AuNPs with a diameter of 13 nm. After cooling to room temperature, 2 mL of a 1% Tween-20 surfactant solution was added to form a protective layer on the AuNP surface and prevent aggregation. The surfactant was adsorbed onto the surface of the gold nanoparticles, providing stability and preventing particle agglomeration.

[0050] Poly(A) molecules of different lengths (A5, A10, A20, A30, and A50) were mixed with AuNPs solution at molar ratios of A5:AuNPs = 1000:1, A10:AuNPs = 500:1, A20:AuNPs = 250:1, A30:AuNPs = 200:1, and A50:AuNPs = 200:1, with the poly(A) concentrations all higher than the actual concentration required to cover the AuNPs solution. The resulting mixture was incubated overnight on a shaker at room temperature. 100 mM phosphate buffer (PB, pH 7.4) was added to a final concentration of 10 mM. 2 M NaCl was added three times at 30-minute intervals to obtain a final concentration of 0.2 M NaCl. After incubation overnight at room temperature, the mixture was centrifuged at 12,000 rpm for 20 minutes at 4°C, and then washed three times with PB (10 mM, pH 7.4). Finally, the precipitate was redispersed in a stock solution (10 mM PB, pH 7.4) to a final concentration of 10 nM, yielding polyA-coated AuNPs (wherein, the electron micrograph of A5-coated AuNPs is shown in Figure 1). Figure 6 (As shown).

[0051] Example 2

[0052] A-covered AuNPs as a test of DNA strand conformational tunability

[0053] 3 μL of FAM-labeled P1 (as shown in SEQ ID NO.1) was added to 100 μL of polyA-coated AuNPs solution, and the solution was frozen at -20°C for 2 h and then thawed at room temperature. 2 μL of 2-mercaptoethanol (diluted 10-fold with deionized water) was added to 100 μL of FAM-labeled P1-modified AuNPs solution. The mixture was then centrifuged at 12000 rpm for 15 min to remove AuNPs. The fluorescence signal of the supernatant was detected using a multi-mode microplate reader. A standard calibration curve was established by diluting known concentrations of P1 with stock solution (10 mM PB, pH 7.4). The average DNA count per AuNP was calculated by dividing the measured P1 concentration by the AuNP concentration.

[0054] like Figure 2 As shown, the results indicate that the number of DNA strands linked to AuNPs covered by poly-A is reduced compared to AuNPs without poly-A coverage, suggesting that poly-A coverage on AuNPs can regulate the conformation of the DNA strand.

[0055] SEQ ID NO. 1: 5'-CACAGAGAGACAACACGTGTTTTTT-SH-3'.

[0056] Example 3

[0057] Preparation of polyA-coated AuNPs viscous flares

[0058] 3 μL of thiol-modified 100 μM P1 was mixed with 100 μL of polyA-coated AuNPs solution and frozen at -20°C for 2 h, then thawed at room temperature. After thawing, the mixture was centrifuged at 12000 rpm for 20 min at 4°C, the supernatant was removed, and the precipitate was redispersed in a stock solution (10 mM PB, pH 7.4) to a final concentration of 10 nM. 0.5 μL of FAM-modified 10 nM F1 (as shown in SEQ ID NO.2) was added to 100 μL of the prepared solution and incubated at 37°C with shaking for 3 h. The mixture was washed three times with 0.1 M PBS, centrifuged at 12000 rpm for 20 min, and resuspended in 100 μL of Tris buffer to obtain polyA-coated AuNPs viscous flares.

[0059] SEQ ID NO. 2: 5'-FAM-CACGTGTTGTCTCTCTGTGTCTCGT-3'.

[0060] Example 4

[0061] Hybridization efficiency detection and optimization

[0062] The prepared viscous flares were added to 5 μL of 1M NaOH solution and incubated for 4 h to allow the bidirectional structure to dissociate. After centrifugation, the supernatant was collected, and the pH was adjusted to neutral by adding an appropriate amount of hydrochloric acid. The fluorescence signal of the supernatant was detected by a microplate reader. The average number of DNA molecules per AuNP was calculated by dividing the measured F1 concentration by the AuNP concentration. The hybridization efficiency was calculated by dividing the number of F1 flares hybridizing with P1 by the total number of P1 molecules assembled on AuNPs.

[0063] like Figure 3 As shown, compared with AuNPs without poly-A coverage, AuNPs with poly-A coverage all exhibited improved hybridization efficiency. Furthermore, among the five different poly-A lengths, AuNPs with A5 coverage showed the highest hybridization efficiency.

[0064] A5 was used to prepare viscous flares of AuNPs covered with polyA. The molar ratio of A5 to AuNPs was changed to 1400:1, 1200:1, 800:1, 600:1, 500:1, 400:1, 200:1 and 100:1 to optimize the hybridization efficiency and determine the optimal ratio of A5-covered AuNPs viscous flares.

[0065] like Figure 4 As shown, the amount of DNA attached to the A5-covered AuNPs gradually increased as the molar ratio of A5 to AuNPs decreased. Furthermore, the hybridization efficiency was highest when the molar ratio of A5 to AuNPs was 600:1; therefore, the sticky flares of poly-A-covered AuNPs prepared under these conditions were subsequently used for further experiments.

[0066] Example 5

[0067] Aflatoxin B1 Detection

[0068] 50 μL of different concentrations of S1 (as shown in SEQ ID NO.3) were added to 50 μL of DNA-covered AuNPs sticky flares and incubated. The concentrations of S1 were 0 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, and 200 nM. The fluorescence spectra were read using a multi-mode microplate reader with an excitation wavelength of 494 nm and an emission wavelength range of 510-600 nm.

[0069] SEQ ID NO. 3: 5'-ACGAGACACAGAGAGACAACACGTG-3'.

[0070] like Figure 5 As shown in Figure b, at 520 nm, the fluorescence intensity of the system increases with increasing aflatoxin B1 concentration. Figure 5 c shows the relationship between fluorescence intensity and aflatoxin B1 concentration, from Figure 5The results show a good linear relationship between the fluorescence intensity of the system and the concentration of AFB1 in the range of 0-75 nM. The detection limit of this method is 1.43 ng / mL.

[0071] In summary, this invention provides a sticky AuNP flare for the detection of aflatoxin B1 (AFB1) based on polyadenine (polyA) coating to regulate DNA conformation and improve hybridization efficiency. This invention focuses on the preparation method and application of the polyA-coated AuNP sticky flare. The sticky flare uses gold nanoparticles as the nanocore, with polyadenine strongly adhering to the surface of the gold nanoparticles through hydrophobic collapse, forming polyA-coated AuNPs. This allows for precise control of the orientation and conformation of surface-modified oligonucleotides and fine-tuning of hybridization ability. Thiol-modified DNA chains are ligated to the polyA-coated AuNPs via a cryo-linking method, and FAM-modified DNA fluorescent chains further hybridize with them, forming the polyA-coated AuNP sticky flare.

[0072] To further optimize the hybridization efficiency of this sticky flare, this invention uses polyaluminum atoms (A5, A10, A20, A30, A50) of different lengths to adhere AuNPs. It was found that the sticky flare of AuNPs covered by A5 exhibited the best hybridization efficiency. Simultaneously, changing the ratio of A5 to AuNPs effectively enhanced the method of using polyaluminum atoms to cover and regulate DNA conformation, thereby improving hybridization efficiency. This invention offers numerous advantages, including low time cost, high specificity, high sensitivity, and the ability to be mass-produced, enabling highly sensitive analysis of AFB1 in the environment or food.

[0073] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of a polyadenine-coated AuNPs viscous flare material in the detection of aflatoxin B1, characterized in that, The preparation method of the polyadenine-coated AuNPs viscous flare material includes the following steps: (1) Mix polyadenine with AuNPs solution, culture and centrifuge to obtain polyadenine-coated AuNPs; (2) P1 is mixed with a polyadenine-coated AuNPs solution, frozen, and then F1 is added for incubation to obtain the polyadenine-coated AuNPs viscous flare material; the concentration of P1 is 30 mM, the concentration of F1 is 15 mM, and the concentration of polyadenine-coated AuNPs is 8-12 nM; the volume ratio of P1, F1 and polyadenine-coated AuNPs is 3:0.5:100; The length of the polyadenine is 5 to 50; the molar ratio of the polyadenine to AuNPs is (100 to 1400): 1; The nucleotide sequence of P1 is shown in SEQ ID NO.1; the nucleotide sequence of F1 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The conditions for the mixed culture include: adding phosphate buffer to a final concentration of 8-12 mM, adding NaCl to a final concentration of 0.1-0.3 M, and incubating overnight at room temperature; The centrifugation conditions were: centrifugation at 12000 rpm for 20 min at 4℃.

3. The application according to claim 1, characterized in that, The conditions for the freezing reaction include: freezing at -20°C for 2-4 hours; The incubation process specifically involves shaking the incubator at 37°C for 3-5 hours.

Images