Lateral flow analysis test strip for detecting vascular endothelial growth factor 165

Abstract

The invention relates to the technical field of biochemistry, in particular to a lateral flow analysis test strip for detecting a vascular endothelial growth factor 165. In the test strip provided by the invention, a detection line and a quality control line are marked on a nitrocellulose membrane, the detection line is coated with a VEGF165 antibody, and the quality control line is coated with a conjugate of streptavidin and a biotinylated VEGF165 aptamer complementary chain. Compared with an existing VEGF165 detection method, the method has the advantages that the VEGF165 aptamer is introduced, compared with a whole antibody system, the cost is reduced, the limitation that the variety of VEGF165 antibodies in the market is limited and most of the VEGF165 antibodies need to be imported is overcome to a certain extent, signal amplification detection can be achieved without a complex and high-cost DNA amplification technology, visual detection is achieved, and the method is suitable for large-scale popularization and application. The detection limit can be as low as 0.12 ng / mL.

Description

Technical Field

[0001] The invention relates to the field of biochemical technology, and in particular to a lateral flow analysis test strip for detecting vascular endothelial growth factor 165. Background Art

[0002] Vascular Endothelial Growth Factor (VEGF) is a type of growth factor that has a highly specific mitogenic effect on vascular endothelial cells and plays a key role in angiogenesis. VEGF165 is an important member of the VEGF family. It is composed of 165 amino acids and has strong biological activity. Its structure contains multiple functional domains that can specifically bind to corresponding receptors, thereby initiating a series of intracellular signal transduction pathways and participating in regulating the proliferation, migration, and increased vascular permeability of vascular endothelial cells.

[0003] VEGF165 plays a vital role in the formation and development of tumors. In the early stages of tumor formation, tumor cells are in a relatively hypoxic environment, which will induce upregulation of VEGF165 expression. VEGF165 can act on vascular endothelial cells, promote the proliferation and migration of endothelial cells, and induce angiogenesis by activating a series of intracellular signaling pathways. These new blood vessels provide sufficient oxygen and nutrients for tumor cells, support the continuous proliferation of tumor cells, and promote the gradual increase in tumor volume. In addition, VEGF165 can also change the tumor microenvironment, making the extracellular matrix around tumor cells more easily degraded, enhancing the invasive ability of tumor cells, helping them to break through the basement membrane and infiltrate into surrounding tissues, and creating favorable conditions for subsequent entry into the blood circulation for distant metastasis. Therefore, the development of VEGF165 detection methods and devices is of great significance for early diagnosis and treatment of tumors.

[0004] At present, people mainly use antibodies as molecular recognition elements to design various VEGF165 detection methods and reagents, including the classic enzyme-linked immunosorbent assay (ELISA) based on the "capture antibody-antigen-signal antibody" sandwich analysis strategy. In the past decade, researchers have screened nucleic acid aptamers for VEGF165, thereby replacing antibodies to construct a variety of detection methods, including fluorescence, electrochemical and other methods, and combined with nucleic acid amplification technology (such as hybridization chain reaction) to achieve a significant improvement in detection sensitivity. However, most methods are complicated to operate and have poor portability, and cannot meet the needs of ordinary people for bedside testing.

[0005] Lateral flow assay (LFA) is a rapid detection method with fast response and easy storage. Lateral flow assay colorimetric test strips have been widely used in human viral infection screening and food safety assessment, etc. There are still no reports on lateral flow assay strips for VEGF165. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a lateral flow analysis test strip for detecting vascular endothelial growth factor 165.

[0007] The lateral flow analysis test strip for detecting vascular endothelial growth factor 165 provided by the present invention comprises: a sample pad, a nitrocellulose membrane, a water-absorbing pad and a back plate;

[0008] The nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is coated with VEGF165 antibody, and the quality control line is coated with a conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain.

[0009] The present invention replaces antibodies with aptamers to design a simple hybrid sandwich principle for detecting VEGF165 based on capture antibodies and signal aptamers, and realizes the application of this principle in test strip preparation and portable and visual detection of VEGF165 for the first time.

[0010] The antibodies used in the present invention are commercially available, and the aptamers used have been screened. Among the multiple aptamers involved in the experiment, the detection signal of aptamer No. 504 used in the present invention is the highest. In the present invention, the complementary strand of the VEGF165 aptamer has a nucleic acid sequence as shown in SEQ ID NO:1.

[0011] The present invention also optimizes the size of the test strip and the setting of the detection line and the quality control line. The results show that under the specific distance and size setting, better detection effect can be obtained. In the present invention, the distance between the detection line and the quality control line is 5mm; in the present invention, the width of the test strip is 3mm, the sample pad is 2.2cm long, the nitrocellulose membrane is 2.5cm long, and the absorbent pad is 1.7cm long.

[0012] The present invention also provides a method for preparing the lateral flow analysis test strip as described above, comprising:

[0013] Soak the sample pad in the treatment solution and then dry it;

[0014] The detection line of the nitrocellulose membrane is sprayed with VEGF165 antibody, and the quality control line is sprayed with a conjugate of streptavidin and the complementary chain of the biotinylated VEGF165 aptamer;

[0015] The sample pad, the nitrocellulose membrane and the absorbent pad are fixed to the back plate in order to obtain the test strip.

[0016] In the present invention, the sample pad is soaked in the treatment solution for 5 to 20 minutes. Preferably, the soaking time is 8 to 12 minutes, for example, soaking for 8 minutes, or 9 minutes, or 10 minutes, or 11 minutes, or 12 minutes.

[0017] After the sample pad is soaked in the treatment liquid, a drying step is further included, and the drying temperature is 30-40° C. For example, the drying temperature is 35-40° C. Preferably, the drying temperature is 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C.

[0018] In the present invention, the pH of the treatment liquid is 7.0 to 8.0. Preferably, the pH of the treatment liquid is 7.5 to 7.7, for example, the pH of the treatment liquid is 7.5, 7.6 or 7.7.

[0019] In the present invention, the treatment solution comprises water and 0.01-0.1 M Tris, 8-12 mM EDTA, 0.20-0.30 wt % Triton X-100 and 0.8-1.2 wt % BSA. In a specific embodiment, the treatment solution comprises water and 0.05 M Tris, 10 mM EDTA, 0.25 wt % Triton X-100 and 1 wt % BSA, with a pH of 7.6.

[0020] In the present invention, the preparation method of the conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain comprises: diluting the biotinylated VEGF165 aptamer complementary chain to a concentration of 10 μM with PBS buffer, heating in a 95°C water bath for 3 minutes, standing at room temperature for 30 minutes, adding an equal volume of 0.1 mg / mL streptavidin, shaking at room temperature for 1 hour, and then incubating at 0-4°C for 8-12 hours; then centrifuging at 14000rcf for 10 minutes with a 30KDa ultrafiltration tube, discarding the supernatant and washing with PBS buffer to obtain the conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain.

[0021] In the present invention, the spraying amount of the VEGF165 antibody is 0.8 μg / cm.

[0022] In the present invention, the spraying amount of the conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain is 4.56 pmol / cm.

[0023] Furthermore, the present invention also provides a lateral flow analysis kit for detecting vascular endothelial growth factor 165, which comprises the test strip and the signal probe solution as described above;

[0024] The signal probe solution includes biotinylated VEGF165 aptamer and streptavidin-modified gold nanoparticles.

[0025] The concentration of the aptamer in the gold nanosignal probe (BVapt / SA-AuNPs) of the present invention is an important parameter affecting the detection performance of the test strip. After optimization, in the present invention, the concentration of the biotinylated VEGF165 aptamer in the signal probe solution in the final detection solution is 100 to 400 nM. For example, the concentration of the VEGF165 aptamer in the final detection solution is 100 nM, 200 nM or 400 nM.

[0026] The concentration of the streptavidin-modified gold nanoparticles in the signal probe solution also has an important influence on the color development effect. In preliminary experiments, it was found that the gold nanoparticle concentration was too low (such as 0.03nM) to be difficult to distinguish with the naked eye; the gold nanoparticle concentration was normal at 0.06nM, 0.08nM, and 0.12nM. Finally, 0.12nM was selected, and the band was very clear. In the present invention, the concentration of the streptavidin-modified gold nanoparticles in the signal probe solution in the final detection solution is 0.06-0.12nM. For example, the concentration of the streptavidin-modified gold nanoparticles in the final detection solution is 0.06nM, 0.08nM or 0.12nM.

[0027] In the present invention, the preparation method of the signal probe solution includes: diluting the biotinylated VEGF165 aptamer to 10 μM with PBS buffer, heating it in a 95°C water bath for 3 minutes and then standing at room temperature for 30 minutes, then mixing it with streptavidin-modified gold nanoparticles and incubating at room temperature for 2 hours to prepare a signal probe solution.

[0028] The present invention provides a method for detecting VEGF165, which comprises mixing a signal probe solution with a sample to be tested, then dripping the solution onto a sample pad of a test strip, and observing the solution after chromatography.

[0029] The present invention performs basic quantitative analysis of VEGF165 based on the LFA test strip of the hybrid sandwich structure of "antibody / VEGF165 / signal probe". When there is no VEGF165 in the sample dripped on the sample pad, the BVapt / SA-AuNPs signal probe in the sample cannot be captured by the VEGF165 antibody (Ab) on the detection line during the chromatography process, so the color of the detection line will not change, but the signal probe will hybridize with the aptamer complementary chain BCVapt modified on the quality control line, so the color of the quality control line will change from colorless to red. When VEGF165 is contained in the sample to be tested, VEGF165 is first recognized by the aptamer on the signal probe, and then captured by the VEGF165 antibody on the detection line during the chromatography process, generating the Ab / VEGF165 / BVapt / SA-AuNPs hybrid sandwich structure, so the color of the detection line changes from colorless to red, and the remaining BVapt / SA-AuNPs signal probe hybridizes with the BCVapt modified on the quality control line, and the color also changes from colorless to red. When one red bar appears on the test strip, it means that the sample does not contain VEGF165; when two red bars appear, it means that the sample contains VEGF165.

[0030] In order to further improve the detection sensitivity of the LFA test strip, the present invention combines a signal amplification strategy of in-situ reduction of chloroauric acid with the basic detection principle.

[0031] In order to achieve signal amplification, the kit of the present invention also includes a cleaning solution, a chloroauric acid solution and a hydroquinone solution.

[0032] In the present invention, the cleaning solution is a PBST solution containing Tween, wherein the Tween is Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Preferably, the cleaning solution contains Tween 20.

[0033] In the present invention, the mass fraction of Tween 20 in the cleaning solution is 0.1% to 1%. Preferably, the mass fraction of Tween 20 in the cleaning solution is 0.3% to 0.7%. For example, the mass fraction of Tween 20 in the cleaning solution is 0.3%, 0.4%, 0.5%, 0.6% or 0.7%.

[0034] In some specific embodiments, the washing solution is a PBST solution containing 0.5% Tween 20.

[0035] In the present invention, the mass fraction of chloroauric acid in the chloroauric acid solution is 0.1% to 1%. Preferably, the mass fraction of chloroauric acid in the chloroauric acid solution is 0.3% to 0.7%. For example, the mass fraction of chloroauric acid in the chloroauric acid solution is 0.3%, 0.4%, 0.5%, 0.6% or 0.7%.

[0036] In the present invention, the concentration of hydroquinone in the hydroquinone solution is 5 to 15 mM. Preferably, the concentration of hydroquinone in the hydroquinone solution is 8 to 12 mM. For example, the concentration of hydroquinone in the hydroquinone solution is 8 mM, 9 mM, 10 mM, 11 mM or 12 mM.

[0037] The present invention utilizes chloroauric acid solution to soak the detection line and the quality control line, and then adds hydroquinone solution to further reduce the chloroauric acid on the gold nanoparticles, thereby achieving the regrowth of gold on the gold nanoparticles. The colors of the two lines will change from red to black. The higher the concentration of VEGF165, the more gold nanoparticles will be bound to the detection line, the more chloroauric acid will be reduced by hydroquinone, and the stronger the signal of the detection line will be.

[0038] The present invention also provides a detection method for detecting VEGF165, which includes mixing a signal probe solution with a sample to be tested, and then dripping it onto a sample pad of a test strip, washing the test strip with a cleaning solution after chromatography, and then soaking the detection line and the quality control line with a chloroauric acid solution, and then soaking the detection line and the quality control line with a hydroquinone solution, and then observing.

[0039] In the present invention, the immersion time of the chloroauric acid solution is 0.5 to 3 minutes, preferably, the immersion time is 0.5 to 2 minutes, for example, the immersion time is 0.5 minutes, 1 minute or 1.5 minutes.

[0040] In the present invention, the immersion time of the hydroquinone solution is 0.5 to 3 minutes, preferably, the immersion time is 0.5 to 2 minutes, for example, the immersion time is 0.5 minutes, 1 minute or 1.5 minutes.

[0041] Preferably, the immersion time of the chloroauric acid solution is 1 minute, and the immersion time of the hydroquinone solution is 1 minute.

[0042] Furthermore, the kit of the present invention also includes a standard solution, wherein the standard solution includes 1 μg / mL VEGF165 and 10% BSA.

[0043] In the test strip provided by the present invention, a detection line and a quality control line are marked on the nitrocellulose membrane, the detection line is coated with a VEGF165 antibody, and the quality control line is coated with a conjugate of streptavidin and a complementary chain of a biotinylated VEGF165 aptamer. Compared with the existing VEGF165 detection method, the method of the present invention introduces a VEGF165 aptamer, which reduces the cost relative to the full antibody system, and overcomes the limitation that the types of VEGF165 antibodies on the market are limited and most of them must be imported to a certain extent. In addition, the method can realize signal amplification detection without complex and high-cost DNA amplification technology, realizes visual detection, and the detection limit can be as low as 0.12ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the schematic diagram of the LFA test strip for detecting VEGF165;

[0045] Figure 2 The responses of different aptamer sequences to VEGF165 (0, 50 ng / mL) based on horseradish peroxidase-linked assay;

[0046] Figure 3 The color development of the test strip to VEGF165 (0, 25 ng / mL) under different aptamer concentrations;

[0047] Figure 4 For the LFA test strips to quantify different concentrations of VEGF165;

[0048] Figure 5 The LFA test strip combined with the gold in situ growth strategy was used to analyze different concentrations of VEGF165;

[0049] Figure 6 The figure shows the change of relative gray value of LFA test strip in response to different concentrations of VEGF165. The inset shows the linear relationship between the concentration of VEGF165 (5-50 ng / mL) and the relative gray value.

[0050] Figure 7 The responses of the LFA test strips in the presence of different markers;

[0051] Figure 8 This is a physical picture of the prepared VEGF165 test strip. DETAILED DESCRIPTION

[0052] The present invention provides a lateral flow analysis test strip for detecting vascular endothelial growth factor 165. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0054] The present invention will be further described below in conjunction with embodiments:

[0055] Example 1

[0056] The test strips and basic detection principles of the present invention are as follows Figure 1shown.

[0057] The test strip consists of three parts: a sample pad, a nitrocellulose membrane, and a water-absorbing pad. The nitrocellulose membrane is marked with a test line and a quality control line. The test line (T line) is loaded with VEGF165 antibodies, while the biotinylated DNA (BCVapt) complementary to the VEGF165 aptamer is modified to the quality control line (C line) through the strong interaction between streptavidin and biotin. The biotinylated VEGF165 aptamer (BVapt) is connected to the streptavidin-modified gold nanoparticles (AuNPs) through the strong interaction between biotin and streptavidin to generate a signal probe.

[0058] When there is no VEGF165 in the sample dropped on the sample pad, the BVapt / SA-AuNPs signal probe in the sample cannot be captured by the VEGF165 antibody on the test line during the chromatography process, so the color of the test line will not change, but the signal probe will hybridize with the modified BCVapt on the quality control line, so the color of the quality control line will change from colorless to red. When the sample to be tested contains VEGF165, VEGF165 is first recognized by the aptamer on the signal probe, and then captured by the VEGF165 antibody (Ab) on the test line during the chromatography process, generating an Ab / VEGF165 / BVapt / SA-AuNPs hybrid sandwich structure, so the color of the test line changes from colorless to red, and the remaining BVapt / SA-AuNPs signal probe hybridizes with the modified BCVapt on the quality control line, and the color also changes from colorless to red. When a red bar appears on the test strip, it means that the sample does not have VEGF165; when two red bars appear, it means that the sample contains VEGF165.

[0059] Example 2 Pairing of VEGF165 Antibody and Aptamer

[0060] The premise of the "capture antibody-antigen-signal aptamer" hybrid sandwich analysis method of the present invention is the successful pairing of the molecular recognition element antibody and the aptamer. Therefore, the inventors used an enzyme-linked immunosorbent assay method to adsorb and fix a well-used VEGF165 antibody (Ab) on a 96-well plate as a capture probe, and screened aptamers that could pair with the antibody from four VEGF165 aptamer DNA sequences (501, 502, 503, 504).

[0061] Sequences of four VEGF165 aptamers:

[0062] 501,5'-TTTTTTCCGTCTTCCAGACAAGAGTGCAGGG-3';

[0063] 502,5'-TTTTTTTGTGGGGGTGGACGGGCCGGGTAGA-3';.

[0064] 503,5'-TTTTTTGGGGCCCGTCCGTATGGTGGGTGTGCTGGCC-3';

[0065] 504(BVapt),5'-TTTTTTCAATTGGGCCCGTCCGTATGGTGGGT-3'

[0066] The 5' ends of the four aptamer sequences were modified with biotin for linking streptavidin-horseradish peroxidase covalent complexes. Horseradish peroxidase can catalyze hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine to generate a blue substance to indicate the content of the target.

[0067] like Figure 2 As shown in a, the four aptamers combined with Ab showed different UV response values ​​at 50 ng / mL and different detection backgrounds (0 ng / mL). In order to more accurately compare the pairing of the four aptamers and antibodies, the inventors calculated the signal ratio R by dividing the UV response value at 50 ng / mL by the signal response value at 0 ng / mL. The results are shown in Figure 2 As shown in b. The signal ratios R of the four aptamers combined with Ab are as follows from high to low: R 504 >R 503 >R 501 >R 502 , where the signal ratio of 504 aptamer combined with Ab was the highest, which was 8.87. Therefore, the present invention uses 504 sequence as aptamer (BVapt) and Ab to pair to construct VEGF165 detection LFA test strip.

[0068] Example 3 Optimization of VEGF165 aptamer concentration

[0069] The concentration of the aptamer in the gold nanosignal probe (BVapt / SA-AuNPs) of the present invention is an important parameter affecting the detection performance of the test strip. Therefore, the inventors optimized the concentration of the aptamer in the LFA method. Figure 3As shown, when the concentration of the aptamer BVapt is lower than 100nM, even if the sample solution does not contain VEGF165, the detection line will have obvious color, that is, a false positive phenomenon occurs. The color of the test strip detection line under the condition of low concentration aptamer indicates that AuNPs are significantly aggregated. As the concentration of the aptamer increases, the false positive phenomenon gradually disappears, and the aggregation phenomenon of AuNPs also gradually weakens, indicating that BVapt / SA-AuNPs gradually tends to be stable in the solution. The results show that high concentrations of VEGF aptamers are conducive to the stability of AuNPs in the solution to be tested. In addition, when the concentration of the aptamer is too high (such as 400nM), the color of the quality control line on the test strip fades, and the detection line signal begins to weaken. This is because the steric hindrance generated by the high density of BVapt on AuNPs affects the hybridization of BVapt with DNA on the C line and the recognition of VEGF165 by BVapt. Therefore, the optimal concentration of the aptamer in the present invention is 200nM.

[0070] Example 4 Preparation of test strips

[0071] 1. Reagents, Materials, and Instruments

[0072] The VEGF165 monoclonal antibody (Ab) and VEGF165 antigen purchased in the present invention were purchased from Abcam (Shanghai) Trading Co., Ltd.; other proteins used were purchased from Shanghai Lingchao Biotechnology Co., Ltd.;

[0073] The sequence of the biotinylated VEGF165 aptamer (BVapt) used is shown in SEQ ID No. 1, specifically:

[0074] 5'-TTTTTTCAATTGGGCCCGTCCGTATGGTGGGT-3',

[0075] The sequence of the biotinylated complementary chain (BCVapt) used is shown in SEQ ID No. 2, specifically:

[0076] 5'-TTTTTTACCCACCATACGGACGGGCCCAATTG-3',

[0077] All were synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.; the test strip materials and film-scratching instruments used were purchased from Shanghai Jinbiao Biotechnology Co., Ltd. The gold nanoparticle (AuNPs) solution used was synthesized by reducing chloroauric acid with sodium citrate.

[0078] 2. Preparation of LFA Test Strips

[0079] The test strip is mainly composed of a sample pad, a nitrocellulose membrane, a water-absorbing pad, and a backing plate. Figure 8 The specific preparation process is as follows:

[0080] First, use a manual paper cutter to cut a 2.2 cm wide, 30 cm long sample pad; a 2.5 cm wide, 30 cm long nitrocellulose membrane; and a 1.7 cm wide, 30 cm long absorbent pad. Soak the sample pad in treatment solution (pH = 7.6, 0.05 M Tris buffer, 10 mM ethylenediaminetetraacetic acid, 0.25% Triton X-100, 1% BSA) for 10 minutes, and then place it in a vacuum drying oven at 37 ° C overnight to dry.

[0081] BCVapt was diluted to 10 μM with PBS buffer, heated in a 95°C water bath for 3 minutes, and then allowed to stand at room temperature for 30 minutes before adding an equal volume of 0.1 mg / mL streptavidin. The mixture was shaken on a vortex mixer at room temperature for 1 hour, and then placed in a refrigerator for overnight incubation at 4°C. The next day, the mixture was centrifuged at 14,000 rcf for 10 minutes using a 30KDa ultrafiltration tube, the supernatant was discarded, and the mixture was washed twice with PBS buffer for 10 minutes each time. Finally, the sample required for the quality control line (SA-BCVapt, 7.6 μM) was obtained after being concentrated 10 times.

[0082] After cleaning the double pumps of the membrane scrubber, 24 μl of VEGF165 antibody Ab (1 mg / mL) and 18 μl of SA-BCVapt (7.6 μM) were sprayed onto the test line (T line) and the quality control line (C line) on the nitrocellulose membrane, with a spacing of 5 mm between the two lines; then dried in a 37°C vacuum drying oven for 4 hours; then the sample pad, nitrocellulose membrane, and absorbent pad were fixed to the back plate in order, and cut into 3 mm wide test strips with a microcomputer cutting machine, which were sealed and stored in a desiccator for subsequent use. Example 5 Simple detection of VEGF165 based on LFA test strips

[0083] BVapt was diluted to 10 μM with PBS buffer, heated in a 95°C water bath for 3 minutes, and then allowed to stand at room temperature for 30 minutes. Then, it was mixed with a certain amount of SA-AuNPs and incubated at room temperature for 2 hours to prepare a signal probe solution, in which the BVapt concentration was 0.52 μM and the SA-AuNPs concentration was 0.31 nM; VEGF165 protein was diluted to 1 μg / mL with PBS buffer, and then different concentrations of VEGF165 protein detection solutions (0-80 ng / mL) were prepared using the 1 μg / mL VEGF165 solution, 10% BSA solution, signal probe solution and PBS buffer, and mixed on a vortex mixer. The mixed solution had a BVapt concentration of 200 nM, a SA-AuNPs concentration of 0.12 nM, and a BSA concentration of 2%; 60 μL of the detection solution was added dropwise to the sample pad of the test strip prepared in Example 4, and photographed with a camera after chromatography for 10 minutes.

[0084] The results are as follows Figure 4As shown in the figure, as the concentration of VEGF165 increases, more gold nanoparticles are bound to the test line of the test strip, and the color gradually deepens. The lowest VEGF165 that can be detected by visual inspection strip is 20ng / mL.

[0085] Example 6 VEGF165 signal amplification detection based on LFA test strips

[0086] In order to further improve the detection sensitivity of the LFA test strip, the present invention combines a signal amplification strategy of in-situ reduction of chloroauric acid on the basis of the basic detection principle of Example 5, that is, after the sample is chromatographed on the test strip for 10 minutes, the detection line and the quality control line are soaked in chloroauric acid solution, and then a hydroquinone solution is added to further reduce the chloroauric acid on the gold nanoparticles to achieve the regrowth of gold on the gold nanoparticles, and the color of the two lines will change from red to black; the higher the concentration of VEGF165, the more gold nanoparticles are bound to the detection line, the more chloroauric acid is reduced by hydroquinone, and the stronger the signal of the detection line.

[0087] like Figure 5 As shown in the figure, after the combined signal amplification strategy, the test strip can be observed by naked eyes at a minimum of 0.5 ng / mL of VEGF165, which is 40 times lower than before the signal amplification. At the same time, the present invention can use imageJ software to analyze the relative grayscale of the detection line ( Figure 6 ), the relative grayscale and VEGF165 concentration showed a good linear relationship in the range of 5-50 ng / mL, and the obtained linear regression equation was: Ai = 6.53098 + 0.73377 × C (VEGF165) / ng·mL -1 (R 2 =0.99542), thus the detection limit was calculated to be 0.12 ng / mL.

[0088] Example 7 Selectivity of VEGF165 Test Strips

[0089] The present invention uses several common disease biomarkers to evaluate the selectivity of the LFA test strip, which are myoglobin (Mb), cardiac fatty acid binding protein (hFABP), carcinoembryonic antigen (CEA) and prostate specific antigen (PSA), and the marker concentration used is 50ng / mL. The above antigens are used for the specific evaluation of VEGF165 detection on the lateral flow analysis test strip prepared in Example 4.

[0090] BVapt was diluted to 10 μM with PBS buffer, heated in a 95°C water bath for 3 minutes, and then allowed to stand at room temperature for 30 minutes. Then, it was mixed with a certain amount of SA-AuNPs and incubated at room temperature for 2 hours to prepare a signal probe solution, in which the concentration of BVapt was 0.52 μM and the concentration of SA-AuNPs was 0.31 nM. Then, different marker detection solutions (50 ng / mL) were prepared using 1 μg / mL of each marker solution, 10% BSA solution, signal probe solution and PBS buffer, and mixed on a vortex mixer. The concentration of BVapt was 200nM, the concentration of SA-AuNPs was 0.12nM, and the concentration of BSA was 2%. 60μL of the mixture was added to the sample pad of the test strip and chromatographed for 10 minutes. Then, the test strip was washed with 200μL of PBST solution containing 0.5% Tween 20, and then the test strip was immersed in 200μL of 0.5% chloroauric acid solution for 1 minute. Then, the test strip was taken out and immersed in 200μL of 10mM hydroquinone solution for 1 minute. Finally, the test strip was taken out and the signal amplified image was recorded with a camera. The gray value of the test line of the test strip was read and analyzed using Image J software ( Figure 7 ).

[0091] The results showed that the test strip only had a significant response to VEGF165, confirming that the test strip had a high specificity for VEGF165.

[0092] Example 8 Evaluation of the suitability of VEGF165 test strips

[0093] The present invention selected normal human serum from a local hospital for the lateral flow analysis test strip prepared in Example 4 to evaluate the suitability of VEGF165 in human body fluids. The difference from Example 6 is that 1% normal human serum is added to the final detection solution, and the serum is provided free of charge by the First Hospital of Jilin University.

[0094] BVapt was diluted to 10 μM with PBS buffer, heated in a 95°C water bath for 3 minutes, and then allowed to stand at room temperature for 30 minutes. Then, it was mixed with a certain amount of SA-AuNPs and incubated at room temperature for 2 hours to prepare a signal probe solution, in which the concentration of BVapt was 0.52 μM and the concentration of SA-AuNPs was 0.31 nM. The serum was centrifuged twice at 10,000 rpm for 20 minutes each time to remove the precipitate in the serum. Then, 1 μg / mL of each marker solution, serum, 10% BSA solution, signal probe solution and PBS buffer were used to prepare VEGF detection solutions of different concentrations. The mixture was mixed on a vortex mixer, wherein the concentration of BVapt was 200 nM, the concentration of SA-AuNPs was 0.12 nM, the concentration of BSA was 2%, and the concentration of serum was 1%; 60 μL of the mixture was added dropwise to the sample pad of the test strip and chromatographed for 10 minutes; the test strip was then washed with 200 μL of PBST solution containing 0.5% Tween 20, and then the test strip was immersed in 200 μL of 0.5% chloroauric acid solution for 1 minute, then the test strip was taken out and immersed in 200 μL of 10 mM hydroquinone solution for 1 minute, and finally the test strip was taken out and the signal amplified picture was recorded with a camera, and the gray value of the test line of the test strip was read and analyzed with Image J software.

[0095] Table 1. Recovery rate of VEGF165 by LFA test strip in 1% serum

[0096]

[0097] As shown in Table 1, the recovery rates of the added VEGF165 at concentrations of 10 ng / mL, 20 ng / mL, and 30 ng / mL in the final test solution were between 98.86% and 107.37%, indicating that the test strip has high reliability and accuracy in actual clinical samples and is expected to be used in actual medical diagnosis in the future.

[0098] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A lateral flow assay test strip for detecting vascular endothelial growth factor 165, comprising: Sample pads, nitrocellulose membranes, absorbent pads, and backing plates; The nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is coated with a VEGF165 antibody, and the quality control line is coated with a combination of streptavidin and a biotinylated VEGF165 aptamer complementary chain; The complementary strand of the VEGF165 aptamer has a nucleic acid sequence as shown in SEQ ID NO:

2.

2. The lateral flow analysis test strip according to claim 1, characterized in that: The distance between the test line and the quality control line is 5 mm; The width of the test strip is 3 mm, the sample pad is 2.2 cm long, the nitrocellulose membrane is 2.5 cm long, and the absorbent pad is 1.7 cm long.

3. A method for preparing a lateral flow analysis test strip according to claim 1 or 2, comprising: Soak the sample pad in the treatment solution and then dry it; The detection line of the nitrocellulose membrane is sprayed with VEGF165 antibody, and the quality control line is sprayed with a conjugate of streptavidin and the complementary chain of the biotinylated VEGF165 aptamer; The sample pad, the nitrocellulose membrane and the absorbent pad are fixed to the back plate in order to obtain the test strip.

4. The preparation method according to claim 3, characterized in that: The treatment solution includes water, 0.05 M Tris, 10 mM ethylenediaminetetraacetic acid, 0.25 wt % Triton X-100 and 1 wt % BSA, with a pH of 7.

6.

5. The preparation method according to claim 3, characterized in that: The preparation method of the conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain comprises: The biotinylated VEGF165 aptamer complementary chain was diluted to a concentration of 10 μM with PBS buffer, heated in a 95°C water bath for 3 minutes, and allowed to stand at room temperature for 30 minutes before adding an equal volume of 0.1 mg / mL streptavidin, oscillated at room temperature for 1 hour, and then incubated at 0-4°C for 8-12 hours; then centrifuged at 14000 rcf for 10 minutes using a 30KDa ultrafiltration tube, discarded the supernatant, and washed with PBS buffer to obtain a conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The spraying amount of the VEGF165 antibody was 0.8 μg / cm; The spraying amount of the conjugate of streptavidin and biotinylated VEGF165 aptamer complementary chain was 4.56 pmol / cm.

7. A lateral flow assay kit for detecting vascular endothelial growth factor 165, comprising the test strip according to claim 1 or 2 and a signal probe solution; The signal probe solution includes biotinylated VEGF165 aptamer and streptavidin-modified gold nanoparticles; The VEGF165 aptamer has a nucleic acid sequence as shown in SEQ ID NO:

1.

8. The lateral flow assay kit according to claim 7, characterized in that In the signal probe solution, The concentration of the biotinylated VEGF165 aptamer was 0.52 μM; The concentration of the streptavidin-modified gold nanoparticles was 0.31 nM.

9. The lateral flow assay kit according to claim 7 or 8, characterized in that: Also included are cleaning solutions, chloroauric acid solutions, and hydroquinone solutions; The cleaning solution is a PBST solution containing 0.5% Tween 20; The mass fraction of chloroauric acid in the chloroauric acid solution is 0.5%; The concentration of hydroquinone in the hydroquinone solution is 10 mM.

10. The lateral flow assay kit according to any one of claims 7 to 9, characterized in that: A standard solution was also included, which included 1 μg / mL of VEGF165 and 10% BSA.

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