Label-free double-color test strip and kit for tumor protein p53 gene detection and preparation method thereof

Abstract

The application belongs to the field of electrochemistry, and relates to tumor protein P53 gene detection, in particular to a label-free double-color test strip, a kit and a preparation method thereof for tumor protein P53 gene detection. The application belongs to a novel LFA method, which combines DNA hybridization chain reaction with the electrostatic adsorption principle of test line / control line, selects common and cost-effective bovine serum albumin as the test line, utilizes the difference in the adsorption capacity of BSA to double-stranded DNA and nano-gold, and the different flow rates of different molecules on the NC membrane to complete the detection of p53 at different concentrations. The test line is constructed by positively charged PDDA to ensure the effectiveness of the LFA. Meanwhile, the detection principle of a liquid colorimetric sensor is combined, and the double-color development greatly reduces the detection limit of the test strip. The sensor is label-free throughout, and has high analytical performance in terms of sensitivity, selectivity and practicability. The p53 gene is detected on the lateral flow test strip for the first time.

Description

Technical Field

[0001] This invention belongs to the field of optical detection and relates to the detection of tumor protein P53 gene, specifically to label-free two-color test strips, reagent kits and their preparation methods for the detection of tumor protein P53 gene. Background Technology

[0002] Cancer is one of the world's most serious health problems, with millions diagnosed and dying from it each year. The p53 gene is an important human cancer suppressor gene that plays a crucial role in many cellular anti-tumor mechanisms, particularly in transcriptional regulation and the function of key proteins. Mutations in the p53 gene occur in approximately 50% of human cancers, making it one of the most common genetic alterations. Therefore, the p53 gene holds great promise as a diagnostic biomarker for early cancer screening and monitoring. Consequently, developing rapid and sensitive p53 gene detection strategies is of great significance in the field of molecular biology.

[0003] Traditional gene detection methods, such as fluorescence, electrochemical, and colorimetric methods, require specialized instruments, complex procedures, and long detection times. Lateralflow analysis (LFA), on the other hand, is widely used in molecular biology due to its speed, ease of operation, and particular affordability. However, the high batch-to-batch variability, error amplitude, and storage requirements of traditional antibody-based LFA significantly hinder its application. Recent advances in aptamer technology have provided an opportunity to create a promising detection method by combining the potential of aptamers and LFA. Wu et al. developed an aptamer-based lateralflow test strip for the detection of zearalenone. This assay is based on aptamer competition between zearalenone and its complementary sequence, and successfully detected zearalenone within a visual detection limit of 20 ng / ml. Zhu et al. developed a sandwich lateralflow test strip for adenosine triphosphate (ATP) detection based on the specific recognition between split aptamer fragments and the target, achieving a good linear logarithmic response in the range of 0.5 μM to 5 mM.

[0004] However, this method requires additional modification of the aptamer and additional complementary DNA sequences, which increases experimental costs and makes it difficult to scale up. Alsager et al. utilized the adsorption-desorption principle of the adapter on a gold nanoparticle surface to detect 17β-estradiol in LFA. This method does not require additional modification of the adapter and uses common proteins and lysozyme as test and detection lines, further reducing costs. Although the visual detection range is 50-5000 nM, the uneven coloring of the test line makes semi-quantitative analysis difficult. LFA's limitations in sensitivity, and its limitation to qualitative or semi-quantitative detection of samples, prevent it from functioning effectively in practical applications. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a label-free two-color test strip, a reagent kit, and a method for preparing the same for detecting the tumor protein P53 gene.

[0006] The technical solution of this invention is implemented as follows:

[0007] A label-free two-color test strip for detecting the tumor protein P53 gene includes a sample pad, an NC membrane, and an absorbent pad, characterized in that: BSA is fixed on the test line and PDDA is fixed on the control line on the NC membrane.

[0008] The NC membrane was obtained after washing with 100 μM trisodium citrate solution; the BSA was a 300 μM BSA solution containing 3% sucrose at pH 9; and the PDDA was a 0.01% (v / v) PDDA solution. Using an adhesive PVC card (6 x 0.5 cm) as a substrate, a 2.5 x 0.5 cm strip of the NC membrane was placed on top, overlapping the strip by 1-2 mm to ensure liquid flow from the sample pad to the absorption pad. Test strips were obtained using a 6 x 0.5 cm shear.

[0009] The sample buffers that come with the above-mentioned unlabeled two-color test strips include H1 solution, H2 solution, HCR buffer (buffer for inducing hybridization chain reaction) and AuNPs solution.

[0010] The nucleotide sequence of H1 is shown in SEQ ID No. 1, and the nucleotide sequence of H2 is shown in SEQ ID No. 2.

[0011] The concentrations of H1 and H2 solutions were both 5 μM; the HCR buffer solution was 16.7 mM CH3COONa, 0.33 M NaCl, and pH 7.5; the gold nanoparticles in the AuNPs solution had a particle size of 40 nm.

[0012] The sample buffer solution that is compatible with the above-mentioned label-free two-color test strip is prepared as follows: Mix 5 μL of H1 solution, 5 μM of H2 solution and 5 μL of the sample to be tested, add 6 μL of HCR buffer, and then add 84 μL of incubated AuNPs solution to the reaction system. Let it stand at room temperature for 30 minutes to obtain the sample buffer solution.

[0013] A test kit for detecting the tumor protein P53 gene includes the aforementioned test strip and sample buffer.

[0014] The detection method for the above test strip kit is as follows: Add 100 μL of sample buffer to the sample pad on the labeled two-color test strip, let it stand at room temperature for 3 minutes, and observe the color change of the test line and the control line.

[0015] When testing low-concentration samples, the detection method of the above test strip is as follows: add 2 μL of 2M sodium chloride to the sample buffer, let it stand at room temperature for 30 minutes, take 100 μL of sample buffer and add it to the sample pad on the test strip, let it stand at room temperature for 3 minutes, and observe the color change of the test line and the control line.

[0016] When the p53 concentration in the sample is greater than 10 nM, the above-mentioned unmarked two-color test strip produces a red band on the test line, with a regression equation of y = 0.00598x + 1.03368 and R² = 0.9958. The y-axis represents the BT / B0 value, where BT and B0 represent the B values ​​of the test line and blank line measured using ImageJ, respectively. When the p53 concentration is less than 10 nM, the test line produces a blue band, with a regression equation of y = -0.0272x + 1.4009 and R² = 0.9978. The y-axis represents the BT / B0 value, where BT and B0 represent the B values ​​of the test line and blank line measured using ImageJ, respectively.

[0017] The detection principle is as follows: When BSA is used as the test line, its adsorption capacity for the former is much greater than that for the latter. When the concentration of the p53 gene in the sample is higher than 10 nM, the double-stranded structure produced by the HCR reaction has fewer base pairs and can be adsorbed onto the surface of gold nanoparticles through its sticky ends, forming a gold nanoparticle double-helix DNA structure, which is then captured by BSA on the test line. As the concentration of p53 decreases, the number of base pairs in the generated double-stranded structure gradually increases. The decrease in the number of sticky ends leads to a decrease in the number of gold nanoparticle double-helix DNA structures. The double-helix structures that are not bound to gold nanoparticles flow faster on the NC membrane, occupying the binding sites of BSA and gold nanoparticles, resulting in a lighter color of the test line. When the concentration of the p53 gene in the sample is lower than 10 nM, the number of gold nanoparticle double-helix DNA structures in the solution is greatly reduced. The lower the concentration of p53, the longer the base pairs of the double-helix structure in the solution, which leads to a slower flow of the double-helix structure on the NC membrane, giving BSA additional binding sites to adsorb gold nanoparticles, and the color of the test line gradually darkens. When the sample does not contain p53, no hybridization reaction occurs, and probes H1 and H2 are directly adsorbed onto the surface of the gold nanoparticles. At this time, the test line is the darkest. The control strain PDDA has a strong positive charge and can effectively adsorb gold nanoparticles and their double helix structures, regardless of the presence of the p53 gene.

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

[0019] 1. This application presents a novel LFA method that combines DNA hybridization chain reaction with the electrostatic adsorption principle of gold nanoparticles. This method constructs a test strip that requires no labeling or modification throughout the entire process. Utilizing the difference in adsorption capacity of BSA for double-stranded DNA and gold nanoparticles, as well as the different flow rates of different molecules on the NC membrane, it enables the detection of p53 at different concentrations. The test line is constructed from positively charged PDDA to ensure the effectiveness of LFA. Simultaneously, combining the detection principle of a liquid colorimetric sensor, the method utilizes dual-color development to significantly lower the detection limit of the test strip. This sensor is completely label-free and exhibits high analytical performance in terms of sensitivity, selectivity, and practicality. It achieves enzyme-free amplification of the target analyte, avoiding the introduction of excessive interfering substances, and solves the problem of low sensitivity in electrostatic adsorption test strips. It is simple to operate and has extremely low preparation costs, making it highly competitive in commercial applications. This is the first time the p53 gene has been detected on a lateral flow test strip.

[0020] By combining the detection principle of a label-free colorimetric sensor with the paper strip detection method, and utilizing dual-color display, the detection limit of the test strip is significantly reduced. This is expected to solve the problem of difficulty in distinguishing similar colored bands in quantitative analysis of test strips, providing a new approach for high-sensitivity detection of test strips. At the same time, this type of test strip has extremely low cost and a very broad market prospect. In the future, it is expected to be more widely used and developed in fields such as medical care, health management, and self-testing. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 ImageJ analysis of test lines at different BSA concentrations (a); effect of pH on BSA (b); and ImageJ analysis of the effect of different sucrose concentrations of BSA on the test strains (c).

[0023] Figure 2(a) Image of a test strip detecting low concentrations of p53 under 20 nm gold nanoparticles; (b) ImageJ analysis of a test line detecting low concentrations of p53 under 20 nm gold nanoparticles; (c) Image of a test strip detecting low concentrations of p53 under 20 nm gold nanoparticles; (d) ImageJ analysis of a test line detecting high concentrations of p53 under 20 nm gold nanoparticles; (e) Image of a test strip detecting low concentrations of p53 under 40 nm gold nanoparticles; (f) ImageJ analysis of a test line detecting low concentrations of p53 under 40 nm gold nanoparticles; (g) Image of a test strip detecting high concentrations of p53 under 40 nm gold nanoparticles; (h) ImageJ analysis of a test line detecting high concentrations of p53 under 40 nm gold nanoparticles; (i) Image of a test strip detecting low concentrations of p53 under 60 nm gold nanoparticles; (j) ImageJ analysis of a test strip for detecting low concentrations of p53 under 60 nm gold nanoparticles; (k) Image of a test strip for detecting high concentrations of p53 under 60 nm gold nanoparticles; (l) ImageJ analysis of a test strip for detecting high concentrations of p53 under 60 nm gold nanoparticles.

[0024] Figure 3 (a) Linearity of the sensor at p53 concentrations of 0.4–200 nM; (b) Image of test strips at p53 concentrations of 0.4–200 nM; (c) Linearity of the sensor at high p53 concentrations after the addition of sodium ions; (d) Linearity of the sensor at low p53 concentrations after the addition of sodium ions; (e) Image of test strips at p53 concentrations of 0.4–200 nM after the addition of sodium ions; (f) Detection of the sample using a UV spectrophotometer (the image shows the color change of the sample).

[0025] Figure 4 This application provides specificity analysis of the sensor and actual sample analysis; (a) is the ImageJ analysis of the strip test line shown in Figure b; (b) is an image of the specificity test strip.

[0026] Figure 5 This application describes the HCR reaction principle and sensor detection principle of the test strip.

[0027] Figure 6 These are samples of the test strips and reagent kits packaged according to this application. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Preparation of reagents for this application:

[0030] 100 μM trisodium citrate solution, and a 300 μM BSA solution containing 3% sucrose.

[0031] PDDA is a 0.01% volume concentration PDDA solution;

[0032] The concentrations of H1 and H2 solutions were both 5 μM, and the concentrations of the HCR buffer were 16.7 mM CH3COONa, 0.33 M NaCl, and pH 7.5.

[0033] The gold nanoparticles in the AuNPs solution have a particle size of 40 nm.

[0034] The nucleotide sequences of the probes involved are as follows:

[0035] H1: 5'-TTTCTCTGGCTTCTGTTCAGGTAAGCCAGAGAAAAGGAAACTGAGT-3';

[0036] H2: 5'- ACCTGAACAGAAGCCAGAGAAAACTCAGTTTCCTTTTCTCTGGCTT-3'.

[0037] Example 1

[0038] Preparation of sample buffer for label-free two-color test strips for detecting tumor protein P53 gene:

[0039] 1. Preparation of sample buffer

[0040] Mix 5 μL of H1 and H2 (5 μM) with 5 μL of p53 (0.25 μM) solution and add 6 μL of HCR buffer. This facilitates the hybridization reaction and the adsorption of single-stranded DNA to gold nanoparticles. Incubate AuNPs at 37°C for 24 hours, then add 84 μL of AuNPs to the solution and incubate at room temperature for 30 minutes.

[0041] 2. Verification of HCR reaction steps using fluorescent dyes

[0042] 60 μL of Hoechst dye was added to 540 μL of HCR buffer as a blank sample. 60 μL of 5 μM H1, H2, and p53 were added as Sample 1, Sample 2, and Sample 3, respectively, to a mixture of 60 μL of Hoechst dye and 534 μL of HCR buffer. 6 μL of 5 μM H1 and p53 were reacted with 528 μL of HCR buffer at 37°C for 20 hours, and then 60 μL of Hoechst dye was added as Sample 4. 6 μL of 5 μM H2 and p53 were reacted with 528 μL of HCR buffer at 37°C for 20 hours, and then 60 μL of Hoechst dye was added as Sample 5. 6 μL of 5 μM H1, H2, and p53 were reacted with 528 μL of HCR buffer at 37°C for 20 hours, and then 60 μL of Hoechst dye was added as Sample 6. After adding Hoechst dye, all samples were left to stand for 30 minutes, and finally the fluorescence intensity of the samples was detected using a fluorescence spectrometer.

[0043] Mix 5 μL of H1 and H2 (5 μM) with 6 μL of HCR buffer. Add different concentrations of p53 gene to the mixture and incubate at 37°C for 20 hours. Then, mix 10 μL of the sample with 2.5 μL of loading buffer and perform electrophoresis on a 4% agarose gel in 1×TAE solution. Perform the electrophoresis at 110 volts for 30 minutes and take pictures using a gel imaging system.

[0044] 3. Test strip detection principle

[0045] In this study, the non-specific adsorption between probes H1 and H2 and gold nanoparticles, as well as the p53 gene-induced HCR, were applied to the test strips. Figure 5 As shown, BSA and PDDA were pre-immobilized on the test and control lines of the NC membrane. BSA is one of the most common proteins in the laboratory and can simultaneously adsorb double-stranded DNA and gold nanoparticles.

[0046] However, through experiments, we found that when BSA is used as the test line, its adsorption capacity for the former is much greater than that for the latter. When the concentration of the p53 gene in the sample is higher than 10 nM, the double-stranded structure produced by the HCR reaction has fewer base pairs and can be adsorbed onto the surface of gold nanoparticles through its sticky ends, forming a gold nanoparticle double-helix DNA structure, which is then captured by BSA on the test line. As the concentration of p53 decreases, the number of base pairs in the generated double-stranded structure gradually increases. The decrease in the number of sticky ends leads to a decrease in the number of gold nanoparticle double-helix DNA structures. The double-helix structures that are not bound to gold nanoparticles flow faster on the NC membrane, occupying the binding sites of BSA and gold nanoparticles, resulting in a lighter color of the test line. When the concentration of the p53 gene in the sample is lower than 10 nM, the number of gold nanoparticle double-helix DNA structures in the solution is greatly reduced. The lower the concentration of p53, the longer the base pairs of the double-helix structure in the solution, which leads to a slower flow of the double-helix structure on the NC membrane, giving BSA additional binding sites to adsorb gold nanoparticles, and the color of the test line gradually darkens. When the sample does not contain p53, no hybridization reaction occurs, and probes H1 and H2 are directly adsorbed onto the surface of the gold nanoparticles. At this time, the test line is the darkest. The control strain PDDA has a strong positive charge and can effectively adsorb gold nanoparticles and their double helix structures, regardless of the presence of the p53 gene.

[0047] Example 2

[0048] Preparation of label-free transverse flow test strips for detecting the tumor protein P53 gene:

[0049] 1. The lateral flow method for detecting p53 gene in serum consists of three components: a sample pad, an NC membrane, and an absorbent pad. The NC membrane is washed with 100 μM trisodium citrate solution to produce clearer and more defined test and control lines. Optimized BSA and PDDA (esi) are electrostatically fixed onto the test and control lines of the NC membrane, respectively. Using an adhesive PVC card (6 x 0.5 cm) as a substrate, a cut NC membrane (2.5 x 0.5 cm) is placed on top, with both overlapping the sample pad and absorbent pad by 1-2 mm to ensure liquid flow from the sample pad to the absorbent pad. Test strips are obtained using a 6 x 0.5 cm shear.

[0050] 2. To obtain optimal experimental results, the adsorption conditions of the probe on the gold nanoparticle surface and the p53-induced probe HCR in the test solution were optimized. The sample solution (100 μL) was added to the sample pad, and the solution migrated through the NC membrane and across the test and control lines via capillary action. The entire detection process took only 3 minutes. Due to the accumulation of AuNP, red bands were observed in the test and control lines, and the test line bands were analyzed using ImageJ. The corresponding integrated band intensity was used for further analysis of the sensor.

[0051] 3. Optimization of experimental conditions

[0052] To obtain clear red bands with appropriate signal intensity and good detection sensitivity on the test and control lines, it is necessary to optimize the solution parameters of BSA and PDDA, as well as the particle size of gold nanoparticles.

[0053] A higher BSA concentration on the test line provides more binding sites, but this may result in a less noticeable color change on the T line as the target analyte concentration decreases. To observe the color change on the T line, probe-coated gold nanoparticles and bare gold nanoparticles were added at the same concentration and volume, and the BSA concentration was adjusted. Figure 1 As shown in Figure a, higher BSA concentrations result in darker test lines and higher corresponding values. A mass concentration of 300 μM BSA was chosen as the optimal concentration because it exhibits the greatest difference in adsorption capacity between probe-encapsulated gold nanoparticles and bare gold nanoparticles. pH significantly affects the surface zeta potential of BSA, which is primarily adsorbed onto the NC film via electrostatic interactions. Changing the pH of BSA alters the width of the T-line. Figure 1 As shown in bc, a BSA solution with a pH of 9 was selected in the experiment to obtain clearer and more aesthetically pleasing test lines.

[0054] Adding sucrose to the solution can protect proteins and enhance hydrogen bonds in water: different amounts of sucrose, such as 3%, 5%, 7%, and 10%, were added to BSA solutions. Figure 3 As shown, the coloring of the test line decreases when the sucrose concentration is between 5% and 10%, indicating that the number of sugar molecules required to protect the protein is limited. Therefore, BSA with pH=9 containing 3% sucrose was chosen as the test line.

[0055] Furthermore, the concentration of PDDA on the control line also significantly affected the color change of the test line. Experimental results showed that high concentrations of PDDA could strip gold nanoparticles from the test line, resulting in a duller color. Therefore, a PDDA concentration of 0.01% was selected as the control line concentration.

[0056] Generally, the smaller the particle size of gold nanoparticles, the stronger their adsorption capacity for BSA; however, smaller-sized gold nanoparticles often exhibit poorer color development. Gold nanoparticles with sizes of 20, 40, and 60 nm were prepared, and p53 was tested at high concentrations (200, 160, 120, 80, 40, and 20 nM) and low concentrations (15, 10, 5, 3.5, 0.95, 0.47, 0.38, 0.19, and 0.05 nM) in these three different sizes. Figure 2As shown, 60 nm gold nanoparticles provided the best colorimetric response to high concentrations of p53. However, the 60 nm gold nanoparticles exhibited poor specificity for low concentrations of p53, leading to particle aggregation and a purple solution. Therefore, 40 nm gold nanoparticles, which showed good performance at all concentrations, were selected as the experimental material.

[0057] Implementation effect analysis

[0058] Under the aforementioned optimal conditions, different concentrations of p53 (200, 100, 40, 20, 10, 4, 2, 1, and 0.4 nM) were set to explore the detection limits of the sensor, such as... Figure 3 As shown in ab, the test line color gradually lightens as the p53 concentration in the sample increases from 0.4 to 10 nM. The intensity of the test line was measured using ImageJ, and the linear relationship between the test line intensity and the p53 concentration was obtained through a regression equation of y = 0.00601x + 1.08094 and R² = 0.99181. Here, the y-axis represents the RT / R0 value, where RT and R0 represent the R values ​​of the test line and blank line measured using ImageJ, respectively. When the p53 concentration increases from 10 to 200 nM, the test line color gradually darkens, and the regression equation is y = 0.0055x + 1.0428 with R² = 0.9882. Although the sensor exhibits good sensitivity at both high and low concentrations, one RT / R0 value may correspond to two p53 concentrations, which will affect the actual detection performance of the sensor.

[0059] To address this issue, it is necessary to differentiate between high and low concentrations in a more intuitive way. Inspired by solution colorimetry, adding 2M sodium ions to the sample can induce the aggregation of exposed gold nanoparticles (e.g., Figure 3 c). This causes the solution color to gradually turn purple as the p53 concentration in the sample decreases, especially when the p53 concentration is less than 10 nM. The sample test results using the test strip are as follows: Figure 3 As shown in the diagram. When the p53 concentration is greater than 10 nM, the test line shows a red band, with the regression equation y = 0.00598x + 1.03368 and R² = 0.9958. When the p53 concentration is less than 10 nM, the test line shows a blue band, with the regression equation y = -0.0272x + 1.4009 and R² = 0.9978. Here, the y-axis represents the BT / B0 value, where BT and B0 represent the B values ​​of the test line and blank line measured using ImageJ, respectively.

[0060] Within these ranges, a linear relationship exists between the intensity of the test line and the p53 concentration, and the high-concentration range can be distinguished from the low-concentration range by whether the test line turns blue. These results indicate that the test strip can effectively distinguish between high and low concentrations of p53, exhibits high sensitivity between 0.4 and 200 nM, and can, to some extent, replace UV-Vis spectrophotometry for the quantitative analysis of p53 in samples. The entire detection process took only 3 minutes after sample addition.

[0061] Application examples

[0062] To verify the sensor's specificity, four different target DNAs (10 nM) were selected as single-mutation, double-mutation, and two random DNA sequences. Figure 4 As shown, only the peak value corresponding to the p53 gene decreased, and the color change of the test line was visible to the naked eye. The experimental results are sufficient to prove that the sensor has good specificity for p53.

[0063] To evaluate the usefulness of the sensor, 10% normal human serum containing different concentrations (0.4, 4, 40, 100, and 200 nM) of p53 was tested, and a spiked recovery experiment was performed. The results are shown in Table 1:

[0064]

[0065] As shown in Table 1, the visual detection limit of the p53 gene in real samples is only slightly higher than that of the standard solution. Therefore, this test strip has great practical application value in detecting p53 in human serum.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A test kit for detecting the tumor protein P53 gene, characterized in that: Includes label-free two-color test strips for detecting the tumor protein P53 gene and accompanying sample buffer; The unmarked dual-color test strip includes a sample pad, an NC membrane, and an absorption pad, characterized in that: BSA is fixed on the test line and PDDA is fixed on the control line on the NC membrane; The sample buffers include H1 solution, H2 solution, HCR buffer and AuNPs solution; the nucleotide sequence of H1 is shown in SEQ ID No. 1 and the nucleotide sequence of H2 is shown in SEQ ID No.

2.

2. The test kit for detecting the tumor protein P53 gene according to claim 1, characterized in that: The NC membrane was obtained by washing with 100 μM trisodium citrate solution; BSA was fixed on the test line using a 300 μM BSA solution containing 3% sucrose at pH 9; PDDA was fixed on the control line using a 0.01% PDDA solution.

3. The test kit for detecting the tumor protein P53 gene according to claim 2, characterized in that: The concentrations of H1 and H2 solutions were both 5 μM, the HCR buffer was 16.7 mM CH3COONa, 0.33 M NaCl, pH 7.5 AuNPs, and the particle size of the gold nanoparticles in the solution was 40 nm.

4. The test kit for detecting the tumor protein P53 gene according to claim 3, characterized in that, The sample solution prepared after sample buffer treatment is as follows: Mix 5 μL of H1 solution, 5 μM H2 solution and 5 μL of sample to be tested, add 6 μL of HCR buffer, and then add 84 μL of AuNPs solution after incubation to the reaction system. Let it stand at room temperature for 30 minutes to obtain the sample solution.

5. The test kit for detecting the tumor protein P53 gene according to claim 4, characterized in that, The detection method of the test strip kit is as follows: take 100 μL of sample solution and add it to the sample pad on the unmarked two-color test strip, let it stand at room temperature for 3 minutes, and observe the color change of the test line and the control line.

6. The kit for detecting the tumor protein P53 gene according to claim 4, characterized in that, The detection method of the test strip is as follows: add 2 μL of 2M sodium chloride to the sample solution, let it stand at room temperature for 30 minutes, take 100 μL of sample solution and add it to the sample pad on the test strip, let it stand at room temperature for 3 minutes, and observe the color change of the test line and the control line.

7. The kit for detecting the tumor protein P53 gene according to claim 5 or 6, characterized in that: When the p53 concentration in the sample is greater than 10 nM, the unmarked dual-color test strip produces a red band on the test line, with a regression equation of y = 0.00598x + 1.03368 and R² = 0.9958. The y-axis represents the BT / B0 value, where BT and B0 represent the B values ​​of the test line and blank line measured using ImageJ, respectively. When the p53 concentration is less than 10 nM, the test line produces a blue band, with a regression equation of y = -0.0272x + 1.4009 and R² = 0.9978. The y-axis represents the BT / B0 value, where BT and B0 represent the B values ​​of the test line and blank line measured using ImageJ, respectively.

Images