Polyadenine-covered AuNPs viscous flare material as well as preparation method and application thereof

Abstract

The invention discloses a polyadenine-covered AuNPs viscous flare material as well as a preparation method and application thereof, and belongs to the technical field of biosensors. Comprising the following steps: (1) carrying out mixed culture on polyadenine and an AuNPs solution, and centrifuging to obtain AuNPs covered by polyadenine; and (2) mixing the P1 with a polyadenine-covered AuNPs solution, carrying out a freezing reaction, and then adding the F1 for incubation to obtain the polyadenine-covered AuNPs viscous flare material. According to the invention, the AuNPs viscous flare covered by DNA (Deoxyribonucleic Acid) is prepared and is used for detecting aflatoxin B1 (AFB1). The high hybridization efficiency of the AuNPs covered by the poly A improves the detection sensitivity of the viscous flare to the aflatoxin B1, realizes the detection of trace aflatoxin B1 in an actual sample, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a polyadenine-covered AuNPs viscous flare material and a preparation method and application thereof. Background Art

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by certain types of fungi such as Aspergillus flavus and Aspergillus parasiticus. It is widely present in contaminated foods, especially agricultural products such as grains, nuts, seeds and spices. AFB1 is one of the most carcinogenic known chemicals. Its acute toxicity is even dozens of times higher than that of potassium cyanide and arsenic. It is classified as a Class 1 carcinogen by the International Agency for Research on Cancer (IARC), posing a serious threat to human health. The main hazard of AFB1 lies in its strong liver toxicity. When the human body ingests food containing AFB1, the toxin is absorbed by the intestinal system, especially by the duodenum, and the unabsorbed part is excreted from the body 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 cause a series of health problems. Therefore, the detection of AFB1 in food and feed is crucial. With the development of science and technology, a variety of advanced detection technologies have emerged to improve the accuracy and efficiency of detection.

[0003] At present, there are many methods widely used for the detection of aflatoxin B1 (AFB1), including thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry, immunoassay technology, electrochemical technology, fluorescence detectors and colorimetric biosensors. These instrumental analysis methods are known for their high sensitivity, accuracy and good repeatability. However, this type of detection equipment is usually expensive, the cost of the detection process is high, and the technical level of the operator is relatively strict. These factors significantly limit its application in large-scale sample processing and on-site detection. Therefore, it is particularly important and of practical significance to develop an AFB1 detection method that is simple, low-cost and highly stable. Summary of the invention

[0004] The purpose of the present invention is to provide a polyadenine-covered AuNPs sticky flare material and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The polyadenine-covered AuNPs sticky flare material has many advantages such as low time cost, strong specificity, high sensitivity and mass production, and can realize highly sensitive analysis of AFB1 in the environment or food.

[0005] To achieve the above object, the present invention provides the following solutions:

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

[0007] (1) Mixing polyadenine and AuNPs solution, incubating, and centrifuging to obtain polyadenine-covered AuNPs;

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

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

[0010] The third technical solution of the present invention is the application of the polyadenine-covered AuNPs sticky 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-covered AuNPs sticky flare material.

[0012] A fifth technical solution of the present invention is a method for detecting aflatoxin B1, which uses the polyadenine-covered AuNPs sticky 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) The present invention synthesizes a poly-A-covered AuNPs for subsequent functionalization of DNA chains. By using poly-A to cover the surface of AuNPs in advance, by shielding part of the functional chain, it is possible to accurately control the orientation and conformation of the surface-modified oligonucleotides and finely adjust the hybridization ability; at the same time, the method has a relatively short preparation time, stable synthesis, and long storage time, and is suitable for mass production and application.

[0015] (2) The present invention prepares DNA-covered AuNPs sticky flares for the detection of aflatoxin B1 (AFB1). The higher hybridization efficiency of poly A-covered AuNPs improves the sensitivity of sticky flares for the detection of aflatoxin B1, realizes the detection of trace amounts of aflatoxin B1 in actual samples, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0018] Figure 2 (a) Schematic diagram of P1 functionalization on poly-A-covered AuNPs and AuNPs not covered with poly-A. (b) Surface density of P1 functionalized on poly-A-covered and not covered AuNPs.

[0019] Figure 3 (a) Schematic diagram of F1 hybridization. (b) Hybridization efficiency of F1 on P1-functionalized polyA-covered and uncovered AuNPs. (c) Number of P1 connections on polyA-covered AuNPs and hybridization efficiency of F1.

[0020] Figure 4 (a) Schematic diagram of the functionalization of P1 on the surface of AuNPs covered with poly A at different densities. (b) The surface density (strips / AuNP) of P1 and the connection efficiency of P1 on AuNPs covered with A5 at different densities. (c) The number of connections of P1 on AuNPs covered with A5 at different densities and the hybridization efficiency of F1.

[0021] Figure 5 (a) Schematic diagram of the detection of S1 by the sticky flare of AuNPs covered with poly A. (b) Fluorescence curve of the sticky flare of AuNPs covered with poly A for detecting different concentrations of S1. (c) Relationship between the fluorescence intensity of the sticky flare of AuNPs covered with poly A and the concentration of S1. Inset: Linear relationship between fluorescence intensity and S1 concentration.

[0022] Figure 6 This is the electron microscopy image of AuNPs covered with A5. DETAILED DESCRIPTION

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

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0028] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0029] The embodiment of the present invention provides a method for preparing a polyadenine-covered AuNPs sticky flare material, comprising the following steps:

[0030] (1) Mixing polyadenine and AuNPs solution, incubating, and centrifuging to obtain polyadenine-covered AuNPs;

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

[0032] In some specific embodiments, the diameter of the AuNPs is 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 length of the polyadenine is 5; the molar ratio of the polyadenine to AuNPs is 600:1

[0035] In some specific embodiments, the mixed culture conditions 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 culturing overnight at room temperature;

[0036] The centrifugation conditions are: centrifugation at 12000 rpm for 20 min at 4°C.

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

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

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

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

[0041] The incubation is specifically shaking incubation at 37° C. for 3-5 hours.

[0042] The embodiment of the present invention also provides a polyadenine-covered AuNPs sticky flare material prepared by the preparation method.

[0043] The embodiment of the present invention also provides the application of the polyadenine-covered AuNPs sticky flare material in the detection of aflatoxin B1.

[0044] An embodiment of the present invention also provides a product for detecting aflatoxin B1, comprising the polyadenine-covered AuNPs sticky flare material.

[0045] The embodiment of the present invention also provides a method for detecting aflatoxin B1, which uses the polyadenine-covered AuNPs sticky flare material or the product to detect aflatoxin B1.

[0046] Apt-AFB1 (the aptamer chain of AFB1) and S1 can hybridize to form a hybrid duplex. When AFB1 is contained in the sample to be detected, Apt-AFB1 will specifically interact with AFB1 and then release S1. Therefore, the present invention detects S1 as a target to determine the content of aflatoxin B1. Therefore, the detection system of aflatoxin B1 provided by the present invention requires a double chain (the aptamer chain of AFB1 and its complementary chain).

[0047] Example 1

[0048] Preparation of polyadenine (poly A)-capped AuNPs

[0049] Heat 50 mL of HAuCl4 solution with a concentration of 1 mM. Under vigorous stirring, bring the solution to a boil, then quickly add 5 mL of sodium citrate with a concentration of 38.8 mM, and continue heating for 20 minutes to obtain AuNPs with a diameter of 13 nm. After cooling to room temperature, 2 mL of 1% surfactant Tween-20 solution was added to form a protective layer on the surface of AuNPs and prevent them from agglomerating. The surfactant is adsorbed to the surface of the gold nanoparticles, providing stability and preventing the particles from agglomerating.

[0050] Poly A of different lengths (A5, A10, A20, A30 and A50) were mixed with AuNPs solution, and the molar ratios were A5:AuNPs=1000:1, A10:AuNPs=500:1, A20:AuNPs=250:1, A30:AuNPs=200:1, A50:AuNPs=200:1, and the concentration of poly A was higher than the actual concentration required to cover the AuNPs solution. The obtained mixture was incubated overnight on a shaker at room temperature. 100mM phosphate buffer (PB, pH 7.4) was added to a final concentration of 10mM. 2M NaCl was added three times with an interval of 30min to obtain a final concentration of 0.2M NaCl. After incubation at room temperature overnight, centrifugation was performed at 12000rpm for 20min at 4°C, and then washed three times with PB (10mM, pH 7.4). Finally, the precipitate was dispersed into a stock solution with a final concentration of 10 nM (10 mM PB, pH 7.4) to obtain poly A-covered AuNPs (among which, the electron microscopy image of A5-covered AuNPs is shown in Figure 6 shown).

[0051] Example 2

[0052] Testing the tunability of AuNPs covered with poly(A) on DNA chain conformation

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

[0054] like Figure 2 As shown, the results showed that the number of DNAs connected to the AuNPs covered with poly A was reduced compared with that of the AuNPs not covered with poly A, indicating that the coverage of poly A on AuNPs can regulate the conformation of the DNA chain.

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

[0056] Example 3

[0057] Preparation of poly-A-capped AuNPs sticky flares

[0058] 3 μL of 100 μM P1 modified with thiol was mixed with 100 μL of AuNPs solution covered with poly A, frozen at -20 °C for 2 h, and then thawed at room temperature. After thawing, 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) with a final concentration of 10 nM. 0.5 μL of 10 nM F1 modified with FAM (as shown in SEQ ID NO.2) was added to 100 μL of the prepared solution and incubated at 37 °C for 3 h with shaking. Washed 3 times with 0.1 M PBS, centrifuged at 12000 rpm for 20 min, and suspended in 100 μL Tris buffer to obtain poly A-covered AuNPs sticky flares.

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

[0060] Example 4

[0061] Testing and optimization of hybridization efficiency

[0062] The prepared sticky flares were added with 5 μL of 1M NaOH solution and incubated for 4 h to dissociate the bidirectional structure. 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 functional microplate reader. The average number of DNA per AuNP was calculated by dividing the measured F1 concentration by the AuNPs concentration. The hybridization efficiency was calculated by dividing the number of F1 flares hybridized with P1 by the total number of P1 assembled on AuNPs.

[0063] like Figure 3 As shown in the figure, the hybridization efficiency of AuNPs covered with poly A is improved compared with that of AuNPs not covered with poly A. At the same time, among the five different lengths of poly A, the hybridization efficiency of AuNPs covered with A5 is the highest.

[0064] A5 was used to prepare poly-A-covered AuNPs sticky flares, and the molar ratio of A5 to AuNPs was varied to 1400:1, 1200:1, 800:1, 600:1, 500:1, 400:1, 200:1, and 100:1, respectively, to optimize the hybridization efficiency and determine the optimal ratio of A5-covered AuNPs sticky flares.

[0065] like Figure 4 As shown in the figure, as the molar ratio of A5 to AuNPs decreases, the amount of DNA attached to the A5-covered AuNPs gradually increases. At the same time, when the molar ratio of A5 to AuNPs is 600:1, the hybridization efficiency is the highest, and the poly A-covered AuNPs sticky flares prepared under this condition were subsequently selected for 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 for incubation, and the concentrations of S1 were 0 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM and 200 nM. The fluorescence spectrum was read using a multifunctional 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 the increase of aflatoxin B1 concentration. Figure 5 Figure c shows the relationship between fluorescence intensity and aflatoxin B1 concentration. Figure 5It can be seen that the fluorescence intensity of the system has a good linear relationship with the concentration of AFB1 in the range of 0-75nM. The detection limit of this method is 1.43ng / mL.

[0071] In summary, the present invention provides an AuNPs sticky flare based on polyadenine (poly A) coverage to adjust DNA conformation and improve hybridization efficiency for the detection of aflatoxin B1 (AFB1). The present invention is based on the preparation method and application of the poly A-covered AuNPs sticky flare, which uses gold nanoparticles as nanocores, and polyadenine strongly adheres to the surface of gold nanoparticles through hydrophobic collapse to form poly A-covered AuNPs, so as to subsequently accurately control the orientation and conformation of surface-modified oligonucleotides and finely adjust the hybridization ability; the thiol-modified DNA chain is connected to the poly A-covered AuNPs by freezing, and the FAM-modified DNA fluorescent chain is further hybridized with it to form a poly A-covered AuNPs sticky flare.

[0072] In order to further optimize the hybridization efficiency of the sticky flare, the present invention uses poly A of different lengths (A5, A10, A20, A30, A50) to adhere AuNPs, and finds that the hybridization efficiency of the AuNPs sticky flare covered with A5 is the best; at the same time, the ratio of A5 to AuNPs is changed, so that the means of poly A coverage to adjust DNA conformation and improve hybridization efficiency are effectively improved. The method of the present invention has many advantages such as low time cost, strong specificity, high sensitivity and mass production, and can realize 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a polyadenine-covered AuNPs sticky flare material, characterized in that: The following steps are involved: (1) Mixing polyadenine and AuNPs solution, incubating, and centrifuging to obtain polyadenine-covered AuNPs; (2) P1 is mixed with the polyadenine-covered AuNPs solution, and the mixture is frozen for reaction, and then F1 is added for incubation to obtain the polyadenine-covered AuNPs sticky flare material.

2. The preparation method according to claim 1, characterized in that: The length of the polyadenine is 5 to 50; the molar ratio of the polyadenine to AuNPs is (100 to 1400):

1.

3. The preparation method according to claim 1, characterized in that: The mixed culture conditions 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 culturing overnight at room temperature; The centrifugation conditions are: centrifugation at 12000 rpm for 20 min at 4°C.

4. The preparation method according to claim 1, characterized in that: The nucleotide sequence of P1 is shown in SEQ ID NO.1; The nucleotide sequence of F1 is shown in SEQ ID NO.

2.

5. The preparation method according to claim 1, characterized in that: The concentration of P1 is 30 mM, the concentration of F1 is 15 mM, and the concentration of the polyadenine-covered AuNPs is 8-12 nM; the volume ratio of P1, F1 and polyadenine-covered AuNPs is 3:0.5:

100.

6. The preparation method according to claim 1, characterized in that: The conditions of the freezing reaction include: freezing at -20°C for 2-4h; The incubation is specifically shaking incubation at 37° C. for 3-5 hours.

7. The polyadenine-covered AuNPs sticky flare material prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the polyadenine-covered AuNPs sticky flare material as claimed in claim 7 in the detection of aflatoxin B1.

9. A product for detecting aflatoxin B1, characterized in that: Comprising the polyadenine-covered AuNPs sticky flare material as described in claim 7.

10. A method for detecting aflatoxin B1, characterized in that: Aflatoxin B1 is detected using the polyadenine-covered AuNPs sticky flare material described in claim 7 or the product described in claim 9.

Images