A dual-signal immunoassay for bisphenol A integrated with a smartphone platform

Abstract

The present invention belongs to the technical field of food safety and environmental monitoring, and discloses a dual-signal immunoassay method for bisphenol A integrated with a smartphone platform. The method comprises the following steps: (1) preparing an immune probe CPNs@ZIF‑8@Ab that can specifically identify bisphenol A; (2) synthesizing an immunomagnetic bead probe coated with a coating original; (3) preparing a fluorescent gel sensor of a gold nanocluster (GSH‑Au NCs) and a colorimetric gel sensor of TMB; (4) assembling the above components to obtain a Lab‑in‑a‑tube device; (5) using the Lab‑in‑a‑tube device to realize ultrasensitive detection of BPA in environmental samples, and obtaining colorimetric and fluorescent dual signals; (6) converting the colorimetric signal and the fluorescent signal into RGB values ​​based on a color recognition application (APP) on a smartphone to quantitatively analyze BPA. The present invention constructs a Lab‑in‑a‑tube device integrated with a smartphone platform to realize on-site rapid detection of BPA, and the device has the advantages of being portable, easy to operate, visible, sensitive, etc., and has great practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety and environmental monitoring, and relates to a dual-signal immunoassay method for bisphenol A integrated with a smart phone platform. Background Art

[0002] In immunoassays, hydrogen peroxide (H 2 O 2 ) is usually added to the reaction solution as a co-reactant to form various reactive oxygen species (ROS) to oxidize substrates and generate detection signals. 2 O 2 Cannot maintain a stable dry state, directly add H 2 O 2 This limits the widespread application of immunoassays and makes them inapplicable to on-site analysis. To solve this problem, a cascade reaction based on natural enzymes to self-supply H 2 O 2 However, the natural defects of natural enzymes (weak tolerance under extreme conditions) hinder their further application. Therefore, a new type of self-produced H 2 O 2 Strategies to improve the stability of immunoassays are particularly important.

[0003] Metal peroxide nanoparticles, composed of metal ions and peroxide, are a promising H 2 O 2 However, peroxide nanoparticles used in detection systems still have the disadvantages of low signal molecule loading and H 2 O 2 Defects such as low local concentration lead to insufficiently satisfactory sensitivity. Introducing metal-organic frameworks (MOFs) into immunoassay systems will help solve the above key problems. Metal-organic frameworks are composed of metal centers and organic bridging ligands. Their inherent properties of high porosity and large internal surface area give MOFs unique carrying capacity, which can encapsulate more signal molecules and contribute to signal amplification of immunoassays. In addition, the excellent confinement effect induced by its porous crystal structure can increase the local concentration of enzymes / nanoparticles and catalytic substrates, accelerate mass transfer, reduce intermediate decomposition, and improve the overall efficiency of the reaction. Summary of the invention

[0004] The purpose of the present invention is to construct a simple, sensitive, low-cost portable Lab-in-a-tube detection device that integrates a smartphone platform to overcome the technical defects existing in existing detection technologies, significantly improve the accuracy and portability of detection, and help achieve low-cost and on-site analysis of bisphenol A.

[0005] Specifically, this project intends to build a Lab-in-a-tube device integrated with a smartphone sensing platform to achieve dual-mode (colorimetric and fluorescence) immunodetection of BPA. CPNs are encapsulated in ZIF-8 and labeled with bisphenol A monoclonal antibodies as immune probes and dual signal indicators to guide competitive immune responses. Under acid treatment, CPNs@ZIF-8@Ab can simultaneously release Cu 2+ and H 2 O 2 , accompanied by a Fenton-like reaction, producing ·OH to oxidize TMB; in addition, the released Cu 2+ The GSH-Au NCs were quenched, and finally a colorimetric and fluorescent dual-mode signal was formed. Subsequently, a Lab-in-a-tube device was developed for the integrated detection of BPA based on immunomagnetic beads, gel sensors, and smartphone platforms. Based on parameter optimization and validation, the new Lab-in-a-tube device based on dual-mode immunoassay was successfully applied to the detection of BPA in actual samples in food and the environment, and has great potential in constructing portable, sensitive, and accurate detection methods.

[0006] The present invention adopts the following steps:

[0007] A dual-signal immunoassay method for bisphenol A integrated with a smartphone platform comprises the following steps:

[0008] (1) Preparation of copper peroxide nanodots CPNs:

[0009] Copper chloride dihydrate CuCl 2 ·2H 2 The copper peroxide nanodots were obtained by ultrafiltration purification and freeze drying, and then stored at room temperature.

[0010] (2) Preparation of immune probe CPNs@ZIF-8@Ab:

[0011] The CPNs obtained in step (1) are dispersed in a methanol solution containing 2-methylimidazole, and then a zinc acetate methanol solution is added, and the mixture is stirred at room temperature. The BPA monoclonal antibody solution and the BSA blocking solution are sequentially added to the mixed solution, and the stirring is continued. Subsequently, the product is collected by centrifugation and washed with water for several times. Finally, the immune probe is dispersed in a PBS buffer solution and stored for later use;

[0012] (3) Preparation of gold nanoclusters GSH-Au NCs:

[0013] The glutathione aqueous solution GSH was mixed with chloroauric acid HAuCl 4The solutions were mixed and stirred at room temperature, then ultrapure water was added and stirred at 70 °C overnight, and the GSH-Au NCs were collected and stored at room temperature for later use;

[0014] (4) Preparation of Antigen-labelled IMBs:

[0015] At room temperature, add immunomagnetic beads to morphine ethanesulfonic acid buffer (MES) and mix well, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups, then remove the excess EDC and NHS, add the antigen solution diluted in PBS buffer, shake gently overnight, wash several times with PBST buffer, collect Antigen-labeled IMBs, and resuspend in PBS buffer containing 2wt.% BSA for future use;

[0016] (5) Preparation of gel sensor:

[0017] Dissolve agarose in HO 2 O and heat it in a microwave oven until the agarose is completely melted, add 3,3',5,5'-tetramethylbenzidine solution TMB to the agarose, stir continuously for several minutes, then transfer the gel to the cap of centrifuge tube A to cool, obtain the colorimetric gel sensor and store it for future use;

[0018] At the same time, the TMB was replaced by the GSH-Au NCs in step (3), and the GSH-Au NCs solution was added to the agarose. After continuous stirring for several minutes, the gel was transferred to the cap of centrifuge tube B and cooled to obtain a fluorescent gel sensor.

[0019] (6) Detection of bisphenol A based on Lab-in-a-tube device:

[0020] The Antigen-labelled IMBs obtained in step (4) are added to centrifuge tube A or centrifuge tube B containing PBS buffer respectively; then different concentrations of BPA standard and CPNs@ZIF-8@Ab immune probe obtained in step (2) are added, and the mixture is incubated at room temperature. After the incubation is completed, the mixture is separated by a magnet and washed several times with PBST buffer. An acidic solution is added to induce the decomposition of CPNs and the pH is adjusted. Centrifuge tube A or centrifuge tube B is inverted to react for several minutes. Finally, the colorimetric and fluorescence results are observed under a fluorescent lamp and a 300 nm ultraviolet lamp respectively, and a qualitative analysis is performed. At the same time, with the support of a color recognition application APP, RGB analysis is performed on the colorimetric and fluorescence signals, with the BPA concentration as the horizontal axis and the Red / Green channel ratio as the vertical axis, to establish a colorimetric and fluorescence dual signal standard curve for BPA detection;

[0021] (7) Determination of BPA in actual samples:

[0022] After centrifuging the actual liquid sample, collect the supernatant, replace the BPA standard in step (6) with the supernatant, repeat the operation of step (6) to obtain the RGB value and Red / Green channel ratio of the supernatant, and bring them into the colorimetric and fluorescence dual signal standard curve to obtain the BPA concentration of the actual liquid.

[0023] In step (1), CuCl 2 ·2H 2 O, PVP, aqueous sodium hydroxide and H 2 O 2 The dosage ratio is 25mL:2.5g:5mL:500μL; among them, CuCl 2 ·2H 2 The concentration of O is 0.01 M, the concentration of sodium hydroxide aqueous solution is 0.02 M, and H 2 O 2 The concentration is 10M.

[0024] In step (2), the dosage ratio of CPNs, methanol solution containing 2-methylimidazole, zinc acetate methanol solution, BPA monoclonal antibody solution, and BSA blocking solution is 20 mg: 2 mL: 2 mL: 50 μL: 10 μL;

[0025] The concentration of 2-methylimidazole in the methanol solution containing 2-methylimidazole is 160 mM, the concentration of zinc acetate methanol solution is 40 mM; the concentration of BPA monoclonal antibody solution is 0.1 mg / mL; the concentration of BSA blocking solution is 1 wt.%; and the concentration of PBS buffer is 10 mM.

[0026] In step (3), GSH aqueous solution, HAuCl 4 The dosage ratio of solution and ultrapure water was 12.0 mL: 0.8 mL: 27.2 mL; the concentration of GSH aqueous solution was 10 mM, and the concentration of HAuCl 4 The concentration of the solution was 100 mM.

[0027] In step (4), the dosage ratio of MES, EDC, NHS, immunomagnetic beads, antigen solution and PBS buffer containing BSA is 1 mL: 30 mg: 10 mg: 50 μL: 10 μL: 10 μL; wherein the concentration of MES is 50 mM; the concentration of immunomagnetic beads is 10 mg / mL; and the concentration of antigen solution is 0.5 mg / mL.

[0028] In step (5), the ratio of agarose to TMB solution is 0.05 g:300 μL, wherein the concentration of TMB solution is 0.4 mM; the ratio of agarose to GSH-Au NCs solution is 0.05 g:300 μL.

[0029] In step (6), the dosage ratio of PBS buffer, Antigen-labelled IMBs, BPA standard, and CPNs@ZIF-8@Ab immune probe is 30μL: 20μL: 50μL: 50μL; wherein the concentration of PBS buffer is 10mM, and the concentration of BPA standard is 0-5ng / mL; the PBST buffer used in the washing process is a PBS solution containing 0.05% Tween-20; the acidic solution is a dilute hydrochloric acid solution, and the pH is adjusted to 5.

[0030] In step (6), the color recognizer application can accurately convert the optical signal into the numerical values ​​of the Red, Green and Blue channels.

[0031] Compared with the prior art, the advantages of the present invention are:

[0032] (1) The present invention introduces self-produced H based on copper peroxide nanomaterials 2 O 2 Replacing the traditional direct addition mode helps to improve the stability and accuracy of the detection system and provides a new idea for building a more stable immunoassay method.

[0033] (2) The present invention simultaneously introduces colorimetric signals and fluorescent signals, which can significantly improve the reliability of the detection method and obtain better accuracy by reducing certain negative interferences. At the same time, the dual-mode signal can make up for the inherent limitations of each sensing modality and integrate their unique advantages, thereby having a synergistic effect on analytical performance.

[0034] (2) The present invention provides a portable Lab-in-a-tube detection device integrated with a smartphone platform, which integrates the detection system into a centrifuge tube, overcomes the technical defects existing in the existing detection technology, significantly improves the portability of the detection and completes rapid quantification during on-site analysis, showing great potential in environmental assessment and biological monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the Lab-in-a-tube device integrated with a smartphone platform for dual-signal immunoassay of bisphenol A;

[0036] Figure 2 (A) TEM image of CPNs; (B) Cu 2p XPS spectrum;

[0037] Figure 3 (A) Colorimetric verification of H 2 O 2 (a) MnO 4- (b) MnO 4- +H + ; (c) MnO 4- +H + +H 2 O 2 ; (d) MnO 4- +H + +CPNs@ZIF-8;

[0038] (B) ICP determination of Cu 2+ release;

[0039] (C) Verification of colorimetric signal induced by acid hydrolysis of CPNs@ZIF-8. (a) TMB; (b) TMB+CPNs@ZIF-8; (c) TMB+H + +CPNs@ZIF-8;

[0040] (D) EPR spectroscopy confirms the presence of OH;

[0041] (E)Cu 2+ Verification of quenching GSH-Au NCs fluorescence. (a) GSH-Au NCs+CPNs@ZIF-8; (b) GSH-Au NCs+CPNs@ZIF-8+acid; (c) GSH-Au NCs+acid; (d) GSH-Au NCs+H 2 O 2 ;

[0042] (F) TEM verification of Cu 2+ Quenching of GSH-Au NCs.

[0043] Figure 4 (A) SEM image of CPNs@ZIF-8; (B) XPS spectrum of CPNs@ZIF-8; (C) SEM image of CPNs@ZIF-8@Ab;

[0044] Figure 5 (A) Schematic diagram of Lab-in-a-tube device for detecting BPA; (B) Colorimetric analysis diagram; (C) Fluorescence analysis diagram; (D) Schematic diagram of RGB analysis of color recognition APP; (E) Linear relationship between Green / Red channel value and BPA concentration in the range of 0-5ng / mL BPA; (F) Linear relationship between the ratio of Green / Red channel value and BPA concentration in the range of 0-5ng / mL BPA. DETAILED DESCRIPTION

[0045] The following is a detailed description of the embodiments of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, which is intended to facilitate the understanding of the technical solution features of the present invention, and does not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent transformation or equivalent replacement falls within the scope of protection of the present invention.

[0046] Embodiment 1:

[0047] Lab-in-a-tube device integrated with smartphone platform for dual-signal immunoassay of bisphenol A

[0048] (1) Synthesis of PVP-coated copper peroxide nanodots:

[0049] CuCl 2 ·2H 2 O aqueous solution (25 mL, 0.01 M) and polyvinyl pyrrolidone (PVP, 2.5 g) were mixed evenly under ultrasonic conditions. Subsequently, NaOH (5 mL, 0.02 M) and H 2 O 2 (500 μL 10 M) and stirred for 0.5 h. Finally, the PVP-coated copper peroxide nanodots were purified by ultrafiltration and freeze-dried and stored in a 4°C refrigerator.

[0050] Result analysis: From Figure 2 From the TEM image (A), we can see that the PVP-coated CPNs have good dispersion and a small particle size of about 5.5 nm, and are surrounded by irregular shadows, which may be related to the PVP coating. Figure 2 (B) XPS spectrum shows that the two peaks at 934.5 and 953.6 eV belong to Cu 2p 1 / 2 and Cu 2p 3 / 2 orbital, indicating that the valence state of copper in PVP-CPNs is +2. The above characterizations provide a basis for the successful preparation of CPNs.

[0051] (2) pH-responsive CPNs acidolysis-induced colorimetric performance and fluorescence quenching verification

[0052] Result analysis:

[0053] (a) CPNs acidolysis induced H 2 O 2 and Cu 2+ As a key step in this study, we evaluated in detail the ability of CPNs in CPNs@ZIF-8 to self-generate Cu under acidic conditions. 2+ and H 2 O2 First, potassium permanganate colorimetry was used to verify the H 2 O 2 The existence of Figure 3 As shown in A, potassium permanganate (MnO 4 - ) solution (curve a) and potassium permanganate (MnO 4 - ) acid solution (curve b) all showed pink; then in potassium permanganate (MnO 4 - ) Add CPNs@ZIF-8 or H 2 O 2 The pink color gradually fades, which is due to the peroxide groups converting MnO 4 - Reduced to colorless Mn 2+ , confirming that CPNs have H 2 O 2 In addition, a large amount of Cu is also released during the acid hydrolysis process. 2+ ( Figure 3 B) The above results indicate that the pH-responsive decomposition of CPNs can trigger H 2 O 2 and Cu 2+ self-supply behavior.

[0054] (b) ·OH-triggered colorimetric ability. The acid-induced decomposition of CPNs@ZIF-8 can oxidize TMB to produce a blue colorimetric signal with a maximum absorption peak at 650 nm. Figure 3 C shows that there is no obvious change in TMB solution when CNPs@ZIF-8 (curve a) or acid solution (curve b) is added alone. In contrast, after adding CPNs@ZIF-8 to the acid solution (curve c), TMB undergoes a significant blue change, which is due to the decomposition of CPNs driving the Cu 2+ and H 2 O 2 The generation of ·OH triggers a Fenton-like reaction to produce ·OH to oxidize TMB and generate a colorimetric signal. Figure 3 The EPR spectrum of D shows that after acid-induced decomposition, CPNs@ZIF-8 has obvious ·OH signal, which confirms that Cu 2+ and H 2 O 2 There is a Fenton-like reaction between them. Therefore, the acid-mediated decomposition of CPNs can induce the generation of colorimetric signals.

[0055] (c)Cu 2+ Trigger fluorescence quenching. Figure 3As shown in E, CPNs@ZIF-8 (curve a), acid (curve c) and H were added to GSH-Au NCs. 2 O 2 After (curve d), the fluorescence of GSH-Au NCs was not quenched. However, the introduction of CPNs@ZIF-8 into acidic solution (curve b) could quench the fluorescence of GSH-Au NCs. This is because the Cu 2+ The fluorescence quenching caused by the aggregation of GSH-Au NCs due to complexation with GSH molecules. The aggregation of GSH-Au NCs in TEM images proves the above hypothesis ( Figure 3 F).

[0056] (3) Preparation of immune probe (CPNs@ZIF-8@Ab):

[0057] The obtained CPNs were dispersed in a methanol solution containing 2-methylimidazole, and then a zinc acetate methanol solution was added. After stirring at room temperature for 2 h, a BPA monoclonal antibody solution and a BSA blocking solution were added in sequence, and stirring was continued for 4 h. Subsequently, the product was collected by centrifugation at 11000 rpm for 5 min and washed with water several times. Finally, CPNs@ZIF-8@Ab was dispersed in PBS and stored at 4 °C for further use.

[0058] Results analysis: First, through SEM images ( Figure 4 A) CPNs@ZIF-8 was characterized, and it showed a typical rhombic dodecahedron structure of pure ZIF-8, indicating that CPNs encapsulated in ZIF-8 had no effect on the crystal formation of ZIF-8; at the same time, the Cu element appeared in the XPS spectrum of CPNs@ZIF-8, indicating that CPNs were successfully encapsulated in ZIF-8 ( Figure 4 B). The above results all confirm the successful preparation of CPNs@ZIF-8. Figure 3 As shown in Figure C, the modification of the antibody has no effect on the crystal formation of ZIF-8. Compared with CPNs@ZIF-8, the surface of CPNs@ZIF-8@Ab is rougher, which also confirms the successful modification of the antibody.

[0059] (4) Preparation of gold nanoclusters GSH-Au NCs:

[0060] Glutathione aqueous solution GSH (12.0 mL, 10 mM) and chloroauric acid HAuCl 4 The solution (0.8 mL, 100 mM) was mixed and stirred at room temperature, and then ultrapure water (27.2 mL) was added and stirred at 70°C overnight, and GSH-Au NCs were collected and stored at room temperature for later use. The obtained GSH-Au NCs had strong orange-yellow fluorescence under 300 nm excitation.

[0061] (5) Preparation of Antigen-labelled IMBs:

[0062] At room temperature, add immunomagnetic beads (50 μL, 10 mg / mL) to morphine ethanesulfonic acid buffer (1 mL, 50 mM) and mix well. Then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC (30 mg) and N-hydroxysuccinimide NHS (10 mg) to activate the carboxyl group. Then, remove the excess EDC and NHS, add antigen solution (10 μL, 0.5 mg / mL) diluted with PBS buffer, shake gently overnight, wash several times with PBST buffer, collect Antigen-labelled IMBs, and resuspend in PBS buffer containing 2 wt.% BSA for future use.

[0063] (6) Preparation of gel sensor:

[0064] Agarose (0.05 g) was dissolved in H 2 O and heated in a microwave oven until the agarose is completely melted, 3,3',5,5'-tetramethylbenzidine solution TMB (300 μL, 0.4 mM) is added to the agarose, and after continuous stirring for several minutes, the gel is transferred to the cap of centrifuge tube A to cool, and the colorimetric gel sensor is obtained and stored for later use;

[0065] At the same time, the TMB was replaced by the GSH-Au NCs in step (3), and the GSH-Au NCs (300 μL) solution was added to the agarose. After continuous stirring for several minutes, the gel was transferred to the cap of centrifuge tube B and cooled to obtain a fluorescent gel sensor.

[0066] (7) Detection of bisphenol A based on Lab-in-a-tube device:

[0067] The Antigen-labelled IMBs (20 μL) obtained in step (4) were added to centrifuge tube A or centrifuge tube B containing PBS buffer (30 μL), respectively; then, different concentrations of BPA standard (50 μL) and CPNs@ZIF-8@Ab immune probe (50 μL) obtained in step (2) were added, and the mixture was incubated at room temperature. After the incubation, the mixture was separated by a magnet and washed several times with PBST buffer. An acidic solution was added to induce the decomposition of CPNs and the pH was adjusted to 5. Centrifuge tube A or centrifuge tube B was inverted for reaction for several minutes. Finally, the colorimetric and fluorescence results were observed under a fluorescent lamp and a 300 nm ultraviolet lamp, respectively, and qualitative analysis was performed. At the same time, with the support of a color recognition application APP, RGB analysis of the colorimetric and fluorescence signals was performed, with the BPA concentration as the horizontal axis and the Red / Green channel ratio as the vertical axis, to establish a colorimetric and fluorescence dual signal standard curve for BPA detection.

[0068] Result analysis: The process of assembling and testing BPA of Lab-in-a-tube equipment is as follows Figure 5 As shown in A. Similar to the above dual-mode ELISA, after adding different concentrations of BPA, a series of colorimetric and fluorescence changes appeared on the gel sensor, realizing the visual analysis of BPA ( Figure 5 B and 5C). In addition, based on a self-compiled color recognition application (APP) on a smartphone platform, the colorimetric and fluorescence images were converted into RGB values ​​for quantitative detection ( Figure 5 D) Figure 5 E and F, as the BPA concentration increases, the Green / Red ratio shows a good linear relationship with the BPA concentration, and the detection limit (LOD) is calculated according to 3σ / k (where σ is the standard deviation of the blank sample and k is the slope of the calibration curve). The detection limit of the colorimetric method is 0.05ng / mL, and the detection limit of the fluorescence method is 0.02ng / mL. In summary, the Lab-in-a- tube device simultaneously realizes semi-quantitative detection by naked eye and quantitative and accurate determination by smartphone platform, which has greater advantages and potential in pollution detection.

[0069] (8) Lab-in-a-tube device integrated with smartphone platform for detecting BPA in real samples

[0070] Results analysis: In order to evaluate its application potential and feasibility in real samples (such as tap water, river water, lake water and milk), a series of 0, 0.5, 2 and 5 ng / mL BPA standards were added. As shown in Table 1, the spiked recoveries of BPA in real samples were 92.8-117.3% (CV: 1.2-3.1%), indicating that the established method can be applied to real samples. In addition, the established immunoassay was used to monitor the BPA levels in real water samples, and the results are shown in Table 2. The test results were consistent with the traditional ELISA method, indicating that this method can be used for the detection of real water samples.

[0071] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to illustrate all the embodiments here. However, obvious changes or modifications derived from this solution are still within the scope of protection of the present invention.

[0072] Table 1. Recovery and relative standard deviation (RSD, n = 3) of BPA detection in different real samples using the Lab-in-a-tube device integrated with a smartphone sensing platform

[0073]

[0074] “-”: Not detected

[0075] Table 2. Concentration determination of BPA in actual water samples

[0076]

[0077] ND: Not detected.

Claims

1. A dual-signal immunoassay method for bisphenol A integrated with a smartphone platform. It is characterized in that The steps include: (1) Preparation of copper peroxide nanodots CPNs: Copper chloride dihydrate CuCl 2 ·2H 2 The copper peroxide nanodots were obtained by ultrafiltration purification and freeze drying, and then stored at room temperature. (2) Preparation of immune probe CPNs@ZIF-8@Ab: The CPNs obtained in step (1) are dispersed in a methanol solution containing 2-methylimidazole, and then a zinc acetate methanol solution is added, and the mixture is stirred at room temperature. The BPA monoclonal antibody solution and the BSA blocking solution are sequentially added to the mixed solution, and the stirring is continued. Subsequently, the product is collected by centrifugation and washed with water for several times. Finally, the immune probe is dispersed in a PBS buffer solution and stored for later use; The dosage ratio of CPNs, methanol solution containing 2-methylimidazole, zinc acetate methanol solution, BPA monoclonal antibody solution, and BSA blocking solution was 20 mg: 2 mL: 2 mL: 50 μL: 10 μL; The concentration of 2-methylimidazole in the methanol solution containing 2-methylimidazole is 160 mM, the concentration of zinc acetate methanol solution is 40 mM; the concentration of BPA monoclonal antibody solution is 0.1 mg / mL; the concentration of BSA blocking solution is 1 wt.%; the concentration of PBS buffer is 10 mM; (3) Preparation of gold nanoclusters GSH-Au NCs: The glutathione aqueous solution GSH was mixed with chloroauric acid HAuCl 4 The solutions were mixed and stirred at room temperature, then ultrapure water was added and stirred at 70 °C overnight, and the GSH-Au NCs were collected and stored at room temperature for later use; (4) Preparation of Antigen-labelled IMBs: At room temperature, add immunomagnetic beads to MES buffer and mix well, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS to activate the carboxyl groups, then remove the excess EDC and NHS, add the antigen solution diluted in PBS buffer, shake gently overnight, wash several times with PBST buffer, collect Antigen-labeled IMBs, and resuspend in PBS buffer containing BSA for future use; (5) Preparation of gel sensor: Dissolve agarose in HO 2 O and heat it in a microwave oven until the agarose is completely melted, add 3,3',5,5'-tetramethylbenzidine solution TMB to the agarose, stir continuously for several minutes, then transfer the gel to the cap of centrifuge tube A to cool, obtain the colorimetric gel sensor and store it for future use; At the same time, the TMB was replaced by the GSH-Au NCs in step (3), and the GSH-Au NCs solution was added to the agarose. After continuous stirring for several minutes, the gel was transferred to the cap of centrifuge tube B and cooled to obtain a fluorescent gel sensor. (6) Detection of bisphenol A based on Lab-in-a-tube device: The Antigen-labelled IMBs obtained in step (4) are added to centrifuge tube A or centrifuge tube B containing PBS buffer respectively; then different concentrations of BPA standard and CPNs@ZIF-8@Ab immune probe obtained in step (2) are added, and the mixture is incubated at room temperature. After the incubation is completed, the mixture is separated by a magnet and washed several times with PBST buffer. An acidic solution is added to induce the decomposition of CPNs and the pH is adjusted. Centrifuge tube A or centrifuge tube B is inverted to react for several minutes. Finally, the colorimetric and fluorescence results are observed under fluorescent light and ultraviolet light respectively and qualitative analysis is performed. At the same time, with the support of a color recognition application APP, RGB analysis is performed on the colorimetric and fluorescence signals, with the BPA concentration as the horizontal axis and the Red / Green channel ratio as the vertical axis, to establish a colorimetric and fluorescence dual signal standard curve for BPA detection; The usage ratio of PBS buffer, Antigen-labelled IMBs, BPA standard, and CPNs@ZIF-8@Ab immune probe is 30 μL: 20 μL: 50 μL: 50 μL; the concentration of PBS buffer is 10 mM, and the concentration of BPA standard is 0-5 ng / mL; The PBST buffer used in the washing process was a PBS solution containing 0.05% Tween-20; The acidic solution is a dilute hydrochloric acid solution, and the pH value is adjusted to 5; (7) Determination of BPA in actual samples: After centrifuging the actual liquid sample, collect the supernatant, replace the BPA standard in step (6) with the supernatant, repeat the operation of step (6) to obtain the RGB value and Red / Green channel ratio of the supernatant, and bring them into the colorimetric and fluorescence dual signal standard curve to obtain the BPA concentration of the actual liquid.

2. The method according to claim 1, It is characterized in that In step (1), CuCl 2 ·2H 2 O, PVP, aqueous sodium hydroxide and H 2 O 2 The dosage ratio is 25mL:2.5g:5mL:500μL; among them, CuCl 2 ·2H 2 The concentration of O is 0.01 M, the concentration of sodium hydroxide aqueous solution is 0.02 M, and H 2 O 2 The concentration is 10M.

3. The method according to claim 1, It is characterized in that In step (3), GSH aqueous solution, HAuCl 4 The dosage ratio of solution and ultrapure water was 12.0 mL: 0.8 mL: 27.2 mL; the concentration of GSH aqueous solution was 10 mM, and the concentration of HAuCl 4 The concentration of the solution was 100 mM.

4. The method according to claim 1, It is characterized in that In step (4), the usage ratio of MES, EDC, NHS, immunomagnetic beads, antigen solution and PBS buffer containing BSA is 1 mL: 30 mg: 10 mg: 50 μL: 10 μL: 10 μL; Wherein, the concentration of MES is 50 mM; the concentration of immunomagnetic beads is 10 mg / mL; the concentration of antigen solution is 0.5 mg / mL; and the concentration of BSA in the PBS buffer containing BSA is 2 wt.%.

5. The method according to claim 1, It is characterized in that In step (5), the ratio of agarose to TMB solution is 0.05 g:300 μL, wherein the concentration of TMB solution is 0.4 mM; The usage ratio of agarose and GSH-Au NCs solution was 0.05 g:300 μL.

6. The method according to claim 1, It is characterized in that In step (6), the color recognizer application can accurately convert the optical signal into the numerical values ​​of the Red, Green and Blue channels.

7. The method according to claim 1, It is characterized in that In step (6), the ultraviolet lamp is a 300nm ultraviolet lamp.

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