Skip to main content
NCBI home page
Food Chemistry: X logoLink to Food Chemistry: X
. 2024 Oct 15;24:101903. doi: 10.1016/j.fochx.2024.101903

Sensitive and rapid detection of bisphenol A using signal amplification nanoparticles loaded with anti-bisphenol A monoclonal antibody

Sumei Ling a,1, Aidi Xu a,1, Menghan Sun a, Xiaoli Li a,b, Yongming Huang a, Yang Xu a, Jianli Huang b, Tingting Xie b, Shihua Wang a,⁎
PMCID: PMC11547962  PMID: 39525053

Abstract

Bisphenol A has been reported to be a ubiquitous contaminant, and exposure to this compound can lead to adverse effects in human health. In the study, monoclonal antibody against BPA (anti-BPA mAb) with high affinity (3.74 × 109 L/mol) secreted by cell line 2E3 was successfully screened. Inspired by the signal amplification of nanoparticles, anti-BPA mAbs were labeled with nano-materials including colloidal gold (AuNP) and gold nanoflowers (AuNF) for preparation of immunoprobes and AuNP-/AuNF-based test strips. The developed AuNP- and AuNF-based test strips achieved the rapid and sensitive detection of BPA within 10 min, with the limit of detection (LOD) of 25 μg/mL and 3.125 μg/mL, respectively. The detection result in BPA spiked samples measured by the proposed methods was consistent with that detected by LC-MS method. The preparation process of as-prepared test strip is time-saving and considered as ideal candidates method for rapid screening BPA in real samples.

Keywords: Bisphenol A, Immunochromatographic assay, Monoclonal antibody, Nanoparticle, Immunoprobe

Highlights

  • •

    Anti-BPA mAb with high affinity secreted by 2E3 cell line was obtained.

  • •

    Colloidal gold (AuNP) and gold nanoflowers (AuNF) were loaded with anti-BPA mAb for immunoprobes preparation.

  • •

    The sensitivity of test strip was significantly enhanced when AuNF was employed as a signal reporter.

1. Introduction

Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane) BPA is a known monomer for organic polymer materials synthesis (Gao et al., 2018; Qiu et al., 2018) and broadly applied for the production of epoxy resins, polycarbonate polymers and polyphenylene oxide resin products (Vandenberg et al., 2007; Rochester et al.,2013; Lorber et al., 2015; Li et al., 2019). Thus, BPA is ubiquitous in many consumer products like water bottles, food packaging and thermal receipts, and so on (Rochester, 2013). However, a large body of evidence have pointed out that human beings exposed to BPA though consumption of the BPA leaching from contamination food result in adverse health outcomes. Previous studies pointed out that BPA is considered to be an endocrine-disrupting chemical that could potentially disrupt the reproductive system and central nervous system, causing adverse effects to human health (Biedermann et al., 2010; Li et al., 2010; Michałowicz, 2014; Mustieles et al., 2018). Therefore, it is extremely imperative to design a rapid, on-site, and sensitive assay for BPA detection.

At present, analytical methods commonly employed for determination of BPA are gas chromatography (Gatidou et al., 2007), liquid chromatography (Inoue et al., 2000), enzyme-linked immunosorbent assays (ELISA) (Feng et al., 2009; Kim et al., 2007; Ohkuma et al., 2002), etc. However, these traditional methods are hindered by the expensive instruments, complex in processes, and proficient operation required for fabrication. Immunoassays rapidly and precisely detecting target contamination analyte have attracted great attention, which was developed based on the mechanisms though the specific reaction between antibody and antigen. However, ELISA also contains some time-consuming steps such as washing step, coating step. Immunochromatographic assay is increasingly regarded as an ideal candidate method for rapid detection, because of its features of simplicity, cheapness and reliability (Wang et al., 2023; Wu et al., 2021; Xiao et al., 2018). For designing the sensitive immunochromatographic strip, signal amplification probe is considered to be a crucial step. Colloidal gold (AuNP) with advantages of good conductivity and biocompatibility was commonly used as a signal reporter for establishment of point-of-care immunochromatographic strip (Ling et al., 2021; Wang et al., 2022). Moreover, gold nanoflower (AuNF) has become a powerful candidate nanoparticles for development of immunosensors, owing to their three-dimensional structural features of large total surface, multi-branched and colloid-stability (Ji et al., 2015; Zhou et al., 2018). Actually, our previous work also used AuNF as antibody labeled tracer to establish test strip for rapidly monitoring different substances including heavy metal and mycotoxin (Ling et al., 2022; Ling et al., 2020). These results also revealed that AuNF as powerful candidate signal reporter could significantly enhance the sensitivity of immunochromatographic strip.

Herein, we designed immunochromatographic strip which combines the advantages of anti-BPA mAb with high affinity and signal amplification reporter. In this study, BPA complete antigens were prepared by coupling with carrier proteins (BSA, OVA, KLH) using NHS and EDC as chemical coupling agents. Anti-BPA mAb with high affinity was obtained though immunization, cell fusion and sub-cloning technology. Then, AuNP and AuNF were taken into consideration to label anti-BPA mAb, and then employed to develop the AuNP-and AuNF-based strip test.

2. Materials and methods

2.1. Materials

Bisphenol A (BPA) was purchased from TCI Corp (Tokyo, Japan). Keyhole limpet hemocyanin (KLH), ovalbumin (OVA), bovine serum albumin (BSA), and 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N-Hydroxysuccinimide (NHS) and 4-Morpholineethanesulfonic acid (MES) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Antibody sub-type kit was purchased from Proteintech Group, Inc. (Wuhan, China). Chloroauric acid was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Nitrocellulose membrane (NC membrane), and PVC plate were obtained from Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China).

2.2. Preparation of complete antigens and hybridoma cells

Complete antigens including BPA-BSA, BPA-OVA and BPA-KLH were prepared as follows. 0.9 mg BPA, 2.98 mg NHS and 4.56 mg EDC were measured and mixed together, and then 300 μL DMSO was added. The reaction solution was labeled as mixture 1. Then, 12 mg BSA, 6 mg OVA and 7.5 mg KLH were completely dissolved in 3 mL PBS (pH 9.0), respectively, and labeled as mixture 2. After mixture 1 completely reacted with mixture 2, the resulting reaction solution was centrifuged at 6000 r/min for 5 min, and the supernatant was collected and dialyzed against 0.01 mol/L PBS. The obtained complete antigens were identified by agarose gel electrophoresis.

Then, Balb/c mice were immunized with BPA-BSA and BPA-OVA emulsified with an equal volume of Freund's complete adjuvant, respectively, by subcutaneous multi-point injection (Moreno et al., 2011). Booster injections immunized with the same complete antigen which was emulsified in Freund's incomplete adjuvant, were given once every two weeks. Five days after the third immunization, mice were tail-bled and the antiserum titer was determined by indirect ELISA using BPA-KLH as the coating antigen. The mice with the highest antibody titer were challenged with 50 μg of pure conjugate without adjuvant and used as spleen donors. Then, hybridoma cells were prepared by the fusion of splenocytes from the immunized mouse and SP2/0 myeloma cells in the presence of PEG 1450, and then cultured in 96-well cell culture plate (37 °C, 5 % CO2). Two weeks after cell fusion, hybridoma cell lines secreting anti-BPA mAb were screened with indirect competitive ELISA. After 4 arounds sub-clone screening by the method of limiting dilution, the table hybridoma cell lines were obtained (Raysyan et al., 2023).

2.3. Characterization of antibody against BPA

The stable hybridoma cell lines secreting anti-BPA mAb were injected intraperitoneally into Balb/c mice pre-injected with 0.5 mL of liquid paraffin. After 10 days injection, the ascites were collected and mAbs were purified from the ascites by ammonium sulfate precipitation and Protein G. SDS-PAGE was carried out to identify the results of the purified mAb. A mouse monoclonal antibody isotyping kit was used to determine the subclass of positive cell lines. The method of Jimsey staining was carried out to measure the chromosome number of the positive cell line. Affinity of anti-BPA mAb secreted by 2E3 and 7A8 cell line were analyzed by indirect ELISA using series of contents of antibody and BPA-KLH coating antigen (Ling et al., 2022).

2.4. Synthesis of AuNP and AuNP probes

AuNPs particles were prepared according to the previously reported method (Wen-de et al., 2017). In brief, 1 mL of 1 % chlorauric acid solution was firstly added into a 250 mL flask containing 100 mL of deionized water and the mixture was heated to boil. Then, 2 mL of 1 % sodium citrate solution was added into the mixture for reaction. The color changes from purple to red in 2 min, and the red obtained solution was heated for an additional 5 min. Then, the solution was allowed to cool down at room temperature. Finally, the obtained AuNPs solution was stored at 4 °C for future use.

The preparation of AuNP probe has been described in previous research (Ji et al., 2015). Briefly, the AuNPs solution was adjusted to optimum PH with K2CO3, and then suitable content anti-BPA mAb was added into 10 mL AuNPs solution under gentle stirring for 1 h at room temperature. Subsequently, 0.102 g BSA was added for another 30 min. After centrifugation, the obtained AuNP probe was redissolved with 1.0 mL ddH2O. The resulting AuNP solution and AuNP probe were characterized using ultraviolet spectrophotometer within a wavelength range of 500–700 nm.

2.5. Preparation of AuNF and AuNF probes

AuNFs particles were synthesized using a gold seed-mediated growth according to the method of Ji et al. with some modification (Ji et al., 2015). The AuNP particles were firstly prepared just as the 2.4 section described, and used as gold seeds. Subsequently, 750 μL 1 % chlorauric acid solution, 500 μL newly as-prepared AuNPs seeds and 300 μL 1 % sodium citrate were added in sequence to 100 mL deionized water. After reaction for 30 s, 1 mL 0.03 M hydroquinone was added, and the color changes from colorless to dark blue. The prepared AuNF solution was obtained and stored at 4 °C for further use.

Anti-BPA mAb labeled with as-prepared AuNF particles was according to the method described previously (Jia et al., 2022; Zhang et al., 2019). The detail was described as follows. AuNFs solution was adjusted with K2CO3 to optimum PH, then optimum anti-BPA mAb was added into 10 mL AuNFs solution with gentle stirring for 1 h. Subsequently, 0.104 g BSA and 0.052 g PEG 2000 were added for another 30 min. After centrifugation, the obtained AuNF probe was redissolved for further use. The prepared AuNF solution and AuNF probe were characterized using ultraviolet spectrophotometer within a wavelength range of 500–700 nm and stored at 4 °C for further use.

2.6. Assemble and principle of test strip

The formation of the immunoassay using the AuNPs and AuNFs as signal amplification are described as follows. The test strip contains four parts, such as conjugate pads, sample pads, the absorbent pad and the NC membrane. The prepared color probes (AuNP-mAbs or AuNF-mAbs) were placed on the pre-treated conjugate pads. The BPA-KLH conjugate and goat anti-mouse IgG were immobilized on the NC membrane as the test and control lines, respectively. Finally, four parts of the proposed test strip were sequentially glued together and cut down into 60 mm long by 4 mm wide test strip (Huang et al., 2023).

The principle of the test strip described as follows. When samples containing BPA, the AuNP or AuNF probe on the conjugate would be react with BPA firstly, resulting in little probe be captured by BPA-KLH antigen coated on the T line. Thus, the color on the T line would be decreased as the content of BPA increased. Nevertheless, the clear color always display on the C line.

2.7. Evaluation of AuNP-and AuNF-based test strip

The sensitivity of the AuNP-and AuNF-based strip were determined by testing different concentrations of BPA with the range of 0–50 μg/mL under optimal experimental condition. The specificity of the test strip was assessed by testing different BPA analog standards including 4,4’-Thiodbisphenol, 4-Cumylphenolm, 4,4’-Dihy oxybenzophenon, 4,4-Dihydroxydiphenylmethane, Doxycycline hyclate, and Minocycline solution into individual test strip (Li et al., 2021). The visual limit of detection (vLOD) was considered as the minimum BPA to produce colorless T line on the NC membrane. The optical densities of T and C line were recorded using optical strip reader. The B/B0 value was defined as the ratio of T/C value with and without competitive BPA in analyzed samples, and concentration which gave 80 % B/B0 values was considered to be quantitative LOD (qLOD) of the proposed method (Ling et al., 2022). Analyzed samples including bottled water, wheat tea, xiancao honey, wangzai milk, milk beer, herbal tea, milk peanut, sprite, coconut water and guava juice were purchased from local market and the sample extracts were filtered with 0.22 μm microfiltration membrane. Then, AuNP-based test strip and AuNF-based strip methods developed in the study were performed for detection of BPA in actual samples. Then, LC-MS method was used to evaluate the reliability of these developed methods. BPA content spiked with bisphenol A at concentrations of 0.5, 1 and 2 μg/mL were measured using the developed methods (AuNP- and AuNF based test strip) and LC-MS method (Pan et al., 2021).

2.8. Data analysis

The titer of the anti-serum, cell supernatant titer, ascites and purified mAb were determined by ELISA method with three triplicates for each experiment, and the obtained data were analyzed using Graphpad Prism 8.0.2 software. All the experiments carried out using proposed immunochromatography methods were also performed three triplicates for each analysis. The color intensity of the T line and C line was scanned by membrane strip reader for quantitative analysis, and Microsoft Excel 2013 and Graphpad Prism 8.0.2 software were used to analyze and plot the data.

3. Results and discussions

3.1. Evaluation of complete antigens and anti-serum against BPA

To obtain antibody against BPA, the most important requirement is to prepare artificial complete antigen, because BPA with small molecular size has no immunogenicity. In the study, BPA was coupled with carrier proteins (BSA, OVA, KLH) using NHS and EDC as chemical coupling agents. Agarose gel electrophoresis was carried out to identify the artificial complete antigens. As shown in Fig. 1A, all the band migrations of complete antigens BPA-BSA, BPA-OVA and BPA-KLH were significant different from that of carrier proteins (BSA, OVA, KLH). The migration velocity of complete antigens was faster than that of carrier proteins, which may due to the more charge of complete antigens after BPA added. The results demonstrated that artificial complete antigen were successfully prepared and could be used for further study. Consequently, BPA-BSA and BPA-OVA complete antigens were performed as immunogens, while BPA-KLH was used as coating antigen.

Fig. 1.

Fig. 1

Open in a new tab

Characterization of complete antigens and anti-serum. (A) Characterization of complete antigens by agarose gel electrophoresis. Lane 1: BPA-BSA, Lane 2: BSA, Lane 3: BPA-OVA, Lane 4: OVA, Lane 5: KLH, Lane 6: BPA-KLH. (B) Determination of the anti-serum titer of mouse 1. (C) Determination of the serum titer of mouse 2. (D) Evaluation of the specificity of the anti-serum titer of mouse 1. (E) Evaluation of the specificity of the anti-serum titer of mouse 2.

The anti-serum titer of the blood collected from each mouse was tested using i-ELISA after four injections. It was seen that both mouse 1 and mouse 2 immunized with complete antigens had higher titer (about 2.56*103) compared to the control mouse, suggesting that the artificial antigens has ability to stimulate a strong immunoreaction of mouse after multiple injections of BPA-BSA or BPA-OVA artificial antigens (Fig. 1B&C). Furthermore, in order to confirm whether the anti-serum has high reactivity with BPA, the ic-ELISA with a series of dilutions of BPA standard antigen was carried out for detection of the supernatant of blood collected from the immunized mice. As shown in Fig. 1D&E, the titer of antibody was inhibited when BPA added and the titer value was corresponded to the concentration of BPA. The ic-ELISA results clearly demonstrated that the antibody of the immunized mice was high specific for BPA. In addition, as compared to the mouse 1, the mouse 2 showed higher specific interaction of BPA with the ani-serum. Therefore, the immunized mouse 2 was selected for subsequent hybridoma cell production.

3.2. Screening of hybridoma cell and characterization of antibody

The immunized mouse 2 was given the booster immunization by intraperitoneal injection. Then, cell fusion had been adopted by the fusion of the spleen cells isolated from immunized mouse 2 with SP2/0 myeloma cells. In screening hybridoma cells step, competitive ELISA was carried out to screen for positive cell clones that has ability to secret antibody against BPA. After several sub-cloning, two cell lines (2E3 and 7A8) reacted strongly with BPA were successfully obtained in our study. The results in Fig. 2A&B showed that the number of chromosomes of 2E3 was 106, while 7A8 was 108, which was consistent with the sum of the number of chromosomes SP2/0 (66 ± 2) and spleen cell (66 ± 2), indicating that both hybridoma cell lines were successfully prepared by cell fusion technology. SDS-PAGE had been used for evaluation of anti-BPA mAb secreted by 2E3 and 7A8. There are two bands with heavy chain of 55 KD and light chain of 25 KD clearly observed on SDS-PAGE gel (date not show), suggesting that anti-BPA mAb was obtained successfully.

Fig. 2.

Fig. 2

Open in a new tab

Characterization of hybridoma cells and antibody. (A) Chromosome analysis of hybridoma cell 2E3. (B) Chromosome analysis of hybridoma cell 7A8. (C) Determination of the isotype of anti-BPA mAb secreted by 2E3. (D) Determination of the isotype of anti-BPA mAb secreted by 7A8. (E) Affinity of anti-BPA mAb secreted by 2E3. (F) Titer of ascites and purified mAb were measured by ic-ELISA.

According to the sub-type results, both cell clones belongs to IgG1 with light chain of kappa type (Fig. 2C&D). Moreover, the affinity constants of antibody produced by 2E3 and 7A8 were measured by indirect ELISA using series of contents of antibody and BPA-KLH coating antigen. The affinity values of antibodies (2E3 and 7A8) were calculated to be 3.74 × 109 L/mol (Fig. 2E) and 3.88 × 106 L/mol (data not show), respectively. Considering that antibody secreted by 2E3 bound tightly to the immobilized BPA-KLH conjugate, the 2E3 monoclonal cell line was selected and expanded for subsequent studies. Additionally, the titer of purified mAb prepared by chromatography from ascites was measured to be 6.4 × 104 (Fig. 2F). Taken together, due to high titer and affinity of antibody produced by 2E3 cell line, it was further employed to establish immunoassay for detection of BPA.

3.3. Establishment and evaluation of the AuNP-based strip

AuNPs were synthesized according to the previously described method (Liu et al., 2012; Wen-de et al., 2017). To evaluate the size and size distribution of particles, transmission electron microscopy (TEM) was carried out in the study. As shown in Fig. 3A, the red color of the AuNP solution was obtained and the resulting particles showed good size distribution with the mean diameter of 20 nm. Then, the prepared AuNPs were employed to couple with anti-BPA mAbs. The PH and the suitable content of anti-BPA mAb play important role in preparation of colloidal gold-mAb conjugate. The optimal PH value and antibody levels were initially optimized using different volume of K2CO3 and series of concentrations of anti-BPA mAbs. The optimal value results showed that PH 6.8 and 10 μg/mL mAb were taken into consideration in the reaction system (data not show). Therefore, AuNP-anti BPA mAbs were prepared under the optimal conditions. The AuNPs solution and prepared AuNP-anti BPA mAb conjugates were ultraviolet (UV) scanned within a wavelength range of 500–700 nm. In Fig. 3B, the maximum characteristic absorption peak of AuNPs and AuNP-anti BPA mAbs were measured to be 520 nm and 525 nm, respectively. The characteristic absorption peak shifts of AuNP-anti BPA mAb conjugates indicated that the AuNP probe was successfully synthesized.

Fig. 3.

Fig. 3

Open in a new tab

Establishment and evaluation of the AuNP-based strip. (A) The color of colloidal gold solution and TEM image of colloidal gold. (B) UV–vis absorption spectrum of colloidal gold (AuNPs) and probe (AuNPs-anti-BPA mAb) with the range of 500 nm–700 nm. (C) Sensitivity of AuNP-based test strip was determined by testing different concentrations of BPA. (D) The optical density values were recorded by optical reader. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Several commonly factors such as resuspension solution, the concentration of BPA-KLH antigen, goat anti-mouse IgG and colloidal gold-mAb would influence the preparation of the immunochromatographic strip. In the study, PB + 1 % BSA, BPA-KLH antigen level of 0.25 mg/mL, goat anti-mouse IgG concentration of 0.02 mg/mL, and AuNP-anti BPA mAb volume of 3 μL were measured to be the optimal conditions for the best performance of the test strip. Furthermore, the sensitivity of the AuNP-based strip was evaluated by testing different concentrations of BPA solution under the optimal conditions. The results are shown in Fig. 3C, with increasing BPA concentration from 0 μg/mL to 50 μg/mL, the red color intensities gradually decreased. When BPA levels of 25 μg/mL added into the individual test strip, the red color completely disappeared on the T line, suggesting that the visual limit of detection (vLOD) of the established test strip for BPA detection was determined to be 25 μg/mL. In addition, an optical reader was applied to record the optical density values of the T line and C line. As shown in Fig. 3D, the qLOD was determined to be 1.56 μg/mL, at which the competitive inhibition rate (B/B0) was approximately 80 %.

3.4. Characterization of the AuNF probe and optimization of the parameters of AuNF-based strip

To enhance the sensitivity, nanoflower was employed as the colored probe for preparation of test strip. Nanoflower with higher brightness and larger total surface area than AuNPs has attracted increasing attention (Ji et al., 2015). In our study, we also synthesized AuNFs by seed growth method using AuNPs as seeds according to the previously reported method (Ling et al., 2022). As shown in Fig. 4A, AuNFs solution exhibits blue color observed by naked eyes. TEM showed that the prepared AuNFs particles were approximately 50 nm in diameter. Subsequently, the resulting AuNFs particles were used to couple with anti-BPA mAb for preparing of AuNF probe. As the PH value was considered to be an important factor that influences the anti-BPA mAb attach on the surface of AuNFs. Thus, the pH of AuNFs solution was optimized by adjusting the contents of K2CO3 and the optimum PH was measured to be 7.0 (data not show). Similarly, the minimum anti-BPA mAb concentration was also determined to be 10 μg/mL (data not show). Then, AuNF probe were synthesized under the optimized condition (PH 7.0, anti-BPA mAb level of 10 μg/mL). UV scanning within a wavelength range of 500–700 nm was carried out to evaluate the resulting AuNF-anti BPA mAb. The characteristic absorption peak of AuNF-anti BPA mAb was measured at 655 nm, which was significantly different from that of AuNFs solution (640 nm) (Fig. 4B). The above result indicated that with the addition of anti-BPA mAb, the surface of AuNFs particles changed, meaning that the AuNF-anti BPA mAb conjugates were successfully prepared under optimized experimental conditions.

Fig. 4.

Fig. 4

Open in a new tab

Characterization of the AuNF probe and optimization of the experimental parameters. (A) The color of nano-flower (AuNF) solution and TEM image of colloidal gold. (B) UV–vis absorption spectrum of AuNFs and probe (AuNFs-anti-BPA mAb) with the range of 500–700 nm. (C) Optimization of resuspension solution. (D) Optimization of goat anti-mouse IgG concentration. (E) Optimization of BPA-KLH conjugate concentration. (F) Optimization of nanoflower labeled mAb (AuNF probe) concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

For best preparing AuNF-based strip, the most important requirement is to optimize the parameters that affect the accuracy of the test strip. The optimal content of experimental parameters was considered to be the lowest concentration that makes the color appearance clear on the NC membrane. These parameters including resuspension solution of AuNF probe, goat anti-mouse IgG, the concentration of BPA-KLH antigen, and AuNF-mAbs were optimized in our study. The analysis results showed that the optimized conditions as follows: Supernate was selected as resuspension solution of AuNF probe, 0.25 mg/mL of goat anti-mouse IgG coated on the test line of NC membrane, 0.015 mg/mL of BPA-KLH antigen spotted on the C line, and 4 μL of AuNF probe (Fig. 4 C—F).

3.5. Evaluation of the AuNF-based strip

Under the optimized reaction system, we developed the immunochromatographic strip based on the AuNF probe, and visible dark-blue bands on the T line and C line were observed. The specificity of the AuNF-based strip was assessed by testing different BPA analog standards. The results showed that blue color on T line was disappeared when BPA (line1) was added into the individual test strip (Fig. 5A). It can be included that anti-BPA mAb was specific to BPA. Moreover, to evaluate the sensitivity of the AuNF-based strip, different concentrations of BPA from 0 to 50 μg/mL were tested in the study. As shown in Fig. 5B, the visual dark-blue color decreased when BPA concentration increased. With further increased BPA concentration to 3.125 μg/mL, the color on the T line completely disappeared. Thus, the vLOD was considered to be about 3.125 μg/mL, which was approximately 8 times more sensitive than that of AuNP-based strip. Additionally, the color intensity of the T line and C line was scanned by membrane strip reader for quantitative analysis and qLOD was determined to be 0.024 μg/mL (Fig. 5C). These results indicated that 50 nm muti-branched AuNF as a colored probe instead of a conventional 20 nm AuNP could enhance the sensitivity for BPA detection.

Fig. 5.

Fig. 5

Open in a new tab

Evaluation of the AuNF-based based strip. (A) Specificity of AuNF-based test strip was determined by different BPA analog standards. 1: BPA, 2: 4,4’-Thiodbisphenol, 3: 4-Cumylphenolm, 4: 4,4’-Dihy oxybenzophenon, 5: 4,4-Dihydroxydiphenylmethane, 6: Doxycycline hyclate, 7: Minocycline, 8: PBS (control). (B) Sensitivity of AuNF-based test strip determined by testing different concentrations of BPA was observed by naked eyes. (C) The optical density values were recorded by optical reader for evaluation of sensitivity of AuNF-based test strip.

3.6. Sample analysis and method comparison

The proposed immunochromatographic strip assay including AuNP-based test strip and AuNF-based strip were employed to detect the analyzed samples (bottled water, wheat tea, xiancao honey, wangzai milk, milk beer, herbal tea, milk peanut, sprite, coconut water and guava juice). PBS was used as negative control, while spiked samples as positive control. As shown in Fig. 6A&C, two lines on the NC membrane were observed clearly by naked eyes, while T line disappeared when the matrix sample spiked with 25 μg/mL BPA were tested. Then, the color intensity of the T line and C line was scanned by membrane strip reader. The results in Fig. 6B&D showed that T/C value of the proposed methods added with bottled water, wheat tea, xiancao honey, wangzai milk, milk beer, herbal tea, milk peanut, sprite, coconut water and guava juice samples exhibited high value, while samples spiked with 25 μg/mL BPA exhibited obvious difference.

Fig. 6.

Fig. 6

Open in a new tab

Real samples were tested by developed AuNP-and AuNF-based immunochromatographic strip assay. (A) Analyzed samples were determined by AuNP-based test strip and (B) the optical density values were recorded by optical reader. (C) Analyzed samples were determined by AuNF-based test strip and (D) the optical density values were recorded by optical reader.

Furthermore, the reliability of the newly developed methods for bisphenol A detection were compared with LC-MS. The samples were spiked with bisphenol A at concentrations of 0.5, 1 and 2 μg/mL. Then, BPA content were detected by the newly developed methods (AuNP- and AuNF based test strip) and evaluated by LC-MS method simultaneously. As shown in Table 1, the recoveries of newly developed methods for 0.5 μg/mL,1 μg/mL and 2 μg/mL BPA spiked samples were in the range of 84.8 %–117.2 %, while those of the LC-MC ranged from 106.15 % to 115.8 %. These data indicated that the BPA content in the spiked samples measured using the newly developed methods was consistent with that detected by LC-MS method, indicating that the proposed methods have potential to apply to assess the risk of BPA leaching from food container for protection of human beings health.

Table 1.

Detection of BPA in the spiked samples by the developed test strip and LC-MS.

Method Spiked
(μg/mL)		 Found
(μg/mL)		 Recovery (%) CV (%)
AuNP-based strip 0.5 0.586 ± 0.052 117.2 % 8.38 %
1 1.142 ± 0.062 114.2 % 5.24 %
2 1.696 ± 0.012 84.8 % 0.69 %
AuNF-based strip 0.5 0.533 ± 0.013 106.6 % 2.43 %
1 0.875 ± 0.097 87.8 % 11.10 %
2 2.048 ± 0.069 102.4 % 3.37 %
LC-MS 0.5 0.579 ± 0.014 115.8 % 2.39 %
1 1.128 ± 0.003 112.8 % 0.30 %
2 2.123 ± 0.060 106.15 % 2.81 %
Open in a new tab

4. Conclusion

BPA artificial antigens were synthesized for preparation of anti-BPA mAb. Positive cell lines 2E3 and 7A8 secreting antibody against BPA were successfully obtained by immunization, cell fusion and sub-screening. In view of high affinity (3.74 × 109 L/mol) of mAb secreted by 2E3, the immunochromatographic strip assay was developed based on the 2E3 cell line. In this work, AuNPs and AuNFs were used as signal amplification probe for loading anti-BPA mAb on their surface. We have successfully demonstrated AuNP- and AuNF-based strip test for monitoring BPA in real samples. Moreover, the employment of AuNFs with large surface area and complex three-dimensional structure could effectively increase the load of anti-BPA mAb, resulting in a great improvement of sensitivity of proposed test strip. Due to the advantages of simplicity, rapid operation, sensitivity and cost effectiveness of the proposed test strip, it is believed that the developed strip assays provide a great application for on-site detection of BPA.

5. Compliance with ethical standards

The procedures for care and use of animals were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the Ethics Committee of the Fujian Agriculture and Forestry University of China. The license number was PZCASFAEU23047.

CRediT authorship contribution statement

Sumei Ling: Writing – original draft, Methodology, Conceptualization. Aidi Xu: Validation, Methodology, Data curation. Menghan Sun: Validation, Data curation. Xiaoli Li: Visualization, Validation, Data curation. Yongming Huang: Software, Methodology. Yang Xu: Visualization, Validation. Jianli Huang: Validation, Methodology. Tingting Xie: Visualization, Methodology. Shihua Wang: Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors are responsible for the content and writing of this article.

Acknowledgments

This work was supported by National Natural Science Foundation of China (32302224), National Key Research and Development Program of China (2023YFD1600503) and the Promotion Marine and fishery industries projects in Fujian Province (FJHYF-L-2023-21).

Data availability

The data that has been used is confidential.

References

  1. Biedermann S., Tschudin P., Grob K. Transfer of bisphenol A from thermal printer paper to the skin. Analytical and Bioanalytical Chemistry. 2010;398(1):571–576. doi: 10.1007/s00216-010-3936-9. [DOI] [PubMed] [Google Scholar]
  2. Feng Y., Ning B., Su P., Wang H., Wang C., Chen F., Gao Z. An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates. Talanta. 2009;80(2):803–808. doi: 10.1016/j.talanta.2009.07.070. [DOI] [PubMed] [Google Scholar]
  3. Gao P., Wang H., Li P., Gao W., Zhang Y., Chen J., Jia N. In-site synthesis molecular imprinting Nb2O5 -based photoelectrochemical sensor for bisphenol A detection. Biosensors and Bioelectronics. 2018;121:104–110. doi: 10.1016/j.bios.2018.08.070. [DOI] [PubMed] [Google Scholar]
  4. Gatidou G., Thomaidis N.S., Stasinakis A.S., Lekkas T.D. Simultaneous determination of the endocrine disrupting compounds nonylphenol, nonylphenol ethoxylates, triclosan and bisphenol A in wastewater and sewage sludge by gas chromatography-mass spectrometry. Journal of Chromatography A. 2007;1138(1–2):32–41. doi: 10.1016/j.chroma.2006.10.037. [DOI] [PubMed] [Google Scholar]
  5. Huang Y., Xu A., Xu Y., Wu H., Sun M., Madushika L., Wang R., Yuan J., Wang S., Ling S. Sensitive and rapid detection of tetrodotoxin based on gold nanoflower-and latex microsphere-labeled monoclonal antibodies. Frontiers in Bioengineering and Biotechnology. 2023;11 doi: 10.3389/fbioe.2023.1196043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Inoue K., Kato K., Yoshimura Y., Makino T., Nakazawa H. Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection. Journal of Chromatography B: Biomedical Sciences and Applications. 2000;749(1):17–23. doi: 10.1016/s0378-4347(00)00351-0. [DOI] [PubMed] [Google Scholar]
  7. Ji Y., Ren M., Li Y., Huang Z., Shu M., Yang H., Xiong Y., Xu Y. Detection of aflatoxin B1 with immunochromatographic test strips: Enhanced signal sensitivity using gold nanoflowers. Talanta. 2015;142:206–212. doi: 10.1016/j.talanta.2015.04.048. [DOI] [PubMed] [Google Scholar]
  8. Jia K., Lin M., Zhao Q., Dong M., Ling S., Wang S. A sensitive and rapid method of lead detection using nanoparticle technology based on monoclonal antibody. Frontiers in Bioengineering and Biotechnology. 2022;10 doi: 10.3389/fbioe.2022.962230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kim A., Li C.R., Jin C.F., Lee K.W., Lee S.H., Shon K.J.…Park J.S. A sensitive and reliable quantification method for Bisphenol A based on modified competitive ELISA method. Chemosphere. 2007;68(7):1204–1209. doi: 10.1016/j.chemosphere.2007.01.079. [DOI] [PubMed] [Google Scholar]
  10. Li D., Zhou Z., Qing D., He Y., Wu T., Miao M.…Yuan W. Occupational exposure to bisphenol A (BPA) and the risk of self-reported male sexual dysfunction. Human Reproduction. 2010;25(2):519–527. doi: 10.1093/humrep/dep381. [DOI] [PubMed] [Google Scholar]
  11. Li L., Ying Y., Zhang C., Wang W., Li Y., Feng Y.…Wang Y. Bisphenol A exposure and risk of thyroid nodules in Chinese women: A case-control study. Environment International. 2019;126:321–328. doi: 10.1016/j.envint.2019.02.026. [DOI] [PubMed] [Google Scholar]
  12. Ling S., Zhao Q., Iqbal M.N., Dong M., Li X., Lin M.…Wang S. Development of immunoassay methods based on monoclonal antibody and its application in the determination of cadmium ion. Journal of Hazardous Materials. 2021;411 doi: 10.1016/j.jhazmat.2020.124992. [DOI] [PubMed] [Google Scholar]
  13. Liu X., Xiang J.J., Tang Y., Zhang X.L., Fu Q.Q., Zou J.H., Lin Y. Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples. Analytica Chimica Acta. 2012;745:99–105. doi: 10.1016/j.aca.2012.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lorber M., Schecter A., Paepke O., Shropshire W., Christensen K., Birnbaum L. Exposure assessment of adult intake of bisphenol A (BPA) with emphasis on canned food dietary exposures. Environment International. 2015;77:55–62. doi: 10.1016/j.envint.2015.01.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Michałowicz J. Bisphenol A--sources, toxicity and biotransformation. Environmental Toxicology and Pharmacology. 2014;37(2):738–758. doi: 10.1016/j.etap.2014.02.003. [DOI] [PubMed] [Google Scholar]
  16. Moreno M.J., D’Arienzo P., Manclús J.J., Montoya A. Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables. Journal of Environmental Science and Health, Part B. 2011;46(6):509–517. doi: 10.1080/03601234.2011.583871. [DOI] [PubMed] [Google Scholar]
  17. Mustieles V., Ocón-Hernandez O., Mínguez-Alarcón L., Dávila-Arias C., Pérez-Lobato R., Calvente I.…Fernández M.F. Bisphenol A and reproductive hormones and cortisol in peripubertal boys: The INMA-Granada cohort. Science of the Total Environment. 2018;618:1046–1053. doi: 10.1016/j.scitotenv.2017.09.093. [DOI] [PubMed] [Google Scholar]
  18. Ohkuma H., Abe K., Ito M., Kokado A., Kambegawa A., Maeda M. Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum. Analyst. 2002;127(1):93–97. doi: 10.1039/b103515k. [DOI] [PubMed] [Google Scholar]
  19. Pan J., Liu Z., Chen J. An amplifying DNA circuit coupled with Mg2+−dependent DNAzyme for bisphenol A detection in milk samples. Food Chemistry. 2021;346 doi: 10.1016/j.foodchem.2020.128975. [DOI] [PubMed] [Google Scholar]
  20. Qiu L., Liu Q., Zeng X., Liu Q., Hou X., Tian Y., Wu L. Sensitive detection of bisphenol A by coupling solid phase microextraction based on monolayer graphene-coated Ag nanoparticles on Si fibers to surface enhanced Raman spectroscopy. Talanta. 2018;187:13–18. doi: 10.1016/j.talanta.2018.05.001. [DOI] [PubMed] [Google Scholar]
  21. Raysyan A., Zwigart S.D., Eremin S.A., Schneider R.J. BPA endocrine disruptor detection at the cutting edge: FPIA and ELISA immunoassays. Biosensors (Basel) 2023;13(6):664. doi: 10.3390/bios13060664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rochester J.R. Bisphenol A and human health: A review of the literature. Reproductive Toxicology. 2013;42:132–155. doi: 10.1016/j.reprotox.2013.08.008. [DOI] [PubMed] [Google Scholar]
  23. Vandenberg L.N., Hauser R., Marcus M., Olea N., Welshons W.V. Human exposure to bisphenol A (BPA) Reproductive Toxicology. 2007;24(2):139–177. doi: 10.1016/j.reprotox.2007.07.010. [DOI] [PubMed] [Google Scholar]
  24. Wang A., Niu Y., Zhao J., Liu H., Ding P., Chen Y., Zhou J., Zhu X., Zhang Y., Liang C., Zhang G. Rapid detection of varicella-zoster virus based on an immunochromatographic strip. Virology. 2023;586:35–42. doi: 10.1016/j.virol.2023.07.008. [DOI] [PubMed] [Google Scholar]
  25. Wang J., He K., Wu Z., Jin W., Wu W., Guo Y., Zhang W., Di W. Development of a colloidal gold immunochromatographic strip for the rapid detection of antibodies against Fasciola gigantica in buffalo. Frontiers in Veterinary Science. 2022;9 doi: 10.3389/fvets.2022.1004932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wen-de W., Min L., Ming C., Li-Ping L., Rui W., Hai-Lan C., Fu-Yan C., Qiang M., Wan-Wen L., Han-Zhong C. Development of a colloidal gold immunochromatographic strip for rapid detection of Streptococcus agalactiae in tilapia. Biosensors and Bioelectronics. 2017;91:66–69. doi: 10.1016/j.bios.2016.11.038. [DOI] [PubMed] [Google Scholar]
  27. Wu H., Xu X., Liu L., Xu L., Kuang H., Xu C. Gold-based immunochromatographic assay strip for the detection of quinclorac in foods. Analyst. 2021;146(22):6831–6839. doi: 10.1039/d1an01748a. [DOI] [PubMed] [Google Scholar]
  28. Xiao M., Fu Q., Shen H., Chen Y., Xiao W., Yan D., Tang X., Zhong Z., Tang Y. A turn-on competitive immunochromatographic strips integrated with quantum dots and gold nano-stars for cadmium ion detection. Talanta. 2018;178:644–649. doi: 10.1016/j.talanta.2017.10.002. [DOI] [PubMed] [Google Scholar]
  29. Zhang W., Duan H., Chen R., Ma T., Zeng L., Leng Y., Xiong Y. Effect of different-sized gold nanoflowers on the detection performance of immunochromatographic assay for human chorionic gonadotropin detection. Talanta. 2019;194:604–610. doi: 10.1016/j.talanta.2018.10.080. [DOI] [PubMed] [Google Scholar]
  30. Zhou Y., Huang X., Zhang W., Ji Y., Chen R., Xiong Y. Multi-branched gold nanoflower-embedded iron porphyrin for colorimetric immunosensor. Biosensors and Bioelectronics. 2018;102:9–16. doi: 10.1016/j.bios.2017.10.046. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that has been used is confidential.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

RESOURCES