A rapid test strip for breast cancer urine metabolites

Abstract

The application discloses a breast cancer urine metabolite rapid detection test strip and belongs to the technical field of in vitro diagnosis. The test strip comprises a PVC bottom plate and a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially connected. The combination pad adsorbs a colloidal gold-aptamer conjugate, and the NC membrane is provided with a detection line T1 (8-OHdG capture aptamer), a detection line T2 (high vanillylmandelic acid capture aptamer), a detection line T3 (creatinine capture aptamer) and a quality control line C (anti-aptamer antibody). The application adopts aptamer as a recognition element, has high specificity and good stability, can synchronously detect two breast cancer core metabolic markers, corrects urine concentration difference through a creatinine internal reference, and has accurate and reliable detection results. The detection process does not need professional equipment, visual interpretation can be completed in 5-10 minutes, and the application has simple operation and low cost. The application is suitable for early screening of breast cancer, population census and family self-monitoring.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and in particular relates to a rapid test strip for detecting breast cancer urinary metabolites based on aptamer-colloidal gold technology. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and early diagnosis is crucial for improving cure rates and reducing mortality. Existing clinical screening methods, such as breast ultrasound, mammography, and MRI, have many limitations: ultrasound relies on operator experience and has a high rate of missed diagnoses of small lesions; mammography involves radiation damage and is not effective for screening dense breasts; MRI is expensive and difficult to widely implement. Therefore, developing non-invasive, convenient, and low-cost early screening technologies is of significant clinical importance.

[0003] Metabolomics studies have confirmed that the levels of metabolic markers such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and homovanillic acid in the urine of breast cancer patients are significantly abnormal, and these markers can serve as potential targets for early diagnosis of breast cancer. Urine samples have the advantages of being non-invasive, readily available, and simple to prepare, making urine-based marker detection technologies a hot research topic.

[0004] Current urinary metabolite detection technologies largely rely on large-scale equipment such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which are complex to operate, have long detection cycles, and are costly, failing to meet the needs of rapid on-site testing. Colloidal gold technology, due to its visualization, speed, and low cost, has been widely used in rapid detection. However, traditional colloidal gold technologies often use antibodies as recognition elements, which suffer from difficulties in preparation, poor specificity, and insufficient stability for small molecule metabolites. Aptamers, as novel recognition elements, offer advantages such as high specificity, high affinity, simple preparation, and good stability, effectively compensating for the shortcomings of antibodies. However, there are currently no mature products that apply aptamer-colloidal gold technology to the simultaneous detection of multiple urinary metabolic biomarkers in breast cancer.

[0005] Therefore, developing a rapid test strip based on aptamer-colloidal gold technology that can simultaneously detect multiple core metabolic markers in breast cancer urine, and which is easy to operate, highly sensitive, and highly specific, is of great significance for filling the existing technological gap and promoting the popularization of early breast cancer screening. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention aims to provide a rapid test strip for breast cancer urinary metabolites, enabling simultaneous detection of 8-OHdG and homovanillic acid in urine, as well as creatinine internal reference calibration. This solves the problems of complex operation, high cost, and poor specificity of existing detection technologies, providing a convenient and accurate detection tool for early breast cancer screening.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide an aptamer assembly, the aptamer assembly comprising: The first aptamer pair for recognizing and binding 8-hydroxy-2'-deoxyguanosine consists of a detection aptamer with the sequence shown in SEQ ID NO.1 and a capture aptamer with the sequence shown in SEQ ID NO.2; A second aptamer pair for recognizing and binding homovanillic acid, comprising a detection aptamer with the sequence shown in SEQ ID NO.3 and a capture aptamer with the sequence shown in SEQ ID NO.4; and The third aptamer pair for recognizing and binding creatinine consists of a detection aptamer with the sequence shown in SEQ ID NO.5 and a capture aptamer with the sequence shown in SEQ ID NO.6.

[0008] The second objective of this invention is to provide the application of the aforementioned aptamer combination in the preparation of a rapid detection product for breast cancer urinary metabolites.

[0009] The third objective of this invention is to provide a method for preparing a rapid test strip for breast cancer urinary metabolites using the aforementioned aptamer combination, comprising the following steps: Step 1: Preparation of colloidal gold particles: Colloidal gold particles with a particle size of 20-40 nm were prepared by the trisodium citrate reduction method. Step 2: Preparation of colloidal gold-aptamer conjugates: The colloidal gold particles obtained in Step 1 are coupled with the detection aptamer of 8-hydroxy-2'-deoxyguanosine, the detection aptamer of homovanillic acid, and the detection aptamer of creatinine, respectively, to obtain colloidal gold-aptamer conjugates. Step 3: Pre-treatment of sample pads: Immerse the sample pads in the pre-treatment solution and dry them for later use; the pre-treatment solution is a pH 7.2-7.6 PBS buffer containing 0.5-1.0% Tween-20 and 0.1-0.2 M NaCl. Step 4: Preparation of the conjugate pad: Spray the colloidal gold-aptamer conjugate obtained in Step 2 onto the conjugate pad and dry it; Step 5: Processing the nitrocellulose membrane: Sequentially set detection lines T1, T2, T3, and control line C on the nitrocellulose membrane. Coat detection line T1 with the capture aptamer of 8-hydroxy-2'-deoxyguanosine, detection line T2 with the capture aptamer of homovanillic acid, and detection line T3 with the capture aptamer of creatinine. The capture aptamer on detection line T1 is used to capture the complex formed by the 8-hydroxy-2'-deoxyguanosine detection aptamer and the target from step 2; the capture aptamer on detection line T2 is used to capture the complex formed by the homovanillic acid detection aptamer and the target from step 2; and the capture aptamer on detection line T3 is used to capture the complex formed by the creatinine detection aptamer and the target from step 2. Coat control line C with anti-aptamer antibody. Step 6, Assembly: The sample pad obtained in Step 3, the bonding pad obtained in Step 4, the nitrocellulose membrane obtained in Step 5, and the absorbent pad are sequentially overlapped and fixed on the PVC base plate to obtain the final product.

[0010] Furthermore, the preparation conditions for the colloidal gold-aptamer conjugate in step two are as follows: pH 7.0-7.5, mass ratio of aptamer to colloidal gold 1:50-1:80, incubation at room temperature for 20-30 min, and then blocking with bovine serum albumin.

[0011] Furthermore, in step five, the capture aptamer coating concentration of detection lines T1, T2, and T3 is 0.5-1.0 mg / mL, and the anti-aptamer antibody coating concentration of control line C is 0.8-1.2 mg / mL; the pore size of the nitrocellulose membrane is 80-120 μm, and the distance between the detection lines and the control lines is 0.5-0.8 cm.

[0012] The fourth objective of this invention is to provide a rapid test strip for breast cancer urine metabolites, the rapid test strip for breast cancer urine metabolites includes a PVC base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped and fixed on the PVC base plate; The conjugate pad is adsorbed with colloidal gold-aptamer conjugates, which include colloidal gold particles and 8-hydroxy-2'-deoxyguanosine detection aptamers, homovanillic acid detection aptamers, and creatinine detection aptamers coupled to the colloidal gold particles. The nitrocellulose membrane is sequentially provided with a detection line T1 for detecting 8-hydroxy-2'-deoxyguanosine, a detection line T2 for detecting homovanillic acid, a detection line T3 for detecting creatinine, and a quality control line C. The detection line T1 is coated with an 8-hydroxy-2'-deoxyguanosine capture aptamer, the detection line T2 is coated with a homovanillic acid capture aptamer, the detection line T3 is coated with a creatinine capture aptamer, and the control line C is coated with an anti-aptamer antibody.

[0013] Furthermore, the detection aptamer sequence of the 8-hydroxy-2'-deoxyguanosine is shown in SEQ ID NO.1, and the capture aptamer sequence is shown in SEQ ID NO.2; the detection aptamer sequence of the homovanillic acid is shown in SEQ ID NO.3, and the capture aptamer sequence is shown in SEQ ID NO.4; the detection aptamer sequence of the creatinine is shown in SEQ ID NO.5, and the capture aptamer sequence is shown in SEQ ID NO.6.

[0014] Furthermore, the colloidal gold particles have a particle size of 20-40 nm; the sample pad is a glass fiber pad that has been soaked in a pretreatment solution and then dried, and the pretreatment solution is a pH 7.2-7.6 PBS buffer containing 0.5-1.0% Tween-20 and 0.1-0.2 M NaCl.

[0015] Furthermore, the nitrocellulose membrane has a pore size of 80-120 μm, and the spacing between the detection lines T1, T2, T3 and the control line C is 0.5-0.8 cm; the capture aptamer coating concentration of the detection lines T1, T2, T3 is 0.5-1.0 mg / mL, and the anti-aptamer antibody coating concentration of the control line C is 0.8-1.2 mg / mL.

[0016] The fifth objective of this invention is to provide the application of the aptamer combination, the method, or the test strip in the preparation of early breast cancer screening products.

[0017] This invention is based on the principle of dual-aptamer sandwich-colloidal gold signal amplification, and the specific process is as follows: 1) The urine sample is dropped onto the sample pad, and the sample migrates sequentially through the conjugate pad and NC membrane under capillary action; 2) When the sample reaches the conjugate pad, the colloidal gold-aptamer conjugate is dissolved. If 8-OHdG, homovanillic acid or creatinine are present in the sample, they will specifically bind to the corresponding detection aptamer to form a "colloidal gold-detection aptamer-target" complex. 3) When the complex migrates to the NC membrane, it is specifically captured by the capture aptamers on the corresponding detection lines, forming a sandwich structure, and the colloidal gold aggregates to form a red band; the conjugates that do not bind to the target continue to migrate to the control line and are captured by the anti-aptamer antibody to form a red control band. 4) The color intensity of the creatinine test line is used to correct for differences in urine concentration, and the color development of the 8-OHdG and homovanillic acid test lines is combined to achieve qualitative / semi-quantitative detection of breast cancer-related metabolic markers.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) Using aptamers as recognition elements has advantages over traditional antibodies, such as high specificity, high affinity, low preparation cost, and good stability, effectively improving the accuracy and repeatability of detection; 2) The design incorporates a three-detection-line + one-quality control line structure, which can simultaneously detect two core metabolic markers of breast cancer, 8-OHdG and homovanillic acid. It also uses creatinine internal reference to correct for differences in urine concentration, significantly improving the reliability of the test results. 3) The testing process requires no complicated equipment or professional operation, and can be completed in 5-10 minutes. It enables visual interpretation and is suitable for primary healthcare institutions, large-scale population screening, and family self-monitoring. 4) The overall structure is reasonably designed, the raw materials are readily available, the preparation process is simple, the cost is low, and it is easy to scale up production and promote application. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the rapid urine metabolite test strip for breast cancer of the present invention; wherein: 1-PVC base plate, 2-sample pad, 3-conjugation pad, 4-NC membrane, 5-absorbent pad, 6-detection line T1 (8-OHdG detection line), 7-detection line T2 (homovanyl acid detection line), 8-detection line T3 (creatinine detection line), 9-control line C; detection line spacing: 0.5–0.8 cm; all lines are solid black lines, and the structural proportions are consistent with the actual test strip.

[0020] Figure 2 This is a schematic diagram illustrating the interpretation of the test results of the test strip of the present invention; wherein: A - negative result (C line is visible, T1, T2, and T3 lines are not visible); B - doubtful result (C line is visible, T1 line is light red); C - positive result (C line is visible, T1 and T2 lines are dark red); D - invalid result (C line is not visible).

[0021] Figure 3 The graph shows the sensitivity test results of the test strip of the present invention; where: A is the detection sensitivity of 8-OHdG, B is the detection sensitivity of homovanillic acid, and C is the detection sensitivity of creatinine, with detection limits of 0.8 ng / mL, 4 ng / mL, and 0.08 mg / dL, respectively.

[0022] Figure 4 This is a diagram showing the specificity test results of the test strip of the present invention; where, from left to right: 1 is the negative control, 2 is the positive control, 3-7 are urea, uric acid, glucose, valine, and isoleucine, respectively, and 8 is a mixed interfering substance. The results show that there is no cross-reactivity among the interfering substances.

[0023] Figure 5 The graph shows the repeatability test results of the test strip of this invention (n=3 batches, 3 replicates per batch). The results show that the intra-batch CV is ≤4.2% and the inter-batch CV is ≤6.8%, indicating good repeatability. Detailed Implementation

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0025] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0026] Example 1: Preparation of test strips 1. Preparation of colloidal gold particles Colloidal gold particles were prepared using the trisodium citrate reduction method: 100 mL of 0.01% (w / v) chloroauric acid solution was placed in a round-bottom flask and heated to boiling. 2-4 mL of 1% (w / v) trisodium citrate solution was quickly added, and boiling continued for 10-15 min until the solution turned a clear wine-red color. Heating was then stopped, and the solution was cooled to room temperature. The solution was then filtered through a 0.22 μm filter and stored at 4°C in the dark for later use. Transmission electron microscopy revealed that the colloidal gold particles were uniform in size, with a diameter of 30 nm.

[0027] 2. Preparation of colloidal gold-aptamer conjugates 1) Adjust the pH of the colloidal gold solution to 7.2 using 0.1 M K₂CO₃ solution; 2) Add 8-OHdG detection aptamer, homovanillic acid detection aptamer, and creatinine detection aptamer to the colloidal gold solution respectively. The mass ratio of aptamer to colloidal gold is 1:60. Stir slowly at room temperature for 25 min. 3) Add BSA to a final concentration of 1% (w / v) and continue incubation for 30 min to block the reaction; 4) Centrifuge at 12000 rpm for 30 min, discard the supernatant, resuspend the precipitate in preservation solution (PBS buffer at pH 7.4, containing 1% BSA and 0.02% NaN3) to 1 / 10 of the original volume, and store at 4℃ in the dark for later use.

[0028] 3. Assembly of test strips 1) Sample pad pretreatment: Immerse the glass fiber sample pad in pretreatment solution (0.8% Tween-20, 0.15 M NaCl, PBS buffer at pH 7.4) and dry at 37°C for 2 h for later use; 2) Preparation of the conjugate pad: The prepared colloidal gold-aptamer conjugate was uniformly sprayed onto the glass fiber conjugate pad at a spraying amount of 5 μL / cm. After vacuum drying at 37℃, it was sealed and stored. 3) NC membrane treatment: 8-OHdG capture aptamer (0.8 mg / mL), homovanillic acid capture aptamer (0.8 mg / mL), creatinine capture aptamer (0.8 mg / mL), and anti-aptamer antibody (1.0 mg / mL, Sigma-Aldrich, cat: MAB3034) were spotted onto the NC membrane using a spotting instrument to form detection lines T1, T2, T3 and control line C. The spotting volume was 1 μL / cm, and the membrane was dried at 37℃ for 3 h. 4) Test strip assembly: The sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially overlapped and fixed on the PVC base plate with an overlap width of 1-2 mm. The strips are then cut into 3-4 mm wide test strips using a cutting machine, sealed and packaged, and stored at 4℃.

[0029] The nucleotide sequence of the 8-OHdG detection aptamer is SEQ ID NO.1: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'; The nucleotide sequence of the 8-OHdG capture aptamer is SEQ ID NO.2: 5'-CCACCACCACCACCACAA CCACCACCACC-3'.

[0030] The nucleotide sequence of the homovanillic acid detection aptamer is SEQ ID NO.3: 5'-TTGGTAGCTCTTGATCCTTTGGT-3'; The nucleotide sequence of the homovanillic acid capturing aptamer is SEQ ID NO.4: 5'-ACCAAAGGATCAAGAGCTACCAA-3'.

[0031] The nucleotide sequence of the creatinine detection aptamer is SEQ ID NO.5: 5'-CGGATCGGGTGGTTGGTGGCGG-3'; The nucleotide sequence of the creatinine capture aptamer is SEQ ID NO.6: 5'-CCGCCACCACCACCCGATCCG-3'.

[0032] The final structure of the prepared test strip is shown in the schematic diagram below. Figure 1 As shown in the diagram, the interpretation of the detection results is as follows: Figure 2 As shown.

[0033] Example 2 Performance Test 1. Sensitivity Test Different concentrations of standard solutions for 8-OHdG, homovanillic acid, and creatinine were prepared and tested using the test strip of this invention, with three replicates for each concentration. The results showed that the detection limit for 8-OHdG was 0.8 ng / mL, the detection limit for homovanillic acid was 4 ng / mL, and the detection limit for creatinine was 0.08 mg / dL, all meeting clinical testing requirements. Figure 3 ).

[0034] 2. Specificity test Common interfering substances in urine (urea, uric acid, glucose, valine, and isoleucine) were selected, and solutions were prepared at concentrations five times higher than those commonly used in clinical settings. These solutions were then tested using the test strip of this invention. Results showed that none of the interfering substances caused color development in the test line, indicating that the test strip of this invention has good specificity and no cross-reactivity. Figure 4 ).

[0035] 3. Repeatability test Test strips from the same batch and different batches (3 batches) were selected to test standard samples of the same concentration, and the intra-batch and inter-batch coefficients of variation (CV) were calculated. The results showed that the intra-batch CV was ≤4.2%, and the inter-batch CV was ≤6.8%, indicating good repeatability. Figure 5 ).

[0036] 4. Stability Test The test strips were subjected to accelerated aging at 37°C and 75% relative humidity for 6 months, and their performance was tested periodically. The results showed that the color intensity, sensitivity, and specificity of the test strips did not change significantly, indicating that they had good stability (Table 1).

[0037] Table 1 Stability Test Results

[0038] 5. Clinical sample testing Five hundred clinical samples were collected, including 150 breast cancer patients, 150 patients with benign breast diseases, and 200 healthy individuals. The test strips of this invention were used for detection, and the results were compared with those obtained by high-performance liquid chromatography (HPLC). The results showed that the test strips of this invention had a sensitivity of 88.7% and a specificity of 92.3% for detecting breast cancer, and good consistency with HPLC (Kappa value = 0.81), meeting the needs of clinical screening (Table 2).

[0039] Table 2 Clinical Sample Test Results

[0040] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An aptamer assembly, characterized in that, include: The first aptamer pair for recognizing and binding 8-hydroxy-2'-deoxyguanosine consists of a detection aptamer with the sequence shown in SEQ ID NO.1 and a capture aptamer with the sequence shown in SEQ ID NO.2; The second aptamer pair for recognizing and binding homovanillic acid consists of a detection aptamer with the sequence shown in SEQ ID NO.3 and a capture aptamer with the sequence shown in SEQ ID NO.4; as well as The third aptamer pair for recognizing and binding creatinine consists of a detection aptamer with the sequence shown in SEQ ID NO.5 and a capture aptamer with the sequence shown in SEQ ID NO.

6.

2. The application of the aptamer combination according to claim 1 in the preparation of a rapid detection product for breast cancer urinary metabolites.

3. A method for preparing a rapid test strip for breast cancer urinary metabolites using the aptamer combination described in claim 1, characterized in that, Includes the following steps: Step 1: Preparation of colloidal gold particles: Colloidal gold particles with a particle size of 20-40 nm were prepared by the trisodium citrate reduction method. Step 2: Preparation of colloidal gold-aptamer conjugates: The colloidal gold particles obtained in Step 1 are coupled with the detection aptamer of 8-hydroxy-2'-deoxyguanosine, the detection aptamer of homovanillic acid, and the detection aptamer of creatinine, respectively, to obtain colloidal gold-aptamer conjugates. Step 3: Pre-treatment of sample pads: Immerse the sample pads in the pre-treatment solution and dry them for later use; the pre-treatment solution is a pH 7.2-7.6 PBS buffer containing 0.5-1.0% Tween-20 and 0.1-0.2 M NaCl. Step 4: Preparation of the conjugate pad: Spray the colloidal gold-aptamer conjugate obtained in Step 2 onto the conjugate pad and dry it; Step 5: Processing the nitrocellulose membrane: Sequentially set detection lines T1, T2, T3, and control line C on the nitrocellulose membrane. Coat detection line T1 with the capture aptamer of 8-hydroxy-2'-deoxyguanosine, detection line T2 with the capture aptamer of homovanillic acid, and detection line T3 with the capture aptamer of creatinine. The capture aptamer on detection line T1 is used to capture the complex formed by the 8-hydroxy-2'-deoxyguanosine detection aptamer and the target from step 2; the capture aptamer on detection line T2 is used to capture the complex formed by the homovanillic acid detection aptamer and the target from step 2; and the capture aptamer on detection line T3 is used to capture the complex formed by the creatinine detection aptamer and the target from step 2. Coat control line C with anti-aptamer antibody. Step 6, Assembly: The sample pad obtained in Step 3, the bonding pad obtained in Step 4, the nitrocellulose membrane obtained in Step 5, and the absorbent pad are sequentially overlapped and fixed on the PVC base plate to obtain the final product.

4. The method according to claim 3, characterized in that, The preparation conditions for the colloidal gold-aptamer conjugate in step two are as follows: pH 7.0-7.5, mass ratio of aptamer to colloidal gold 1:50-1:80, incubation at room temperature for 20-30 min, and then blocking with bovine serum albumin.

5. The method according to claim 3, characterized in that, In step five, the capture aptamer coating concentration of detection lines T1, T2, and T3 is 0.5-1.0 mg / mL, and the anti-aptamer antibody coating concentration of control line C is 0.8-1.2 mg / mL; the pore size of the nitrocellulose membrane is 80-120 μm, and the distance between the detection lines and the control lines is 0.5-0.8 cm.

6. A rapid test strip for detecting breast cancer metabolites in urine, characterized in that, It includes a PVC base plate, and a sample pad, a bonding pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and fixed on the PVC base plate; The conjugate pad is adsorbed with colloidal gold-aptamer conjugates, which include colloidal gold particles and 8-hydroxy-2'-deoxyguanosine detection aptamers, homovanillic acid detection aptamers, and creatinine detection aptamers coupled to the colloidal gold particles. The nitrocellulose membrane is sequentially provided with a detection line T1 for detecting 8-hydroxy-2'-deoxyguanosine, a detection line T2 for detecting homovanillic acid, a detection line T3 for detecting creatinine, and a quality control line C. The detection line T1 is coated with an 8-hydroxy-2'-deoxyguanosine capture aptamer, the detection line T2 is coated with a homovanillic acid capture aptamer, the detection line T3 is coated with a creatinine capture aptamer, and the control line C is coated with an anti-aptamer antibody.

7. The rapid test strip for breast cancer urine metabolites according to claim 6, characterized in that, The detection aptamer sequence of the 8-hydroxy-2'-deoxyguanosine is shown in SEQ ID NO.1, and the capture aptamer sequence is shown in SEQ ID NO.2; the detection aptamer sequence of the homovanillic acid is shown in SEQ ID NO.3, and the capture aptamer sequence is shown in SEQ ID NO.4; the detection aptamer sequence of the creatinine is shown in SEQ ID NO.5, and the capture aptamer sequence is shown in SEQ ID NO.

6.

8. The rapid test strip for breast cancer urine metabolites according to claim 6, characterized in that, The colloidal gold particles have a particle size of 20-40 nm; the sample pad is a glass fiber pad that has been soaked in a pretreatment solution and then dried, and the pretreatment solution is a pH 7.2-7.6 PBS buffer containing 0.5-1.0% Tween-20 and 0.1-0.2 M NaCl.

9. The rapid test strip for detecting breast cancer urinary metabolites according to claim 6, characterized in that, The nitrocellulose membrane has a pore size of 80-120 μm, and the distance between the detection lines T1, T2, T3 and the control line C is 0.5-0.8 cm; the capture aptamer coating concentration of the detection lines T1, T2, T3 is 0.5-1.0 mg / mL, and the anti-aptamer antibody coating concentration of the control line C is 0.8-1.2 mg / mL.

10. The use of the aptamer combination of claim 1, the method of any one of claims 3-5, or the test strip of any one of claims 6-9 in the preparation of a breast cancer early screening product.

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