Lateral flow chromatography test strip for colorimetric detection of perfluorooctanoic acid

Abstract

The invention discloses a lateral flow chromatography test strip for detecting perfluorooctanoic acid by combining gold nanoparticles, a nucleic acid aptamer and an antibody. According to the test strip, a nucleic acid aptamer marked by gold nanoparticles is combined with PFOA molecules to form a nucleic acid aptamer with a G-quadruplex structure, and then the nucleic acid aptamer with the G-quadruplex structure is specifically recognized and captured by an anti-BG4 antibody, so that specific visual detection of perfluorooctanoic acid is realized. After a sample is injected, a G-quadruplex structure formed by the PFOA and the nucleic acid aptamer acts with the anti-BG4 antibody coated on the test strip, so that the gold nanoparticles are agglomerated to generate a chromogenic reaction, the signal intensity can be detected by naked eyes or reading equipment, the visual detection limit is 10 mu M, and the equipment detection limit is 304 nM. The test strip has the advantages of high detection speed, high specificity and high sensitivity, and is suitable for field detection and rapid screening. Compared with a traditional method, the method realizes simple, convenient and economical detection of PFOA, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to a nucleic acid aptamer test strip for detecting perfluorooctanoic acid, belonging to the field of molecular biology. Background Art

[0002] As a typical representative of perfluorocarboxylic acids, perfluorooctanoic acid (PFOA) was developed by an American company in 1951. Due to including a perfluorinated carbon chain that is both hydrophobic and lipophobic and a hydrophilic charged functional group, PFOA has surface activity and hydrophobic and lipophobic properties. Therefore, it is widely used in industrial production and consumer applications, such as in clothing and antifouling coatings; oil-resistant coatings for food contact paper; aviation hydraulic oil; fire-fighting foams; paints, adhesives, waxes, polishing agents and other products; and industrially used as additives and coatings. With the development of society, the demand for these products is increasing day by day, and PFOA is also released into the environment in large quantities. Unfortunately, however, their unique C-F bonds also make them resistant to typical degradation pathways (such as photolysis and biodegradation). Therefore, PFOA is called "forever chemicals". Humans are exposed to PFOA in a very wide range of ways in life, which can affect human health, including prostate cancer, thyroid diseases and the development of fetuses. Therefore, in addition to restricting the use of perfluorooctanoic acid at the production source, it is urgent to quickly and accurately detect perfluorooctanoic acid in the environment.

[0003] Currently, the traditional detection methods for PFOA are gas chromatography and high-performance liquid chromatography. This method requires collecting samples on-site and then taking them to a laboratory with professional instruments. This method can achieve high sensitivity and can find the exact molecular structure of any component present in the sample, but the biggest limitation of this method is its cost and complex operation process. It is affected by instrument and power requirements, and the cost of each sample is very high, and the analysis time is several hours. This is not ideal for the cost-effectiveness and rapid detection of PFOA. Therefore, there is an urgent need to establish a sensitive, simple and low-cost on-site rapid detection technology for perfluorooctanoic acid.

[0004] In the case where it is difficult to prepare high-affinity antibodies for PFOA, nucleic acid aptamers, as a new type of biorecognition element, provide a new perspective for the development of rapid detection of environmental pollutants. As a shorter single-stranded DNA or RNA, aptamers can bind to a variety of target substances with high specificity and high selectivity by forming hairpin, loop and G-quadruplex structures. Moreover, the DNA or RNA fragments screened by this systematic evolution of ligands by exponential enrichment (SELEX) are lower in cost, more stable in performance, and more portable in the synthesis and modification process, and are potential recognition elements for target detection.

[0005] In recent years, nucleic acid aptamers have been widely used to construct various biosensors for environmental detection. Therefore, replacing the antigen-antibody in LFS with aptamers can effectively expand the application range of the rapid detection technology of test strips and improve its sensitivity, and make the shelf life of LFS longer and the price lower. In summary, the aptamer-based sensing analysis method has become an important means for the detection of PFOA residues. Summary of the Invention

[0006] To solve the above problems, the present invention proposes a nucleic acid aptamer test strip for detecting perfluorooctanoic acid, which has a simple preparation process, low cost, and high accuracy and sensitivity.

[0007] The technical solution adopted by the present invention: A nucleic acid aptamer test strip for detecting perfluorooctanoic acid is realized as follows.

[0008] The present invention proposes a nucleic acid aptamer test strip for detecting perfluorooctanoic acid. The nucleic acid aptamer used is obtained through specific screening, can efficiently bind to perfluorooctanoic acid and produce a specific reaction to form a G-quadruplex structure. The sequence of the nucleic acid aptamer used is 5'-ctctcgggacgacGGCGTGGGGTGGTAGGCTGTAAAGGGGGTCgtcgtccc-3′, and the DNA sequence captured on the control line of the NC membrane is 5'-CCGTCGTCCCGAGAGTTTT-3'.

[0009] Preferably, after the 3'-end of the nucleic acid aptamer is modified with a thiol group, it is mixed evenly with the concentrated AuNPs solution and incubated at room temperature for 24 h to prepare a gold nucleic acid aptamer conjugate molecular recognition probe. Using BSA solution as a blocking agent can block the excess sites on the surface of the gold nucleic acid aptamer conjugate probe and avoid non-specific binding of the probe.

[0010] Preferably, the 3'-end of the captured DNA is modified with biotin and fixed on the NC membrane using streptavidin.

[0011] Preferably, the sample pad is used to remove impurities in the reaction solution and promote the chromatography process of the solution on the NC membrane. At the same time, the absorbent paper is assembled at the top of the test strip, and the capillary action provides a siphon driving force to enable the sample to uniformly penetrate to the detection line on the NC membrane.

[0012] Preferably, the conjugate pad is uniformly covered with the gold nucleic acid aptamer conjugate; the nitrocellulose membrane is provided with a detection line, and the BG4 antibody is sprayed on the detection line to recognize and capture the G-quadruplex structure formed by PFOA and the gold nucleic acid aptamer. After drying it at room temperature, it is cut with an automatic cutting machine, and the width of each Apt-LFS is 3 mm, and it is stored in a self-sealing bag containing a desiccant for standby.

[0013] Preferably, the processed sample pad, absorbent pad, conjugate pad, NC membrane and polyvinyl chloride bottom plate are assembled in sequence to prepare a complete lateral flow test strip.

[0014] Preferably, when using Apt-LFS to detect PFOA, directly add 5 μL of PFOA solution to the sample pad of the prepared lateral flow test strip, and the color change of the C / T line on the test strip can be observed with the naked eye within 10 minutes. After the color stabilizes, take a photo and record the image with a camera, and then use Image J and Origin 2021 software to read the gray value of the T line and perform quantitative analysis of PFOA.

[0015] The present invention proposes an application of a nucleic acid aptamer test strip for detecting perfluorooctanoic acid in environmental monitoring.

[0016] The present invention has the following beneficial effects.

[0017] (1) The present invention establishes a new rapid detection technology based on nucleic acid aptamer lateral flow chromatography strip (LFS), which is used for the first time in the on-site rapid detection of PFOA in the environment.

[0018] This method uses gold nanoparticles (AuNPs) modified with specific aptamers of perfluorooctanoic acid as probes, BG4 antibody as the capture molecule for the test line (T line), and capture DNA as the quality control line (C line). By observing the color change in the test area, qualitative detection of PFOA can be achieved with the naked eye within 10 minutes, and the visual limit is 1 μM. At the same time, through the analysis of the gray value of the T-line, a linear detection range of 1-250 μM can be achieved, and it has high accuracy and sensitivity.

[0019] (2) The nucleic acid aptamer test strip proposed by the present invention can be used to detect different types of perfluorinated pollutants by replacing different recognition elements (other types of aptamers), thereby expanding the scope of such detection technologies and applications. This technology provides technical support for the rapid and sensitive detection of various perfluorinated compounds in the environment. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of APT-LFS for detecting PFOA.

[0021] Figure 2 It is the characterization of AuNPs and gold nucleic acid aptamer conjugates. (A, B) TEM characterization images of AuNPs; (C) Gel electrophoresis diagram of AuNPs and gold nucleic acid aptamer conjugates.

[0022] Figure 3Feasibility verification of APT-LFS: Feasibility diagram of testing PFOA with test strips.

[0023] Figure 4 APT-LFS standard curve: (A) Detection images of PFOA at different concentrations; (B) Relationship between the gray value of line C and the concentration of PFOA.

[0024] Figure 5 APT-LFS detection performance test: (A) Detection images of different types of perfluorinated pollutants; (B) Selectivity of APT-LFS. (C) Detection images of PFOA before and after one month; (D) Stability of APT-LFS. Specific implementation mode

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments do not limit the present invention in any form.

[0026] Example 1: A nucleic acid aptamer test strip for detecting perfluorooctanoic acid, the specific steps are as follows.

[0027] The schematic diagram of APT-LFS for detecting PFOA is as Figure 1 shown. The LFS is successively composed of a PVC substrate, an NC membrane, a water absorption pad, a conjugate pad and a sample pad. The blotting paper is assembled at the top of the test strip to provide a siphon driving force by means of capillary action. The sample pad is used to remove impurities in the reaction solution and promote the chromatography process of the solution on the NC membrane. During the detection of PFOA, the sample solution is directly dropped onto the sample pad of the lateral flow chromatography test strip. Under the action of capillary force, the solution flowing through the sample pad reacts with the gold nucleic acid aptamer on the conjugate pad to form a G-quadruplex structure. The BG4 antibody on the test line on the NC membrane can specifically recognize the G-quadruplex structure, and the capture DNA on the quality control line binds to the excess gold nucleic acid aptamer, and the quantitative detection of PFOA is completed by using the color development of the C / T line. When the sample solution does not contain PFOA, no substance forms a G-quadruplex structure with the gold nucleic acid aptamer, resulting in the inability of the BG4 antibody to capture the gold nucleic acid aptamer, and no color is shown on the T line on the NC membrane, while the excess gold nucleic acid aptamer is captured by the capture DNA, and a red band will be shown. When PFOA is present, the G-quadruplex structure formed by the gold nucleic acid aptamer and PFOA specifically binds to the BG4 antibody, and the enrichment of AuNPs results in a clear red band that can be observed on the T line. At the same time, the excess gold nucleic acid aptamer is captured by the capture DNA, and a red band will also be shown. As the concentration of PFOA increases, the colorimetric brightness on the T line continuously increases until it stabilizes. In short, the color development degree on the T line is proportional to the concentration of PFOA.

[0028] The morphology of the prepared AuNPs particles was characterized by using TEM. Figure 2(A) shows the AuNPs particles captured under a transmission electron microscope with a scale bar of 100 nm and 50 nm. The results indicate that the prepared AuNPs are uniformly dispersed particles, and according to the scale bar, the particle size of AuNPs can be calculated to be about 15 nm. Using ssDNA as the recognition element, it is coupled with AuNPs to prepare the gold aptamer conjugate, and its successful preparation is the key to quickly and accurately detecting PFOA. Therefore, 3% agarose gel electrophoresis is used to verify the successful preparation of the gold aptamer. As shown in Figure (2)C, due to the binding of the aptamer, the overall charge, size, and surface charge density of the gold aptamer conjugate will be different, and usually, it will show a different migration rate from that of pure gold nanoparticles in gel electrophoresis. The aptamer is negatively charged, which will make the conjugate overall negatively charged, thus affecting its migration speed in gel electrophoresis.

[0029] The feasibility of the prepared test strip for detecting PFOA is verified again by assembling a lateral flow chromatographic test strip to detect different sample solutions. The results are as Figure 3 shown. First, PBS buffer is dropped on the test strip to study the interference signal of the gold aptamer. At this time, there is no color development on the T line, and color development occurs on the C line (Sample 1), indicating that the interference signal is extremely weak. When a PFOA solution (Sample 2) is dropped on the test strip, both the C / T lines show color development. The results show that the gold aptamer conjugate can specifically recognize PFOA, and thus the formed G-quadruplex structure can be specifically recognized by the BG4 antibody. All of the above results can illustrate that the designed lateral flow chromatographic test strip can effectively achieve the real-time detection of PFOA, and the detection results can be visually distinguished by the naked eye on-site.

[0030] Under the optimal conditions, by analyzing the detection range and the lowest detection limit, the sensitivity of APT-LFS to PFOA is investigated. Different concentrations of PFOA solutions are dropped on the test strip, and the color development of the T line at 10 min is photographed and the gray value is analyzed. The results are as Figure 4 (A) shown. As the concentration of PFOA increases, it can be clearly seen by the naked eye that the colorimetric brightness on the T line increases significantly. In addition, Figure 4 (B) shows that the gray value at the T line increases with the increase in the concentration of PFOA and tends to be stable when the concentration reaches 250 μM. In the linear range of 1 - 250 μM, there is a strong linear relationship between the gray value of the T line and the concentration of PFOA. The regression equation is y = 18.5152x + 272, R 2 = 0.995, and its detection limit is 304 nM.

[0031] Under the optimal conditions, by comparing the color development of other perfluorinated pollutants and perfluorooctanoic acid on the test strip, its selectivity for PFOA is investigated. Perfluorooctanoic acid with a concentration of 250 μM is selected for testing. Figure 5 (A) and Figure 5(B) shows that the gray value of perfluorooctanoic acid is significantly higher than that of other perfluorinated compounds, and the color difference from other test strips is visible to the naked eye. This verifies the high affinity and specificity of the gold nucleic acid aptamer for PFOA. In addition, the stability of the APT-LFS on-site detection was evaluated by comparing the PFOA test strips stored at room temperature before and after one month. The results are shown in 5(C) and 5(D). The colorimetric brightness of the T line changed very little before and after one month and was not easily observable by the naked eye. The gray value of the T line remained almost unchanged. This indicates that the developed method has good stability in practical applications.

[0032] The above-described embodiments only represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. A lateral flow chromatography test strip for colorimetric detection of PFOA, characterized in that: The lateral flow chromatography test strip comprises a nucleic acid aptamer that specifically recognizes PFOA, an anti-BG4 antibody that specifically recognizes a G-quartet structure, and capture DNA.

2. The lateral flow chromatography test strip according to claim 1, characterized in that: The nucleic acid aptamer is selected through specific screening, can efficiently and specifically combine with PFO A to form a G-quartet structure, and its sequence is: 5'-ctctcgggacgacGGCGTGGGG TGGTAGGCTGTAAAGGGGGTCgtcgtccc-3'.

3. The lateral flow chromatography test strip according to claim 1, 2, characterized in that: The nucleic acid aptamer is modified with a thiol group at the 3' end to bind to the gold nanoparticles.

4. The lateral flow chromatography test strip according to claim 1, characterized in that: The anti-BG4 antibody can specifically recognize a nucleic acid having a G-quartet structure.

5. The lateral flow chromatography test strip according to claim 1, 3, characterized in that: The diameter of the gold nanoparticles ranges from 10 to 30 nm.

6. The lateral flow chromatography test strip according to claim 1, 5, characterized in that: The diameter of the gold nanoparticles is 15 nm.

7. The lateral flow chromatography test strip according to claim 1, characterized in that: The capture DNA is used to bind to the redundant gold nanoparticle-labeled nucleic acid aptamer, and its sequence is: 5'-CCGTCGTCCCGAGAGTTTT-3'.

8. The lateral flow chromatography test strip according to claim 1, 7, characterized in that: The capture DNA is modified with biotin at the 3' end so that it can be fixed on the nitrocellulose membrane through reaction with streptavidin.

9. The lateral flow chromatography test strip according to claims 1-8, characterized in that: The nucleic acid aptamer labeled with gold nanoparticles is embedded in the conjugate pad, the anti-BG4 antibody is embedded in the T line of the detection pad, the capture DNA is embedded in the C line of the detection pad, and finally the sample pad and the absorption pad are added to assemble into a lateral flow chromatography test strip.

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