Colloidal gold test strip and kit for quantitatively detecting perfluorooctanoic acid in human serum and use method of colloidal gold test strip
By developing colloidal gold test strips and kits based on monoclonal antibodies, rapid and accurate detection of perfluorooctanoic acid (PFOA) in human serum has been achieved, solving the problem of expensive and complex traditional testing equipment. This technology is suitable for testing needs in grassroots units and developing countries.
Patent Information
- Application Number
- CN202610102784.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-26
- Publication Date
- 2026-05-15
AI Technical Summary
Current technologies lack rapid detection methods for perfluorooctanoic acid (PFOA) in human serum, and traditional detection equipment is expensive and complex, making it difficult to meet the needs of large-scale screening.
Colloidal gold test strips and kits were prepared using monoclonal antibodies that specifically bind to perfluorooctanoic acid (PFOA). The kits included a PVC plate, sample pad, gold label pad, nitrocellulose membrane, and absorbent pad, and quantitative detection was achieved using colloidal gold immunochromatography.
This invention provides a low-cost, simple-to-operate, and highly sensitive detection method suitable for grassroots units and developing countries. It can quickly and accurately detect perfluorooctanoic acid in human serum and is applicable to public health emergency responses.
Smart Images
Figure CN122042957A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a colloidal gold test strip and reagent kit for the quantitative detection of perfluorooctanoic acid in human serum and its usage method. Background Technology
[0002] Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated compound with extremely strong chemical stability and hydrophobic and oleophobic properties. It was once widely used in industries such as non-stick coatings, waterproof materials, and food packaging. However, PFOA is extremely difficult to degrade in the environment, can accumulate through the food chain, and has been proven to have various toxic effects, including disrupting the endocrine system, damaging liver and immune function, increasing cancer risk, and even affecting fetal development. The International Agency for Research on Cancer (IARC) classifies it as a Group 2B possible carcinogen, and countries in Europe and the United States have gradually restricted its use. However, residual PFOA in the environment still poses a persistent threat to global ecosystems and public health. Establishing a rapid immunoassay method for PFOA is urgent and of multiple significances.
[0003] Traditional detection relies on liquid chromatography-mass spectrometry (LC-MS), which offers high accuracy but is expensive and involves complex pretreatment, making it unsuitable for large-scale screening. Immunoassays (such as ELISA or colloidal gold test strips) can achieve rapid on-site detection by specifically recognizing PFOA with antibodies, aiding in pollution source tracing and public health emergency response. PFOA exposure is often insidious and cumulative; rapid screening can promptly assess population exposure levels, especially in high-contamination areas, providing a basis for early intervention. Immunoassay test strips offer rapid detection without complex instruments, with low cost and high throughput per test, making them suitable for deployment in grassroots units or developing countries, addressing the shortcomings of existing technologies in terms of coverage.
[0004] However, existing technologies lack rapid detection techniques for perfluorooctanoic acid (PFOA) in human serum. Based on this, we propose a colloidal gold test strip and kit for the quantitative detection of PFOA in human serum, along with its usage method, in order to address the shortcomings of existing technologies. Summary of the Invention
[0005] To address the lack of rapid detection technology for perfluorooctanoic acid (PFOA) in human serum in existing technologies, this invention provides a low-cost, simple-to-operate, and highly efficient colloidal gold test strip and kit for the quantitative detection of PFOA in human serum. By selecting a monoclonal antibody that specifically binds to PFOA, a colloidal gold test strip was prepared, enabling efficient and rapid detection of whether PFOA levels in human serum exceed the standard.
[0006] This invention is achieved through the following technical solution: The first objective of this invention is to provide a colloidal gold test strip for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum, comprising a PVC board, a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad. The gold-labeled pad is prepared by spraying anti-perfluorooctanoic acid mouse monoclonal antibody onto a glass fiber membrane; The nitrocellulose membrane is provided with a control line and a test line. The control line is formed by spraying goat anti-mouse secondary antibody, and the test line is formed by spraying perfluorooctanoic acid-bovine serum albumin (BSA) solution. The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially pasted onto the PVC board; The murine monoclonal antibody against perfluorooctanoic acid (PFOA) is a murine monoclonal antibody against PFOA obtained by immunizing mice with a hybridoma cell line with accession number CGMCC No. 46526.
[0007] Furthermore, the detection limit of the colloidal gold test strip is 2.4 μg / mL.
[0008] A second objective of this invention is to provide a kit for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum, comprising PFOA extract and the colloidal gold test strip as described in claim 1 or 2.
[0009] Furthermore, the perfluorooctanoic acid extract is acetonitrile.
[0010] A third objective of this invention is to provide a method for quantitatively detecting perfluorooctanoic acid (PFOA) in human serum using the aforementioned kit, comprising the following steps: (1) Take the human serum to be tested into a centrifuge tube, add perfluorooctanoic acid extraction solution, shake at 3000 r / min for 10 min to obtain mixture A; (2) Centrifuge mixture A at 3000 r / min for 5 min, and take the supernatant to obtain solution B; (3) Take 100 μL of solution B and add it to the sample pad of the colloidal gold test strip that has been preheated to 37°C for detection.
[0011] Furthermore, the amount of human serum sample added in step (1) is 1~1.5 mL.
[0012] Further, the amount of perfluorooctanoic acid extract added in step (1) is 5 mL.
[0013] A fourth objective of this invention is to provide a method for preparing the colloidal gold test strip, comprising the following steps: S1. Preparation of colloidal gold-labeled antibody: Colloidal gold solution was prepared by the trisodium citrate reduction method, and then labeled with the anti-perfluorooctanoic acid monoclonal antibody produced by the hybridoma cell line with accession number CGMCC No. 46526. After blocking and purification, the colloidal gold-labeled antibody complex was obtained. S2. Preparation of gold-labeled pad: The colloidal gold-labeled antibody complex obtained in step S1 is sprayed onto a glass fiber membrane and dried to obtain a gold-labeled pad; S3. Coating with nitrocellulose membrane: Spray goat anti-mouse secondary antibody as a quality control line and spray perfluorooctanoic acid-BSA solution as a detection line onto the nitrocellulose membrane. S4. Assembly: The sample pad, the gold label pad obtained in step S2, the nitrocellulose membrane coated in step S3, and the absorbent pad are sequentially pasted onto the PVC base plate. After drying, they are cut to obtain the final product.
[0014] The present invention has the following advantages over the prior art: 1. Traditional instruments for detecting perfluorooctanoic acid (PFOA) in human serum suffer from complex operation and reliance on specialized equipment. This invention innovatively develops a colloidal gold test strip and reagent kit based on a special monoclonal antibody, greatly improving detection efficiency and convenience. This monoclonal antibody has a unique gene sequence, significantly enhancing the detection sensitivity for PFOA in human serum. In use, simply drop the sample onto the sample pad of the test strip. The sample moves towards the absorbent pad under capillary action, interacting with the colloidal gold label as it flows over the gold label pad. If the sample contains PFOA, it will specifically bind to the antigen on the detection line (T line), causing the T line to lighten in color; if the sample does not contain the target analyte, the T line will be relatively darker, meaning the color intensity of the T line is inversely proportional to the target analyte concentration. The control line (C line) serves as a process verification tool. Regardless of the sample condition, colloidal gold labels that have not bound to the T line will migrate to the C line, forming a red band through non-specific binding. If the C line does not develop color, it indicates an error in the detection process, and the result is invalid.
[0015] 2. Compared with traditional instrument detection, the colloidal gold immunochromatography technology of this invention does not require professional personnel to operate or complex equipment. The detection process is simple and fast, maintaining high sensitivity and enabling rapid on-site detection of perfluorooctanoic acid (PFOA) content in human serum. This provides an efficient and practical new solution for responding to public health emergencies and managing health risks from pollutants. Attached Figure Description
[0016] Figure 1 This is a top view schematic diagram of a colloidal gold test strip for quantitative detection of perfluorooctanoic acid in human serum according to the present invention. Figure 2 This is a curve showing the fitting relationship between perfluorooctanoic acid concentration and absorbance value according to the present invention. Figure 3The figure shows the experimental results of this invention. Detailed Implementation
[0017] The technical solutions 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.
[0018] Example: This example provides a colloidal gold test strip for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum, as shown in the attached image. Figure 1 As shown, the assembly includes a sample pad, a gold-labeled pad, a nitrocellulose membrane (chromatographic membrane), and an absorbent pad. The gold-labeled pad is prepared by spraying anti-perfluorooctanoic acid (PFOA) mouse monoclonal antibody onto a glass fiber membrane. The nitrocellulose membrane has control lines and detection lines. The control lines are sprayed with goat anti-mouse secondary antibody, and the detection lines are sprayed with PFOA-BSA (bovine serum albumin) solution. The sample pad, gold-labeled pad, nitrocellulose membrane, and absorbent pad are then sequentially adhered to a PVC board (backing). The overlap length between the sample pad and the gold-labeled pad is 17 mm, the overlap length between the gold-labeled pad and the nitrocellulose membrane is 3 mm, and the overlap length between the nitrocellulose membrane and the absorbent pad is 17 mm. Both the sample pad and the absorbent pad are aligned with the top and bottom edges of the PVC board.
[0019] This embodiment also provides a method for preparing a colloidal gold test strip for quantitative detection of perfluorooctanoic acid in human serum, comprising the following steps: (1) Preparation of monoclonal antibodies: Perfluorooctanoic acid (PFOA) was conjugated with the carrier protein hemocyanin (KLH) to obtain a complete antigen, which was then used to immunize mice. Unlike existing technologies, this example added 0.01 M phosphate buffer containing 10% acetonitrile as a screening matrix during the serum screening stage. The control group mouse serum matrix did not contain PFOA or its structural analogs. The serum of the experimental group mice was evaluated, and the absorbance value at 450 nm was used as the evaluation standard. At the same time, the screening matrix was used to detect the cross-reactivity of mouse serum to PFOA and its structural analogs. Mice with high sensitivity to PFOA and no obvious cross-reactivity to other structural analogs were selected for cell fusion. In this example, a total of 5 mice were screened for mouse serum. The screening results are shown in Table 1 below. PFOA (50 μg / mL) and its structural analogs were added to the experimental group respectively. The inhibition rate of mouse serum was calculated using the following formula: ; Table 1: Mouse serum screening results ; The antibody produced by mouse #1 showed an inhibition rate of 63% against perfluorooctanoic acid (PFOA) at a concentration of 50 μg / mL, and an inhibition rate of less than 10% against other structural analogs at a concentration of 500 μg / mL. It showed high sensitivity to PFOA and no significant cross-contamination with other antioxidants. Therefore, this mouse was selected for cell fusion. After fusion, the cells were screened again using the above-mentioned matrix containing 10% acetonitrile and 0.01 M phosphate buffer to obtain a hybridoma cell line that could produce monoclonal antibodies tolerant to the matrix. This hybridoma cell line was named.
[0020] Hybridoma cell lines were expanded and cultured, then injected into mice to induce ascites. 1 mL of ascites was taken and 1 mL of sodium acetate buffer was added. 33 μL of caprylic acid was added dropwise while stirring at room temperature. The mixture was shaken for 30 min and centrifuged at 9000 r / min for 10 min to obtain the supernatant. 2 mL of saturated ammonium sulfate was added and the mixture was allowed to stand at 4℃ for 2 h. After centrifugation at 8000 r / min for 5 min, the supernatant was discarded. The precipitate was dissolved in PBS and dialyzed for 3 days to obtain a monoclonal antibody against perfluorooctanoic acid.
[0021] In human serum matrix, the IC50 of perfluorooctanoic acid monoclonal antibody was detected. 50 Value, IC 50 The value is 2.4 μg / mL. This method is suitable for the detection of perfluorooctanoic acid (PFOA) in human serum. The applicant has deposited the PFOA hybridoma cell line at the China General Microbiological Culture Collection Center (CGMCC), classifying it as a monoclonal cell line with accession number CGMCC No. 46526, and the deposit date is April 17, 2025.
[0022] (2) Preparation of colloidal gold pads: Using trisodium citrate as a reducing agent, chloroauric acid was reduced to prepare colloidal gold particles with a particle size of 20 nm: First, 1000 mL of 0.01% chloroauric acid aqueous solution was taken and heated to boiling with a constant temperature electromagnetic stirrer. 2 mL of 20% trisodium citrate aqueous solution was added while stirring continuously. Stirring and heating continued for 10-15 min. When the above mixed solution was stably transparent wine red, it was cooled to room temperature to obtain gold nanoparticles with a particle size of 20 nm.
[0023] In this embodiment, the pH of the colloidal gold solution was first adjusted to 9 using K2CO3 solution. Then, a monoclonal antibody with a pH of 7.4 was added and allowed to react fully, wherein the concentration of the perfluorooctanoic acid (PFOA) monoclonal antibody was 100 μg / mL. BSA solution was added to the above mixture to block excess sites, and the precipitate was collected by centrifugation to obtain the monoclonal antibody-colloidal gold label. Subsequently, the monoclonal antibody-colloidal gold label was sprayed onto a glass fiber membrane.
[0024] (3) Spray control lines and test lines onto the nitrocellulose membrane. In this embodiment, goat anti-mouse secondary antibody and perfluorooctanoic acid-BSA solution were first sprayed onto a nitrocellulose membrane to form three lines, which served as the quality control line and the detection line (two lines), respectively. Then, the membrane was dried at 45°C for 48 h.
[0025] (4) Assemble the PVC board, sample pad, gold label pad, nitrocellulose membrane and absorbent pad.
[0026] (5) Cut the colloidal gold test strip assembled in step (4) into strips to obtain colloidal gold test strips. In this embodiment, the specifications of the colloidal gold test strips are 8 cm long × 4 mm wide.
[0027] This embodiment also provides a kit for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum. The kit includes human PFOA extract, diluent, and the colloidal gold test strip prepared above. The PFOA extract is 5 mL of acetonitrile.
[0028] This embodiment also provides a method for using a kit for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum, comprising the following steps: Take 1.0 g of the sample to be tested into a 15 mL centrifuge tube, add acetonitrile, and vortex to obtain mixture A. Centrifuge mixture A at 3000 r / min for 5 min, then take 100 μL of the supernatant and add it to the sample pad of a colloidal gold test strip preheated to 37℃ for detection, timing for 8 min. After timing, remove the test strip and read the result within 20 s.
[0029] Example 1: Optimization of Reagent Kit Sample Content. Following the reagent preparation method of a reagent kit for the quantitative detection of perfluorooctanoic acid (PFOA) in human serum as described in the example, samples were tested according to the prepared amounts shown in Table 3 below. The detection method was the same as the method described above for using the reagent kit for the quantitative detection of PFOA in human serum. Based on the ratio of the T line to the C line on the test strip, the optimal solution formulation was selected as the solution combination that yielded the highest T / C value between 2.0 and 4.0 when PFOA was added at 0 μg / mL, and the highest T / C value ratio when PFOA was added at 50 μg / mL. The experimental results are shown in Table 3 below: Table 2. Reagent dosage combinations and results analysis of the reagent kit ; The experimental results above show that when the acetonitrile volume is 5 mL, the ratio of the T line to the C line on the test strip is the largest, and the T / C value is between 2.0 and 4.0.
[0030] Example 2: Comparison of the effects of this invention with different detection methods. The samples for the experimental and control groups were prepared as follows: human serum samples without perfluorooctanoic acid (PFOA) were taken, and PFOA was added to a concentration of 50 μg / mL. The control group was detected using quantitative HPLC-MS. The experimental method was as follows: 5 mL of the human serum sample to be tested was taken, 50 mL of acetonitrile was added, and the mixture was ultrasonically extracted for 1 h. After cooling to room temperature, the supernatant was centrifuged at 9000 r / min for 5 min, filtered, and then used by the liquid chromatography instrument.
[0031] The experimental group used the detection kit and detection method provided by this invention for detection.
[0032] The experimental results are shown in Table 3 below: Table 3. Results of perfluorooctanoic acid (PFOA) in human serum detected by different methods ; Analysis of experimental results: The experimental results show that the colloidal gold test strip detection method established in this study can detect perfluorooctanoic acid in human serum with a recovery rate between 97.74% and 99.44%, which is not much different from the detection results of HPLC-MS in human serum. The detection kit and detection method provided by this invention are accurate and reliable.
[0033] Meanwhile, compared with other traditional instrumental detection methods (gas chromatography, refer to GB / T 17813-2022), the detection method provided by this invention not only has similar accuracy and repeatability to instrumental methods, but also is safer, less expensive, and faster.
[0034] Experiment Example 3: Specificity Detection Experiment for Perfluorooctanoic Acid (PFOA). Human serum samples lacking PFOA were taken, and perfluorohexadecanoic acid, perfluorooctanoic acid, and perfluorosebacic acid were added to a concentration of 50 μg / mL, respectively. The experimental results were as follows: Figure 3 .
[0035] The test strip method was used for detection. The test results showed that the test strip did not recognize perfluorohexadecanoic acid, perfluorooctanoic acid, or perfluorosebaic acid. The test results of this method are specific.
[0036] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A colloidal gold test strip for quantitative detection of perfluorooctanoic acid (PFOA) in human serum, characterized in that, Includes PVC board, sample pad, gold label pad, nitrocellulose membrane and absorbent pad; The gold-labeled pad is prepared by spraying anti-perfluorooctanoic acid mouse monoclonal antibody onto a glass fiber membrane; The nitrocellulose membrane is provided with a control line and a test line. The control line is formed by spraying goat anti-mouse secondary antibody, and the test line is formed by spraying perfluorooctanoic acid-bovine serum albumin (BSA) solution. The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially pasted onto the PVC board; The murine monoclonal antibody against perfluorooctanoic acid (PFOA) is a murine monoclonal antibody against PFOA obtained by immunizing mice with a hybridoma cell line with accession number CGMCC No. 46526.
2. The colloidal gold test strip for quantitative detection of perfluorooctanoic acid in human serum according to claim 1, characterized in that, The detection limit of the colloidal gold test strip is 2.4 μg / mL.
3. A kit for quantitative detection of perfluorooctanoic acid (PFOA) in human serum, characterized in that, It includes perfluorooctanoic acid extract and the colloidal gold test strip as described in claim 1 or 2.
4. The kit for quantitative detection of perfluorooctanoic acid in human serum according to claim 3, characterized in that, The perfluorooctanoic acid extract is acetonitrile.
5. A method for quantitatively detecting perfluorooctanoic acid (PFOA) in human serum using the kit described in claim 3 or 4, characterized in that, Includes the following steps: (1) Take the human serum to be tested into a centrifuge tube, add perfluorooctanoic acid extraction solution, shake at 3000 r / min for 10 min to obtain mixture A; (2) Centrifuge mixture A at 3000 r / min for 5 min, take the supernatant to obtain solution B; (3) Take 100 μL of solution B and add it to the sample pad of the colloidal gold test strip that has been preheated to 37°C for detection.
6. The method for quantitatively detecting perfluorooctanoic acid in human serum according to claim 5, characterized in that, The amount of human serum sample added in step (1) is 1~1.5 mL.
7. The method for quantitatively detecting perfluorooctanoic acid in human serum according to claim 5, characterized in that, The amount of perfluorooctanoic acid extract added in step (1) is 5 mL.
8. A method for preparing the colloidal gold test strip of claim 1, characterized in that, Includes the following steps: S1. Preparation of colloidal gold-labeled antibody: Colloidal gold solution was prepared by the trisodium citrate reduction method, and then labeled with the anti-perfluorooctanoic acid monoclonal antibody produced by the hybridoma cell line with accession number CGMCC No. 46526. After blocking and purification, the colloidal gold-labeled antibody complex was obtained. S2. Preparation of gold-labeled pad: The colloidal gold-labeled antibody complex obtained in step S1 is sprayed onto a glass fiber membrane and dried to obtain a gold-labeled pad; S3, Coating with nitrocellulose membrane: Spray goat anti-mouse secondary antibody as a quality control line and spray perfluorooctanoic acid-BSA solution as a detection line onto the nitrocellulose membrane; S4. Assembly: The sample pad, the gold label pad obtained in step S2, the nitrocellulose membrane coated in step S3, and the absorbent pad are sequentially pasted onto the PVC base plate. After drying, they are cut to obtain the final product.