Pyriminostm lateral flow assay method and test strip based on triple helix bivalent aptamer and nano-zyme

Abstract

The application discloses a pyridaben lateral flow analysis method and test paper based on triple helix bivalent aptamer and nano enzyme, and belongs to the fields of analytical chemistry, medicine, environment, food safety detection and nano biosensing. By constructing a triple helix bivalent aptamer, the binding rate of the aptamer and the target is greatly improved, and the response time is shortened to 1 / 6 of that of a conventional aptamer. With the excellent peroxidase-like activity of the Au@Pt nano enzyme, the sensitivity of the Apt-LFA test for detecting pyridaben is improved by 5 times. The streptavidin and streptavidin-biotin-DNAc sprayed in the detection zone and the control zone do not need to be changed, and only the nucleic acid chain part on the probe Au@Pt NPs@poly-DNA needs to be changed to detect another substance. A new lateral chromatography method for rapid, sensitive and low-cost detection is developed, and the nano enzyme triple helix aptamer test paper has great application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to a chlorantraniliprole lateral flow analysis method and test paper based on triple helix bivalent aptamer and nano enzyme, and belongs to the fields of analytical chemistry, medicine, environment, food safety detection and nanobiological sensing. BACKGROUND

[0002] Lateral flow assay (LFA) technology is a widely used paper-based biosensing detection platform, which can obtain test results within a few minutes without the need for trained personnel to operate expensive and precise instruments. Therefore, it has attracted extensive attention of researchers with the advantages of low cost, simple operation and fast detection speed, and lateral flow assay technology has been widely used for on-site detection and widely applied in various fields, including biomedical, food safety, quality control and environmental health. Recognition elements are one of the key components of LFA, and for a long time, LFA for small molecule compounds has always relied on antibodies. However, the application of antibodies is hindered by various factors, such as irreversible temperature-induced denaturation, prolonged preparation time, and poor controllability of chemical modification. Today, nucleic acid aptamers screened through the in vitro iterative process of SELEX are expected to be utilized to overcome these limitations.

[0003] LFA technology mainly includes competitive, sandwich and adsorption desorption types. For the detection of low molecular weight compounds (such as mycotoxins, etc.), the competitive type is usually used. There are currently two types of competitive assays, one is based on oligonucleotides partially complementary to aptamers competing with aptamers for binding to targets; the other is lateral flow assay technology in which the target in the sample solution competes with the target fixed in the test area for the binding of aptamers. The mode based on complementary sequences and targets for competition is more commonly used.

[0004] Nucleic acid aptamers have been the focus of analytical chemistry and other fields due to their unique properties. Although it has been proven that it has great application potential in biosensing, few people pay attention to the improvement of the response rate of aptamer sensors, which is an important performance indicator for the practical application of aptamer sensors. So far, most of the aptamer-based sensors have a relatively obvious shortcoming, which is the long response time. In the literature (Analytical Chemistry, 2013, 85(17):8397-8402.), the aptamer needs 3h of incubation time to achieve the best signal sensitivity in the aptamer sensor based on target-induced strand displacement. Aptamer presents a relatively sensitive and flexible conformation, and the unstable conformation sensitive to the environment is also an obstacle to achieve fast response. Several strategies have been implemented to optimize aptamers, such as in the literature (Toxicon, 2015, 101:41-47.), a chemical modification method was used; in the literature (Bioorganic and Medicinal Chemistry Letters, 2009, 19(23):6585-6587) length truncation and mutagenesis methods were used; in the literature (Biosensors and Bioelectronics, 2018, 103:39-44) the nucleic acid aptamer was optimized by multivalent effect and synergistic binding, multiple binding motifs simultaneously bind to multiple targets, reduce off-target effects, and significantly enhance the binding affinity. The binding interaction of aptamer can be strengthened by minimizing their structural flexibility. The target binding affinity is determined by their self-adapting folding into a unique 3D structure. Although the aptamer should have a certain degree of flexibility when binding to the target to undergo conformational changes, the excessive flexibility of the aptamer hinders the folding process. In the literature (Journal of the American Chemical Society, 2012, 134(49):19957-19960) is to use triple helix to fix the end of the aptamer strategy to limit the flexibility of the aptamer and increase the binding force to the protein molecule; In the literature (Journal of the American Chemical Society, 2019, 141(44):17493-17497) synthesized triple helix DNA, which has 10 times higher affinity than the original aptamer, and the triple helix structure fixed at the end has been proved to enhance the target binding force of the aptamer by stabilizing the conformation structure.

[0005] The detection sensitivity has an important influence on the practical application of lateral flow assay technology. The enzyme catalytic enhanced lateral flow assay technology is a method for amplifying the colorimetric contrast by adding a color developing reagent to the test area after normal determination. In the literature (Biosensors & Bioelectronics, 2019, 142: 111498), a bidirectional immunochromatographic method based on platinum nanoparticle-anchored nickel hydroxide nanomaterials (Pt-Ni(OH)2NSs) is designed. Since Pt-Ni(OH)2NSs have strong peroxidase-like activity, the detection signal is significantly enhanced after the color developing substrate is added, and this method can simultaneously realize the low detection limit detection of ethyl acetate and methyacrylate; in the literature (Biosensors & Bioelectronics, 2020, 169: 112610), a magnetic Prussian blue nanoscale enzyme (MPBN) mediated immunochromatographic detection technology (MLFIA) for clenbuterol and ractopamine is established. By using the peroxidase activity of MPBN, the detection signal is enhanced after the color developing substrate is introduced, and the detection range is also widened. The use of nanoscale enzyme lateral flow assay test paper for the detection of small molecule compounds can improve the sensitivity of target detection, and some can also widen the detection range, but methods that can improve both properties are rarely reported.

[0006] In general, due to the conformation of the nucleic acid aptamer sensitive to the environment, a long time is needed to respond to the target, which limits the rapid development of the aptamer LFA. Compared with a large number of small molecule antibody LFA methods, the aptamer LFA method is still very few. At the same time, the detection method of the LFA with Au NPs as the marker has a relatively low sensitivity, which limits the application of the method in the scene with an ultra-low detection limit. Therefore, it is of important application value to develop a new aptamer LFA method and test paper that can improve both properties. SUMMARY

[0007] [TECHNICAL PROBLEM]

[0008] The existing nucleic acid aptamer sensing method usually needs a long time to respond to the target, which limits the development of the aptamer lateral flow assay (LFA) test paper, especially the rapid detection application of small molecule compounds. The method of using nanoscale enzyme labeled nucleic acid chains for LFA is still relatively few.

[0009] [TECHNICAL SCHEME]

[0010] The nanometer gold platinum particles (Au@PtNPs) used in the application are prepared by the seed-mediated method in the reference (Talanta, 2021, 225: 121961). First, gold nanoparticles (AuNPs) are prepared, and the prepared Au NPs solution is used as a seed to grow a Pt shell on the surface of the Au NPs to prepare nanometer gold platinum particles.

[0011] The application adopts polyA-cDNA as a probe, the probe adopts polyA as an anchor block, and Au@Pt NPs@polyA-cDNA conjugates are prepared by anchoring polyA-cDNA on the gold platinum nanoparticle. The detection area (T line) of the nucleic acid test strip is sprayed with streptavidin (SA), the control area (C line) is sprayed with an oligonucleotide chain (SA-Bio-DNAc) complementary to the polyA-cDNA part of the Au@Pt NPs@polyA-DNA conjugate, and a universal nano-enzyme labeled nucleic acid test strip is constructed. The test strip sprays SA on the T line and SA-Bio-DNAc on the C line without changing, only changing the part complementary to the aptamer on the polyA-cDNA, so as to detect another substance, which provides convenience for future detection.

[0012] The triplex bivalent aptamer used in the application has 10 bases complementary to the gold platinum nanoprobe (Au@Pt NPs@PolyA-cDNA). After the biotinylated triplex bivalent aptamer is combined with the nanoprobe, the Au@Pt NPs probe can be captured by the SA on the T line. When there is a small molecule substance, the small molecule substance competes with the gold platinum nanoprobe to bind the triplex bivalent aptamer, inhibits the hybridization of the gold platinum nanoprobe and the triplex bivalent aptamer, and thereby inhibits the color development signal of the T line.

[0013] The rapid detection characteristic of the application is reflected in the response rate of the triplex bivalent aptamer to the target. The conventional aptamer needs to be incubated with the target for about 60 min to achieve a good signal response, and the triplex bivalent aptamer only needs 10 min to achieve even more than the effect of the conventional aptamer incubated for 60 min.

[0014] The first object of the application is to provide a triplex bivalent aptamer for detecting acetamiprid, which is composed of two identical monovalent aptamers and a bridge nucleic acid chain, the nucleotide sequence of the monovalent aptamer is shown in SEQ ID NO. 10, and the nucleotide sequence of the PolyA bridge nucleic acid chain is shown in SEQ ID NO. 20.

[0015] In an embodiment, the triplex bivalent aptamer is a monovalent aptamer with a nucleotide sequence shown in SEQ ID NO. 10 and a PolyA bridge nucleic acid chain with a nucleotide sequence shown in SEQ ID NO. 20 mixed at a molar ratio of 2:1, incubated at 90-95 DEG C for 5 min and cooled to room temperature.

[0016] A second object of the present application is to provide a lateral chromatographic test strip for detecting acetamiprid, which contains the triple helix bivalent aptamer, the nano-gold platinum particles, the probe polyA-cDNA, and the streptavidin-biotin-DNAc.

[0017] In an embodiment, the probe polyA-cDNA contains a polyA fragment, a fragment complementary to the DNAc, and a fragment complementary to the nucleic acid aptamer of the small molecule substance.

[0018] In an embodiment, the nucleotide sequence of the probe polyA-cDNA is shown in SEQ ID NO. 3.

[0019] In an embodiment, the 5' end of the triple helix bivalent aptamer is labeled with biotin; and the nucleic acid aptamer can specifically bind to the small molecule substance to be detected.

[0020] In an embodiment, the streptavidin-biotin-DNAc is prepared by mixing equal volumes of streptavidin and 5' end biotin-labeled DNAc, and incubating at 3-5°C for 2-4 hours.

[0021] In an embodiment, the nucleotide sequence of the DNAc is shown in SEQ ID NO. 2.

[0022] In an embodiment, the concentration of the streptavidin is 2.0-3.0 mg / mL, and the concentration of the DNAc is 240-260 μM.

[0023] In an embodiment, the test strip comprises a sample pad, a conjugate pad (gold standard pad), a nitrocellulose membrane (NC membrane), a water absorption pad, and a PVC backing plate; the sample pad, the gold standard pad, the NC membrane, and the water absorption pad are sequentially pasted on the PVC backing plate; the NC membrane is provided with a detection zone and a control zone in sequence, and the distance between the detection zone and the control zone is 4-6 mm; the detection zone is provided with streptavidin, and the control zone is provided with streptavidin-biotin-DNAc; and the gold standard pad contains Au@Pt NPs@polyA-cDNA conjugates.

[0024] In an embodiment, the overlapping part between the sample pad and the gold standard pad has a length of 1-2 mm, and the sample pad is placed above the gold standard pad; the overlapping part between the gold standard pad and the NC membrane has a length of 1-2 mm, and the gold standard pad is placed above the NC membrane; and the overlapping part between the NC membrane and the water absorption pad has a length of 1-3 mm, and the water absorption pad is placed above the NC membrane.

[0025] In an embodiment, the Au@Pt NPs@polyA-DNA conjugates are obtained by anchoring the probe polyA-DNA on nano-gold platinum particles.

[0026] In an embodiment, the nano gold platinum particle has a particle size of 10-30 nm; and the polyA-DNA has a concentration of 80-120 μM.

[0027] The test of the test strip is based on a competition mode: when the sample solution to be tested does not contain small molecule substances, the 5' end biotinized triple helix bivalent aptamer is combined with the Au@Pt NPs@polyA-cDNA coupling agent, and then is captured by streptavidin on the T line to form an Au@Pt NPs@polyA-cDNA-Apt-bio complex. When the color developing solution is added, a clear blue band can be observed on the T line due to the nanozyme catalysis of Au@Pt NPs, and the detection result is negative. When the sample solution to be tested contains small molecule substances, the small molecule substances are combined with the 5' end biotinized triple helix bivalent aptamer, which inhibits the combination of the triple helix bivalent aptamer and the Au@Pt NPs@polyA-cDNA probe, thereby reducing the accumulation of the probe in the T line region. When the color developing solution is added, the T line is lighter or does not develop color, and the detection result is positive. The color intensity on the T line region is negatively correlated with the concentration of the small molecule substances.

[0028] A third object of the present application is to provide a method for rapidly detecting small molecule substances, which is tested by using the above-mentioned lateral chromatographic test strip. 50-100 μL of the sample solution to be tested is added to the sample pad after being mixed with the 5' end biotin-labeled triple helix bivalent aptamer and incubated, and is incubated for 3-5 min and the color developing solution is added. Qualitative analysis is performed by naked eye or using a colloidal gold reading and photographing instrument, and quantitative analysis is performed according to the standard curve after software analysis.

[0029] In an embodiment, the preparation method of the standard curve is that the standard solution of the small molecule substances is diluted to different concentrations, the diluted solutions with different concentrations are mixed with the 5' end biotin-labeled triple helix bivalent aptamer and incubated, and then are added to the sample pad, incubated for 3-5 min, and quantitative analysis is performed by using a colloidal gold test paper quantitative analyzer, and the data is fitted to obtain the standard curve.

[0030] In an embodiment, the mixing and incubation time is 10-15 min.

[0031] In an embodiment, the volume ratio of the 5' end biotin-labeled triple helix bivalent aptamer to the sample solution to be tested or the diluted solution is 1:99.

[0032] In an embodiment, the initial concentration of the 5' end biotin-labeled triple helix bivalent aptamer is 5-50 nM.

[0033] A fourth object of the present application is to provide a preparation method of the above-mentioned lateral chromatographic test strip, and the specific steps are as follows:

[0034] (1) Cut the sample pad and gold pad, then add PBS for soaking and dry.

[0035] (2) Spray the probe Au@Pt NPs@polyA-cDNA on the gold pad and dry.

[0036] (3) Spray streptavidin on the detection area of the NC membrane, and spray streptavidin-biotin-DNAc on the control area, the distance between the detection area and the control area is fixed at 4-6 mm, and dry at 35-39℃ for 2-3 h.

[0037] (4) Paste the sample pad, gold pad, NC membrane and water absorption pad prepared in steps (1)-(3) on the PVC plate in sequence to obtain the lateral chromatographic test strip for detecting acetamiprid.

[0038] In an embodiment, the Au@Pt NPs@polyA-cDNA conjugate is obtained by anchoring the probe polyA-cDNA as an anchor block on the gold platinum nanoparticles; the probe polyA-cDNA contains a polyA fragment, a fragment complementary to DNAc and a fragment complementary to a nucleic acid aptamer of a small molecule substance.

[0039] In an embodiment, the particle size of the gold platinum nanoparticles is 10-30 nm.

[0040] In an embodiment, the concentration of the polyA-cDNA is 80-120 μM.

[0041] In an embodiment, the nucleotide sequence of the DNAc is shown in SEQ ID NO. 2.

[0042] In an embodiment, the nucleotide sequence of the probe polyA-cDNA is shown in SEQ ID NO. 3.

[0043] In an embodiment, the overlapping part between the sample pad and the gold pad has a length of 1-2 mm, and the sample pad is placed above the gold pad; the overlapping part between the gold pad and the NC membrane has a length of 1-2 mm, and the gold pad is placed above the NC membrane; the overlapping part between the NC membrane and the water absorption pad has a length of 1-3 mm, and the water absorption pad is placed above the NC membrane.

[0044] The beneficial effects of the present application are:

[0045] (1) A novel lateral chromatographic test method based on nano-enzyme and triple helix aptamer is invented herein, which is rapid, high sensitivity and high specificity. The test strip is sensitive (the naked eye detection limit is 5 ng / mL), rapid (10 min) and has high repeatability of detection results.

[0046] (2) The detection method proposed in the application realizes high-sensitivity colorimetric detection of small molecules (the application is acetamiprid). In a concentration range of 1-150 ng / mL, the T line and the concentration of acetamiprid show a good linear relationship, and the detection limit is 0.068 ng / mL.

[0047] (3) In the design of the aptamer of the test strip, the T base is extended by using a PolyT linker at both ends of the aptamer (PolyT-apt), and is combined with a PolyA bridge nucleic acid chain to form a triple helix bivalent aptamer, so that the response rate of the aptamer to the target is greatly improved.

[0048] (4) The application introduces gold platinum nanoparticles (Au@PtNPs) as a signal tag, uses the excellent nano-enzyme activity to realize signal amplification of the aptamer lateral flow analysis test paper, significantly improves the detection sensitivity, and also widens the detection range. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is the detection principle diagram of the lateral flow method; (a) comparison of response time of original aptamer and triple helix bivalent aptamer to target; (b) mechanism of Au@Pt nano-enzyme lateral flow test strip mediated by triple helix bivalent aptamer; (c) comparison diagram of sensitivity and range of acetamiprid detection under uncatalyzed and catalyzed modes

[0050] Figure 2 is the characterization of gold platinum nanoparticles: (a) TEM image of Au@PtNPs; (b) EDS element analysis diagram of Au@PtNPs; (c) absorption spectrum diagram of AuNPs and Au@PtNPs; (d) absorption spectrum diagram of TMB before and after catalysis by Au@PtNPs.

[0051] Figure 3 is the optimization of the length of the complementary sequence of the bivalent double helix aptamer. The data points represent the average value and standard deviation of three technical repeats using the Apt-LFA strip.

[0052] Figure 4 is the optimization of the length of the polyT arm in the triple helix univalent aptamer. The data points represent the average value and standard deviation of three technical repeats using the Apt-LFA strip.

[0053] Figure 5 is the optimization of the length of the polyT arm in the triple helix univalent aptamer. The data points represent the average value and standard deviation of three technical repeats using the Apt-LFA strip.

[0054] Figure 6Figure is the inhibition rate comparison chart of different structure aptamers in acetamiprid detection, (a) from left to right, the structure is original aptamer, double helix bivalent aptamer, triple helix monovalent aptamer, triple helix bivalent aptamer, (b) electrophoresis analysis of triple helix bivalent aptamers with different PolyT arm length.

[0055] Figure 7 Figure is the response signal comparison of original aptamer and triple helix bivalent aptamer under different incubation time, (a) color development picture, (b) data chart *T 1 / 2 Half-time refers to the time to reach half of the reaction endpoint.

[0056] Figure 8 Figure (a) is the colorimetric detection result chart of different concentrations of acetamiprid based on gold nanoparticles (AuNPs) and gold platinum nanoparticles (Au@Pt NPs); (b) is the calibration curve of signal intensity and logarithm of acetamiprid concentration. DETAILED DESCRIPTION

[0057] Example 1: Preparation of acetamiprid rapid detection test strip based on triple helix nucleic acid aptamer

[0058] The specific steps are as follows:

[0059] The lateral chromatographic test strip provided by the application contains gold platinum nanoparticles, nucleic acid aptamer, probe polyA-cDNA and streptavidin-biotin-DNAc.

[0060] 1. Design and optimization of nucleic acid aptamer sequence

[0061] When the aptamer recognizes the target, it needs to adaptively fold into a specific conformational structure to bind to its target. However, the inherent flexibility of oligonucleotides is highly sensitive to the working environment, making the conformational structure unstable, resulting in weak affinity and selectivity. End fixation design can limit the flexibility of aptamer to stabilize the conformational structure and achieve stronger binding capacity. Different structures of aptamers are selected to explore their effects in acetamiprid detection. The length of the complementary sequence will affect the stability of the constructed nucleic acid aptamer, and also affect the feasibility of acetamiprid competition.

[0062] Preparation of double-helix divalent aptamer (BDHA) solution: The base fragment of nucleotide sequence SEQ ID NO.1 was mixed with the base fragments of nucleotide sequences such as SEQ ID NO.4 to SEQ ID NO.7 at a molar ratio of 2:1 in acetamiprid binding buffer (2.5 mmol / L KCl, 1.5 mmol / L KH2PO4, 0.5 mmol / L MgCl2, 137 mmol / L NaCl, 8 mmol / L Na2HPO4, pH 7.4). The mixed solution was heated to 95℃ and held for 5 min, then naturally cooled to room temperature and diluted to 1 nM with binding buffer. The response of aptamer solutions prepared with different complementary sequence lengths to acetamiprid was investigated.

[0063] Preparation of triple-helix monovalent aptamer (MTHA) solutions: The base fragments of nucleotide sequences such as SEQ ID NO.10 and SEQ ID NO.13 to SEQ ID NO.17 were mixed in binding buffer at a 1:1 molar ratio. The mixture was heated to 95°C and held for 5 min, then naturally cooled to room temperature and diluted to 1 nM with binding buffer. The response of aptamer solutions prepared with different PolyT arm lengths to acetamiprid was investigated.

[0064] Preparation of triple-helix divalent aptamer (BTHA) solution: The base fragment of nucleotide sequence such as SEQ ID NO.10 was mixed with the base fragment of nucleotide sequence such as SEQ ID NO.18 to SEQ ID NO.22 at a molar ratio of 2:1 in binding buffer. The mixed solution was heated to 95℃ and held for 5 min, then naturally cooled to room temperature and diluted to 1 nM with binding buffer. The response effect of aptamer solutions prepared under different PolyT arm lengths to acetamiprid was investigated.

[0065] like Figure 3 , 4 As shown in Figure 5, this invention first investigated the response effects of double-helix bivalent aptamers, triple-helix monovalent aptamers, and triple-helix bivalent aptamers to acetamiprid under structures with different complementary sequence lengths or PolyT arm lengths.

[0066] Mix 50 μL of 50 ng / mL acetamiprid standard solution with 50 μL of acetamiprid aptamer solution and incubate for 10 min to obtain mixture A. Add 20 μL of mixture A to 80 μL of running buffer to obtain mixture B. Add 70 μL of mixture B to the sample pad for detection. After chromatography for 3 min, measure the (T) signal intensity using a colloidal gold reader. The running buffer composition is: 4×SSC, 0.5% Tween-20.

[0067] The signal intensities (T0 and C0) of the T and C lines in the absence of acetamiprid solution were used to calculate the inhibition rate of different aptamers.

[0068] The results showed that all three aptamers with different structures achieved optimal performance with complementary sequences or a PolyT arm length of 10 nt, but the triple-helix monovalent and triple-helix bivalent aptamers showed significantly better response than the double-helix bivalent aptamer. Longer complementary arm lengths could also lead to excessively strong binding forces, thus hindering aptamer-target binding. Figure 6 As shown, the original aptamer (SEQ ID NO. 1) had an inhibition rate of only 7%; the double-helix bivalent aptamer (SEQ ID NO. 1, SEQ ID NO. 6) with a complementary arm length of 10 nt could achieve an inhibition rate of 27%; while the triple-helix monovalent aptamer (SEQ ID NO. 10, SEQ ID NO. 15) and the triple-helix bivalent aptamer (SEQ ID NO. 10, SEQ ID NO. 20) with complementary arms length of 10 nt had inhibition rates of 47% and 55%, respectively. These results demonstrate that the bivalent and triple-helix structures are very effective in improving the binding effect between the target and the aptamer.

[0069]

[0070] T0 and C0 refer to the signal intensity of the T and C lines in the absence of acetamiprid, while T1 and C1 represent the signal intensity of the T and C lines in the presence of 50 ng / mL acetamiprid.

[0071] Electrophoretic analysis also confirmed the optimization results of the aptamers, such as Figure 6 As shown in b, extending the triple-helix aptamer end by 10 nt can induce the disintegration of the triple-helix aptamer probe after the addition of acetamiprid. Although longer base pairings can produce more stable triple-helix aptamers, they also inhibit the binding of the aptamer to the target, thus leading to a decrease in sensor sensitivity. Therefore, the triple-helix bivalent aptamers used in the following embodiments are (SEQ ID NO.10, SEQ ID NO.20).

[0072] Table 1. Nucleic acid sequences used for acetamiprid triple-helix aptamer LFA

[0073]

[0074]

[0075] (Italicized sequences indicate complementary sequences; bolded sequences indicate aptamer portions or their complementary sequences.)

[0076] Design of oligonucleotide DNAc: As shown in Tables 1 and 2, the DNAc sequence underlined at the point of intersection is complementary to polyA-cDNA.

[0077] Table 2 DNA sequences of acetamiprid rapid detection test strip

[0078]

[0079] (italicized sequence represents the complementary sequence; bold sequence represents the aptamer partial sequence or its complementary sequence.)

[0080] 2. Preparation and functionalization of gold-platinum nanoparticles (Au@PtNPs)

[0081] (1) Preparation of Au@PtNPs

[0082] The glassware used for the synthesis and storage of nanomaterials was immersed in aqua regia (hydrochloric acid: nitric acid = 3:1) for 24-48 h and washed with ultrapure water before use.

[0083] The preparation of Au@PtNPs used a seed-mediated method. The specific steps are as follows:

[0084] 1) Add 100 mL of 0.01% HAuCl4 to a 250 mL conical flask, heat and stir until the solution boils, and maintain for 1-2 min.

[0085] 2) Quickly add 2 mL of 1% trisodium citrate solution to the conical flask, continue to heat and stir. The color of the solution gradually changes from light yellow to deep purple and finally to wine red. Maintain heating for 10 min to prepare Au NPs seed solution with a particle size of 15 nm.

[0086] 3) Add 1 mL of 0.1 mol / L ascorbic acid to the Au NPs seed solution, then add 1.5 mL of 0.01 g / mL H2PtCl6 and mix. Heat the above mixture solution at 90°C for 30 min to obtain Au@PtNPs, which is cooled to room temperature and stored at 4°C for standby.

[0087] (2) Functionalization of Au@PtNPs

[0088] 1) Add 5-15 μL of 100 μM polyA-DNA to 1 mL of Au@PtNPs (10 nM) prepared in step (1) and mix well, then add 20 μL of 500 mM citric acid buffer (pH 3.0). After mixing, incubate at room temperature for 3 min.

[0089] 2) After incubation, add 60 μL of 500 mM HEPES buffer at pH 7.6 to adjust the pH of the Au@PtNPs solution to neutral, then incubate at room temperature for 5-10 min.

[0090] 3) After the incubation, centrifuge at 10000 r / min for 20 min, remove the supernatant, add resuspension to the precipitate, repeat the centrifugation at 10000 r / min for 20 min three times to remove the unreacted nucleic acid, and finally add 400 μL of resuspension to obtain functionalized Au@Pt NPs, namely probe Au@Pt NPs@polyA-cDNA, and store at 4°C for standby.

[0091] The Au@Pt NPs prepared in step (1) and the Au@Pt NPs@polyA-DNA prepared in step (2) were characterized by transmission electron microscopy, and the results are shown in Figure 2 a, which shows that the synthesized Au@Pt has a sea urchin shape. The TEM results confirm that Pt particle clusters are formed on the Au NP seeds, and there are no separate Pt particles. The average hydrated particle size of the Au@Pt NPs is 28.43 nm. The spectrum of Au@Pt is significantly different from that of the initial Au NP seeds. The growth of the Pt layer causes the disappearance of the plasmon resonance peak at 520 nm characteristic of Au NPs, and the absorbance in the entire UV-Vis wavelength range increases significantly. Figure 2 b, which shows that the nanoparticles contain Au and Pt elements, which also indicates that Pt is deposited on the surface of the Au NPs. As shown in Figure 2 d, the Au@Pt NPs can catalyze the oxidation-reduction reaction of the chromogenic substrate TMB and H2O2, and the reaction solution has a strong ultraviolet-visible light absorption peak at 650 nm. The Au NPs with a particle size of 15 nm prepared have a single characteristic absorption peak at 520 nm, and when combined with polyA-cDNA, the maximum absorption wavelength shifts to 530 nm, which preliminarily proves that the Au@Pt NPs are successfully synthesized.

[0092] Composition of resuspension: 20 mM Na3PO4, 5% BSA, 10% sucrose, 0.25% Tween-20.

[0093] 3. Preparation of streptavidin-biotin-DNAc

[0094] (1) 100 μL of 2.5 mg / mL streptavidin was mixed with 100 μL of 250 μM 5'-end biotin-labeled DNAc and incubated at 4°C for 1 h to obtain a mixture.

[0095] (2) The mixture was treated with an ultrafiltration tube (MWCO 30 kDa), centrifuged at 6000 r / min for 20 min three times, and resuspended in 300 μL of 10 mM PBS to obtain streptavidin-biotin-DNAc, which was stored at 4°C for standby.

[0096] 4. Assembly of nucleic acid aptamer test strip

[0097] (1) The sample pad and the conjugate pad (gold pad) were cut to appropriate size, soaked in 10 mM PBS for 30 min, and then dried at 45°C.

[0098] (2) The probe Au@Pt NPs@polyA-cDNA prepared in step 1 was uniformly sprayed on the conjugate pad, and dried at 37°C for 2 h.

[0099] (3) The streptavidin and streptavidin-biotin-DNAc prepared in step 3 were uniformly sprayed on the NC membrane with a three-dimensional spray instrument at a speed of 0.9 μL / cm, as the detection zone (T line) and the control zone (C line), respectively, and the distance between the detection zone and the control zone was fixed at 5 mm, and dried at 37°C for 2 h.

[0100] (4) The sample pad, conjugate pad, NC membrane, and water absorption pad prepared in steps (1)-(3) were sequentially pasted on a PVC plate, and the assembled test strip was uniformly cut to a width of 4 mm and placed in a dispensing bag for sealed storage. Figure 1 b The assembled test strip was uniformly cut to a width of 4 mm and placed in a dispensing bag for sealed storage.

[0101] Example 2: Optimization of incubation time of nucleic acid aptamer with target

[0102] The triplex bivalent aptamer with the sequence of (SEQ ID NO. 10, SEQ ID NO. 20) was selected for dynamic response and incubation time optimization with the original aptamer with the sequence of SEQ ID NO. 1.

[0103] Preparation of triplex bivalent aptamer solution: The acetamiprid aptamer polyT-Apt with the nucleotide sequence of SEQ ID NO. 10 and the polyA bridge nucleic acid chain with the nucleotide sequence of SEQ ID NO. 20 were prepared in a 2:1 molar ratio in a binding buffer, and the mixed solution was heated to 95°C for 5 min, naturally cooled to room temperature, and diluted to 1 nM with the binding buffer.

[0104] The standard solution of acetamiprid was diluted with ultrapure water to a final concentration of 500 ng / mL, the triple helix bivalent aptamer solution was diluted with acetamiprid binding buffer to 1 nM, and the original aptamer with nucleotide sequence of SEQ ID NO. 1 was diluted with acetamiprid binding buffer to 40 nM. Subsequently, 50 μL of the triple helix bivalent aptamer and 50 μL of the original aptamer were mixed with 50 μL of acetamiprid to obtain mixture A. After mixture A was incubated for 0, 5, 10, 15, 20, 40, 60 and 80 min, 20 μL of mixture A was added to 80 μL of running buffer to obtain mixture B. 70 μL of mixture B was added dropwise to the sample pad for detection. After 3 min of reaction, color developing solution was added, and the (T) signal intensity was recorded.

[0105] As shown in Figure 7 a, the original aptamer needs to be incubated with the target for 60 min before a significant difference in detection signal can be observed on the test strip; while the triple helix bivalent aptamer only needs to be incubated for 10 min to achieve even exceed the response effect of the original aptamer incubated for 60 min; as shown in Figure 7 b, the half-saturation reaction time (T 1 / 2 ) required to reach half of the reaction endpoint was determined. The T 1 / 2 of the conventional aptamer is 26 min, while the T 1 / 2 of the triple helix bivalent aptamer is only 3 min. For the triple helix bivalent aptamer, it only takes 10 min to reach the reaction endpoint, while the conventional aptamer takes 60 min. The triple helix bivalent aptamer accelerates the recognition speed and binding process of the aptamer and the target, significantly shortening the detection time of the Apt-LFA. Therefore, the incubation time of the triple helix aptamer and the target in the following examples is determined to be 10 min.

[0106] Example Three: Determination of acetamiprid standard solution by test strip

[0107] (1) Preparation of acetamiprid standard solution

[0108] The acetamiprid standard solution was diluted with ultrapure water to a final concentration of 0, 10, 30, 50, 100, 250, 500, 1000 and 1500 ng / mL, respectively. The acetamiprid aptamer polyT-Apt with nucleotide sequence of SEQ ID NO. 10 was combined with the polyA bridge nucleic acid chain with nucleotide sequence of SEQ ID NO. 20 in a molar ratio of 2:1 in binding buffer, and the mixed solution was heated to 95°C for 5 min, naturally cooled to room temperature and diluted to 1 nM with binding buffer.

[0109] (2) Establishment of acetamiprid nucleic acid test strip detection standard curve:

[0110] The 50 μL different concentrations of acetamiprid standard solution in step (1) was mixed with 50 μL acetamiprid triple helix aptamer solution for 10 min to obtain mixed solution A. 20 μL mixed solution A was added to 80 μL running buffer to obtain mixed solution B, and 70 μL mixed solution B was added dropwise to the sample pad for detection. The (T) signal intensity was measured after chromatography for 3 min, and the standard curve of the corresponding relationship between the (T) light signal intensity and different acetamiprid concentrations was established.

[0111] The results are shown in Figure 8 a. When the concentration of acetamiprid was 5 ng / mL, the color of the T line on the test strip could be clearly distinguished from the color of the T line on the test strip without acetamiprid (0 ng / mL acetamiprid). When the concentration of acetamiprid was 5-150 ng / mL, the color of the T line decreased with the increase of the concentration of acetamiprid, and when the concentration of acetamiprid was 150 ng / mL, the color of the T line was basically unchanged, so the lower limit of naked eye detection was 5 ng / mL and the upper limit was 150 ng / mL.

[0112] The ZJ-600 colloidal gold reader was used to take pictures and record the gray value, and the Image J software was used to process to obtain the intensity of the T line light signal of different concentrations of acetamiprid, and the relationship curve between the (T) light signal intensity and the concentration of acetamiprid is shown in Figure 8 b. The lowest detection line was 0.068 ng / mL. Within the concentration range of 5-150 ng / mL, the (T) light signal intensity and the concentration had a linear relationship, and the linear regression equation was y=-62.05x+144.9, R 2 =0.9773, where y is the (T) light signal intensity and x is the concentration of acetamiprid (ng / mL).

[0113] Example Four: Detection of acetamiprid residues in tomato samples

[0114] Tomato simulation samples were used to test the recovery rate, and the steps were as follows:

[0115] (1) Au@Pt NPs@polyA-cDNA solution pretreatment: 0.8-1.4 μL volume of prepared and stored Au@Pt NPs@polyA-cDNA was added to the gold label pad of the test strip, and stored at 4°C.

[0116] (2) Sample pretreatment: for positive samples, 10.0 g of tomato sample was accurately weighed and placed in a centrifuge tube, 2 mL of acetamiprid standard (200, 500 and 1000 ng / mL) was added to the sample, after homogenization, 8 mL of binding buffer (i.e. 5-fold dilution) was added, then centrifuged at 8000 r / min for 10 min to take the supernatant, and the supernatant was filtered through a 0.22 μm membrane, thus obtaining positive samples with a spiked amount of 40, 100 and 200 ng / g; for negative samples: 10.0 g of tomato sample was accurately weighed and placed in a centrifuge tube, 10 mL of binding buffer was added to the sample, then centrifuged at 8000 r / min for 10 min to take the supernatant, and the supernatant was filtered through a 0.22 μm membrane, thus obtaining negative samples.

[0117] (3) Preparation of triple helix bivalent aptamer solution: the acetamiprid aptamer polyT-Apt with nucleotide sequence as shown in SEQ ID NO. 10 was combined with polyA bridge nucleic acid chain with nucleotide sequence as shown in SEQ ID NO. 20 in a 2:1 molar ratio in binding buffer, and the mixed solution was heated to 95℃ for 5 min, naturally cooled to room temperature and diluted to 5 nM with binding buffer.

[0118] (4) Determination of the recovery rate of acetamiprid in tomatoes: 1 μL of triple helix bivalent aptamer solution (5 nM) was mixed with 99 μL of tomato solution containing different concentrations of acetamiprid in step (2) for 10 min of incubation, and then the test strip was used for detection after the mixing reaction.

[0119] The nucleic acid aptamer test strip prepared in application example one was used to detect the content of acetamiprid in tomatoes, and the results are shown in Table 3.

[0120] Table 3 Detection of acetamiprid in tomato samples

[0121]

[0122] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for rapid detection of acetamiprid, characterized in that, The lateral chromatographic test strip is used for testing, 50-100 μL of the solution to be tested is added to the sample pad after the solution to be tested is mixed with biotinylated triple helix bivalent aptamer, incubated for 3-5 min, and the color developing solution is added dropwise, and qualitative analysis is performed by naked eye or using a colloidal gold reading and photographing instrument, and quantitative analysis is performed according to the standard curve after software analysis. The lateral chromatographic test strip contains a triple helix bivalent aptamer for detecting acetamiprid, nano gold platinum particles, a probe polyA-cDNA and a streptavidin-biotin-DNAc; the probe polyA-cDNA contains a polyA fragment, a fragment complementary to the DNAc and a fragment complementary to the nucleic acid aptamer of a small molecule substance; The triple helix bivalent aptamer is composed of two identical monovalent aptamers and a polyA bridge nucleic acid chain; the nucleotide sequence of the monovalent aptamer is shown as SEQ ID NO. 10, and the nucleotide sequence of the polyA bridge nucleic acid chain is shown as SEQ ID NO. 20; The 5' end of the triple helix bivalent aptamer is labeled with biotin; The streptavidin-biotin-DNAc is prepared by mixing equal volumes of streptavidin and 5' end biotin-labeled DNAc, and incubating at 3-5 DEG C for 2-4 h, and the nucleotide sequence of the DNAc is shown as SEQ ID NO. 2; The nucleotide sequence of the probe polyA-cDNA is shown as SEQ ID NO. 3; The test strip includes a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad and a PVC plate; the sample pad, the gold label pad, the nitrocellulose membrane and the water absorption pad are sequentially pasted on the PVC plate; the nitrocellulose membrane is provided with a detection zone and a control zone in sequence, and the distance between the detection zone and the control zone is 4-6 mm; the detection zone is provided with streptavidin, and the control zone is provided with streptavidin-biotin-DNAc; the gold label pad contains Au@Pt NPs@polyA-cDNA conjugate; The Au@Pt NPs@polyA-DNA conjugate is obtained by anchoring the probe polyA-DNA on the nano gold platinum particles.

2. The method of claim 1, wherein, The concentration of the streptavidin is 2.0-3.0 mg / mL, and the concentration of the DNAc is 240-260 μM.

3. The method of claim 1, wherein, The particle size of the nano gold platinum particles is 10-30 nm, and the concentration of the polyA-DNA is 80-120 μM.

4. The method according to claim 1, characterized in that, The preparation method of the standard curve is that the standard solution of acetamiprid is diluted to different concentrations, the diluted solutions with different concentrations are mixed with biotinylated triple helix bivalent aptamer, and then are added dropwise to the sample pad, and quantitative analysis is performed using a colloidal gold test strip quantitative analyzer, and the data are fitted to obtain a standard curve.

5. The method of claim 1, wherein, The mixing incubation time is 10-15 min, and the initial concentration of the 5' end biotin-labeled triple helix bivalent aptamer is 5-50 nM.

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