Aptamer test strip for HCR and click chemistry combined signal amplification

Abstract

The invention belongs to the field of pesticide detection, and discloses an aptamer test strip for HCR and click chemistry combined signal amplification, which comprises a test strip assembly and an aptamer signal enrichment probe, a nucleic acid skeleton is self-assembled through HCR, and a high-density signal enrichment probe is formed by inducing cross-linking of gold azide nanoparticles through click chemistry reaction. According to the method, the detection limit of procymidone reaches 0.054 ng / mL, the color development response time is greatly shortened to be within 2 seconds, and the detection efficiency and sensitivity are remarkably improved. In actual vegetable sample detection, the recovery rate of the method is 93.44%-104.00%, and a specificity experiment shows that the method only responds to procymidone and is excellent in anti-interference capability. The detection capability of trace procymidone pesticide residues is remarkably improved, and a new idea of dual signal amplification is provided for the aptamer lateral flow chromatography technology.

Description

Technical Field

[0001] The present invention relates to an aptamer test strip with signal amplification by combining HCR and click chemistry, and belongs to the field of pesticide detection. Background Art

[0002] Many researchers are attempting to develop aptamer-based lateral flow chromatography test strips. Gold nanoparticles (AuNPs) are classic colorimetric materials for constructing aptamer lateral flow chromatography test strips. The test strips based on AuNPs have been widely used in disease diagnosis, food safety, environmental monitoring, etc. However, due to low molar absorptivity, poor colloidal stability, etc., the trace detection ability of lateral flow chromatography test strips based on AuNPs is often limited. The particle size of AuNPs is usually 20-40 nm, their molar extinction coefficient is relatively small, and the sensitivity is limited, which is suitable for qualitative detection. Larger-sized AuNPs tend to exhibit better sensitivity but will cause a decrease in structural stability. In addition, compared with using dispersed AuNPs as labeling signals, the detection sensitivity using aggregated AuNPs is significantly increased by 40 times. Therefore, using aggregated AuNPs probes in lateral flow chromatography test strips may be an effective solution to improve detection sensitivity and structural stability. "Click chemistry" reactions, including copper-catalyzed azide-alkyne cycloaddition reactions, are effective means to obtain larger-sized aggregated AuNPs. The team of Zhao Chao from Jilin University functionalized AuNPs respectively to obtain azide-functionalized AuNPs and alkyne-functionalized AuNPs. Under the catalysis of Cu(I), -N3 and -C≡CH undergo alkyne-azide cycloaddition reaction, causing AuNPs to form larger-sized AuNPs aggregates. Similarly, the research group of Li Dawei from East China University of Science and Technology constructed a single-particle dark-field microscopy platform for detecting inorganic pyrophosphatase. This platform uses AuNPs-ALK as the core and AuNPs-N3 as the satellite. PPase can reduce Cu(II) to Cu(I), and then catalyze the cross-linking between the core AuNPs and the satellite AuNPs to form aggregated AuNPs. However, the cross-linking of AuNPs based on "click chemistry" is random and disordered, and it is still a challenge to stably and uniformly cross-link AuNPs into aggregated AuNPs.

[0003] This limitation has prompted the combination of nucleic acid nanotechnology and click chemistry to achieve programmable nanoparticles. HCR is an enzyme-free assisted isothermal amplification technology that can form linear nucleic acid polymers based on the Watson-Crick base pairing principle. The research group led by Qiao Zhaohui at Nanchang University used multivalent aptamers constructed by the HCR reaction, which simultaneously served as the structural backbone and signal carrier. The affinity of this multivalent aptamer was 33 times that of the monovalent aptamer. Similarly, Li Ming et al. from Jiangsu University achieved signal cascade amplification through the self-assembly of aptamer nucleic acid structures by CHA and HCR reactions, without the need for complex procedures or instruments. In addition, the research team led by Academician Tan Weihong assembled silver nanoclusters of DNA templates by triggering the HCR reaction to form multi-branched linear structures. This strategy had a 20-fold increase in sensitivity compared to DNA-AgNPs coupled with individual aptamers. Collectively, the nucleic acid nanostructures constructed by HCR can not only achieve the cascade amplification of nucleic acid signals but also potentially induce the cross-linking and aggregation of nanomaterials. Summary of the Invention

[0004] The object of the present invention is to propose an aptamer test strip with combined signal amplification of HCR and click chemistry, providing reference and technical support for improving the detection sensitivity of aptamer test strips.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: Prepare an aptamer test strip with combined signal amplification of HCR and click chemistry, which is prepared by the following method:

[0006] (1) Design a procymidone probe sequence, hairpin primer, capture probe, and quality control probe containing the initiation sequence respectively;

[0007] (2) Self-assemble the core nucleic acid backbone through HCR, and induce the cross-linking of azide gold nanoparticles by click chemical reaction to form high-density signal probes;

[0008] (3) Fix the capture probe and the quality control probe on the NC membrane respectively, and assemble the test strip;

[0009] Further, in the step (1), the procymidone probe sequence is 5’-TTTTTTTTTTTAGGGAATTCGTCGACGTAGCGAACGCATGGGCCGGCCGCGGCGCATGCGTCGACCTGACTAAAAGGGTCTGAGGG-3’, the sequence of hairpin primer 1 is 5’-ALK-TTTTTTTACTCCCCCAGGTGCCCCTCAGACCCTTTTAGT-3’, the sequence of hairpin primer 2 is 5’-ALK-TTTTTTGCACCTGGGGGAGTAACTAAAAGGGTCTGAGGG-3’, the sequence of capture probe is 5’-Biotin-GTTCGCTACGTCGACGAATT-3’, and the sequence of quality control probe is 5’-Biotin-AAAAAAAAAA-3’.

[0010] Further, in the step (2), the method for forming the nucleic acid nanoscaffold is as follows: the self-assembly of the core architecture of the signal enrichment probe is achieved through HCR. The hairpin sequence 1 and hairpin sequence 2 modified with alkynyl groups are mixed with the procymidone probe sequence and incubated at 37 °C for 1 h. The hairpin structures of hairpin sequence 1 and hairpin sequence 2 are successively opened. After a cascade reaction of multiple hybridization events, the nucleic acid nanoscaffold is formed.

[0011] Further, in the step (2), the method for preparing gold nanoparticles is as follows: 0.01% HAuCl4·3H2O is heated and shaken evenly, 1% sodium citrate solution is quickly added thereto and shaken evenly, and then the solution is continuously heated and cooled to room temperature.

[0012] Further, in the step (2), the method for preparing azide-functionalized gold nanoparticles is as follows: 0.1 M K2CO3 is added dropwise to AuNPs, the pH is adjusted to 8.5, the solution is concentrated to 5× concentration by centrifugation. Meanwhile, tris(2-carboxyethyl)phosphine is added to SH-PEG-N3 in an equal volume, and after mixing, it is added to 5× AuNPs and reacted overnight at room temperature.

[0013] Further, in the step (2), the method for preparing the signal enrichment probe is as follows: the nucleic acid nanoscaffold and an equal amount of azide-functionalized gold nanoparticles are mixed evenly, and then 10 mM sodium ascorbate and an equal amount of CuSO4·5H2O are added simultaneously to initiate a click chemical reaction to form the signal enrichment probe.

[0014] Further, the assembly method of the test strip in step (3) is as follows: The capture probe and the quality control probe are respectively mixed with streptavidin and incubated at 4°C for 1 h. The concentration of NaCl in the incubation system needs to be maintained at 1 M. After incubation, the formed capture probe-streptavidin and quality control probe-streptavidin are respectively sprayed onto the positions of the T line and the C line. The sample pad is immersed in the blocking buffer. After all the components are dried at 37°C, they are assembled onto the surface of the bottom plate in the order of "sample pad-NC membrane-absorbent pad", and then cut according to a width of 3.8 mm / strip to obtain the finished test strip.

[0015] An aptamer test strip with signal amplification by combining HCR and click chemistry prepared based on the above method, the aptamer test strip is strip-shaped, the sample addition position is at one end of the sample pad, and the detection area is located in the NC membrane area. It is characterized in that: from one end of the sample pad to one end of the absorbent pad, there are successively a T line and a C line.

[0016] The above-mentioned aptamer test strip with signal amplification by combining HCR and click chemistry is applied to the rapid detection of procymidone in agricultural products.

[0017] Further, the detection sensitivity of the procymidone is ≥0.054 ng / mL.

[0018] The present invention has the following beneficial effects:

[0019] The present invention designs a signal amplification strategy by combining click chemistry and HCR, which significantly improves the aggregation density and signal intensity of AuNPs, reduces the detection cost. The present invention has good affinity and specificity, and the recovery rate and stability in actual vegetable samples both meet the detection requirements. The present invention solves the problem of low sensitivity of aptamer test strips through a dual signal amplification mechanism, and effectively improves the efficiency of qualitative detection and the sensitivity of trace detection. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the design drawing of the aptamer test strip of the present invention.

[0022] Figure 2 It is the characterization diagram of the self-assembly process of the nucleic acid nanoskeleton of the present invention.

[0023] Figure 3 It is the characterization diagram of the preparation process of the signal enrichment probe of the present invention.

[0024] Figure 4 It is the optimization of the process parameters of the aptamer test strip of the present invention.

[0025] Figure 5 It is the detection principle and detection calibration curve of the aptamer test strip of the present invention.

[0026] Figure 6 It is the graph of the specificity and stability results of the aptamer test strip of the present invention. Detailed implementation manners

[0027] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0028] Example 1: Self-assembly of nucleic acid nanoskeleton based on HCR

[0029] The hairpin sequence 1 and hairpin sequence 2 modified with alkynyl groups are successively added to the amplification buffer (0.3 M NaCl, 10 mM Tris-HCl, pH 7.4), and the final concentration is 50 mM for both. The procymidone probe sequence is added, and the reaction concentration is 10 mM. Incubate at 37°C for 1 h. After adding the procymidone probe sequence, HCR is triggered, which will successively trigger the opening of the hairpin structures of hairpin sequence 1 and hairpin sequence 2. After a cascade reaction of multiple hybridization events, the nucleic acid nanoskeleton is formed.

[0030] Example 2: Preparation of AuNPs

[0031] All glass products need to be soaked in aqua regia for 24 h. First, heat 200 mL of HAuCl4·3H2O (0.01%) at high power of the microwave oven for 4 min, shake 2 - 3 times to make the solution temperature uniform, quickly add 5.4 mL of C6H5Na3O7 solution (1%) and shake well. Secondly, continue to heat the solution at medium-high power for 4 min. After cooling the solution to room temperature, make up the total volume to 200 mL.

[0032] Example 3: Preparation of azide-functionalized AuNPs

[0033] 0.1 M K2CO3 was added dropwise to 25 mL of AuNPs, and the pH was adjusted to 8.5. The solution was concentrated to 5× concentration by centrifugation (13,000 rpm, 20 min). Equal volume of tris(2-carboxyethyl)phosphine (5 mg / mL) was added to 15 μL of SH-PEG-N3 (100 μM, dissolved in methanol), and after mixing for 5 min, it was added to 5× AuNPs and reacted overnight at room temperature. The synthesized azide-functionalized AuNPs (AuNPs-N3) were centrifuged (13,000 rpm, 20 min) and washed to remove excess SH-PEG-N3, NaCl, and tris(2-carboxyethyl)phosphine. Both the supernatant buffer and the resuspension solution were methanol-water (2:1, v / v).

[0034] Example 4: Assembly of signal enrichment probe HCR-AuNPs

[0035] After 100 μL of nucleic acid nanoskeleton was mixed evenly with an equal amount of AuNPs-N3, 10 mM sodium ascorbate and an equal amount of CuSO4·5H2O were added simultaneously. Among them, sodium ascorbate needs to be freshly prepared. Sodium ascorbate can reduce Cu(II) to obtain Cu(I). The azide groups on the surface of AuNPs and the terminal alkyne groups of the nucleic acid nanoskeleton undergo a click reaction catalyzed by Cu(I). At the same time, the addition of sodium ascorbate can also effectively reduce the generation of oxidative coupling products. The above reactions were characterized by TEM, SEM, multi-functional microplate reader, and FTIR, etc.

[0036] Example 5: Assembly of test strip

[0037] 2.5 nmol of capture probe and 2.5 nmol of quality control probe were respectively mixed evenly with 175 μg of streptavidin and incubated at 4°C for 1 h. The concentration of NaCl in the incubation system needs to be maintained at 1 M. After incubation, the formed capture probe-streptavidin and quality control probe-streptavidin were respectively sprayed onto the positions of the T line and the C line. The sample pad was soaked in the blocking buffer (containing 0.15 M NaCl, 0.25% Triton-100, 0.02 M Tris-HCl). After all components were dried at 37°C, they were assembled onto the surface of the bottom plate in the order of "sample pad - NC membrane - absorbent pad" and cut according to a width of 3.8 mm / strip to obtain the finished test strip.

[0038] Example 6: Detection steps

[0039] 20 μL of the signal enrichment probe was added to 80 μL of the sample solution. The test strip was inserted into the sample pool for chromatography and incubation. The color development of the strip was observed to qualitatively analyze the sample. The picture data of the blank group and the experimental group were simultaneously acquired using a smartphone, and quantitative analysis was performed using ImageJ software.

Claims

1. An aptamer test strip with signal amplification by combining HCR and click chemistry, characterized in that, The steps are as follows: (1) Design a procymidone probe sequence containing a primer sequence, hairpin primers, capture probes, and quality control probes respectively; (2) Self-assemble the core nucleic acid backbone through HCR, and induce the cross-linking of azide-functionalized gold nanoparticles to form high-density signal probes through click chemistry reaction; (3) Fix the capture probe and the quality control probe onto the NC membrane respectively, and assemble the test strip; In the step (1), the procymidone probe sequence is 5’-TTTTTTTTTTTAGGGAATTCGTCGACGTAGCGAACGCATGGGCCGGCCGCGGCGCATGCGTCGACCTGACTAAAAGGGTCTGAGGG-3’, the sequence of hairpin primer 1 is 5’-ALK-TTTTTTTACTCCCCCAGGTGCCCCTCAGACCCTTTTAGT-3’, the sequence of hairpin primer 2 is 5’-ALK-TTTTTTGCACCTGGGGGAGTAACTAAAAGGGTCTGAGGG-3’, the sequence of the capture probe is 5’-Biotin-GTTCGCTACGTCGACGAATT-3’, and the sequence of the quality control probe is 5’-Biotin-AAAAAAAAAA-3’.

2. The aptamer test strip with signal amplification by combining HCR and click chemistry according to claim 1, characterized in that In the step (2), the method for forming the nucleic acid nanostructure is: realizing the self-assembly of the core architecture of the signal enrichment probe through HCR, mixing the hairpin sequence 1 modified with alkynyl and the hairpin sequence 2 with the procymidone probe sequence, incubating at 37 °C for 1 h, sequentially triggering the opening of the hairpin structures of the hairpin sequence 1 and the hairpin sequence 2, and forming the nucleic acid nanostructure after a cascade reaction of multiple hybridization events.

3. The aptamer test strip with signal amplification by combining HCR and click chemistry according to claim 1, characterized in that, In the step (2), the method for preparing gold nanoparticles is: heating 0.01% HAuCl4·3H2O and shaking evenly, quickly adding 1% sodium citrate solution thereto and shaking evenly, continuing to heat the solution and then cooling it to room temperature.

4. The aptamer test strip with combined signal amplification of HCR and click chemistry according to claim 1, wherein In the step (2), the method for preparing azide-functionalized gold nanoparticles is: dropwise adding 0.1 M K2CO3 to AuNPs, adjusting the pH to 8.5, concentrating the solution to 5× concentration by centrifugation, and at the same time adding an equal volume of tris(2-carboxyethyl)phosphine to 15 μL SH-PEG-N3, mixing evenly, and adding it to 5× AuNPs, and reacting overnight at room temperature.

5. The aptamer test strip for signal amplification by combining HCR and click chemistry according to claim 1, wherein In the step (2), the method for preparing the signal enrichment probe is: mixing the nucleic acid nanostructure with an equal amount of azide-functionalized gold nanoparticles evenly, and simultaneously adding 10 mM sodium ascorbate and an equal amount of CuSO4·5H2O, and forming the signal enrichment probe by triggering click chemistry reaction.

6. The aptamer test strip for combined signal amplification of HCR and click chemistry according to claim 1, characterized in that, The assembly method of the test strip in step (3) is as follows: The capture probe and the quality control probe are respectively mixed with streptavidin and incubated at 4 °C for 1 h. The concentration of NaCl in the incubation system needs to be maintained at 1 M. After incubation, the formed capture probe-streptavidin and quality control probe-streptavidin are respectively sprayed onto the positions of the T line and the C line. The sample pad is immersed in the blocking buffer. After all components are dried at 37 °C, they are assembled onto the surface of the bottom plate in the order of "sample pad - NC membrane - absorbent pad". After cutting according to the width of 3.8 mm / strip, the finished test strip is obtained.

7. An aptamer test strip with signal amplification by combining HCR and click chemistry, prepared by the method according to any one of claims 1-6, wherein the aptamer test strip is strip-shaped, the sample addition position is at one end of the sample pad, and the detection area is located in the NC membrane area, and is characterized in that: From one end of the sample pad to the absorbent pad end, there are the T line and the C line in sequence.

8. The method for using an aptamer test strip with signal amplification by combining HCR and click chemistry according to claim 7, characterized in that: 20 μL of the signal enrichment probe is mixed with 80 μL of the sample solution. The aptamer test strip is inserted into the sample pool for chromatography and incubation, and the color development of the strip is observed to qualitatively and quantitatively analyze the sample.

9. Application of an aptamer test strip with combined signal amplification of HCR and click chemistry according to claim 8 in the rapid detection of procymidone in agricultural products.

10. The application according to claim 9, characterized in that: The detection sensitivity of the procymidone is ≥ 0.054 ng / mL.

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