Graphene oxide and gold nanoparticles-based dual amplification method for immunomagnetic beads-derived ELISA of parvalbumin

Food Control 110 (2020) 106989

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Graphene oxide and gold nanoparticles-based dual amplification method for immunomagnetic beads-derived ELISA of parvalbumin

T

Yanbo Wang, Qinqin Qi, Jinru Zhou, Huan Li, Linglin Fu∗ Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: ELISA Parvalbumin Food allergy Signal amplification

Fish has been recognized as one of the “Big-Eight” allergenic food sources with parvalbumin (PV) being one of the major allergens. Here, we report a novel immunomagnetic beads-derived enzyme-linked immunosorbent assay (ELISA) method which employs antibody functionalized graphene oxide (GO) and gold nanoparticles (AuNPs) to amplify the detection signal of PV. The GO and AuNPs are modified with monoclonal antibody (mAb) and HRP-labeled secondary antibody respectively to detect the PV captured by amino-functionalized magnetic beads. With the amplification effect of the antibody modified GO and AuNPs, the proposed ELISA could selectively detect PV with a detection limit as low as 4.29 ng/mL when using pure PV. Moreover, the recovery rates are found to range from 89.82% to 115.37% in the spiked samples. The feasibility in real-world fish sample analysis further shows that the proposed immunomagnetic beads-derived ELISA is a highly promising approach for PV detection and pave a new way for food safety analysis.

1. Introduction Food allergy has risen as a worldwide public health concern along with the greatly improved economy and living standard. The incidence of food allergy has increased dramatically over the past decades with approximately 5%–10% children and 3%–4% adults being affected (Calvani et al., 2012). According to the International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee, 342 kinds of food allergens have been identified for now. Fish, one of the “Big Eight” allergenic food types, plays a vital role in our daily diet because of the highly assimilated proteins and the incontestable nutritional value (Fernandes, Costa, Carrapatoso, Oliveira, & Mafra, 2015). The mass consumption of fish and its derivatives made the related allergy to be a severe issue to be addressed. In fact, fish allergy is estimated to affect 0.2–2.29% of the general population, and the number could even reach up to 8% for fish processing workers (Sharp & Lopata, 2014). Allergy to fish is known to trigger severe immunoglobulin E (IgE)-mediated allergic reactions, such as atopic dermatitis, emesis, diarrhea, urticaria, and angioedema (Jeebhay, Robins, Lehrer, & Lopata, 2001). In the most serious cases, anaphylaxis shocks could even threaten one's life. The major cross-reactive fish allergen has been identified as parvalbumin (PV), an EF-hand small calcium binding protein found in the muscles of vertebrates to which 95% of fish allergy

patients are related (Kumari et al., 2010). PV, 10–13 kDa, has a globular 3D structure and acidic isoelectric points (3.9–5.5). PV exhibits low concentration in dark muscle tissues, while is abundant in white muscle tissues of fish (Saptarshi, Sharp, Kamath, & Lopata, 2014). PV could be found as one or two distinct isoform lineages: α and β. Fish often contains both α and β lineage, and the β lineage with well conserved sequences is considered to be very important for the allergenicity of proteins (Sharp & Lopata, 2014). In general, PV presents a remarkable resistance to high temperatures, denaturing chemicals and proteolytic enzymes (Fernandes, Costa, Oliveira, & Mafra, 2015), and is considered to be well conserved among different fish species (Sharp & Lopata, 2014). In this sense, great efforts have been made to develop analytical methods for detection of PV in fish related foodstuffs. Among them, ELISA is the most widely used method. There are two commercial ELISA kits for fish PV detection, the “Fish Protein ELISA kit” with the detection limit being 1 mg/kg and the “AgraQuant® Fish” giving a limit of quantification (LOQ) of 4–100 mg/kg (Fernandes, Costa, Carrapatoso et al., 2015). However, traditional ELISA assays are likely to present false positive results. Chen et al. (Chen & Hsieh, 2014) and Shibahara’ group (Yusuke, Yoshihiko, Jun, Shoichi, & Kazuo, 2013) developed a sandwich and competitive ELISA with the detection limit of 0.1 ppm and 0.04 mg/kg, respectively. Carrera, Cañas, & Gallardo (2012)

∗ Corresponding author. School of Food Science and Biotechnology, Zhejiang Gongshang University, 18 Xue Zheng Street, Hangzhou, 310018, Zhejiang Province, China. E-mail address: [email protected] (L. Fu).

https://doi.org/10.1016/j.foodcont.2019.106989 Received 19 September 2019; Received in revised form 6 November 2019; Accepted 8 November 2019 Available online 09 November 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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Technology Company (Nanjing, China). Amino magnetic beads with a diameter of 500 nm were bought from BioMag Scientific Inc (Wuxi, China). Mouse anti-frog PV monoclonal antibody (PV-mAb) were purchased from JieShengjiekang Biotechnology Co., Ltd (Wuxi, China). Goat anti-mouse IgG2a heavy chain (HRP) were purchased from Abcam, Chloroauric acid (HAuCl4) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and all other reagents of analytical grade were purchased from Aladdin. All solutions were prepared using ultrapure water (18.2 MΩ) purified by a Milli-Q system (Millipore, Billerica, Massachusetts).

applied liquid chromatography-coupled mass spectrometry (LC-MS) to detect PV. The method took less than 2 h but skilled technicians and sophisticated equipment were required. Polymerase chain reaction (PCR) provided a fast, simple and sensitive way for PV detection. But it mainly focused on specie-specific identification for authentication purposes (Prado, Boix, & von Holst, 2013; Rencova, Kostelnikova, & Tremlova, 2013). Capillary electrophoresis (L. Fu et al., 2018), cellbased fluorescence biosensor (Jiang et al., 2014) and surface plasmon resonance immunosensor (Lu, Ohshima, & Ushio, 2015) were also explored to detect PV. These methods gave a low detection limit but expensive instruments were often needed. Hence, a sensitive yet easy to conduct method is highly required for PV detection, which remains as a big challenge. Here we developed an immunomagnetic beads-derived ELISA of PV, by using antibody functionalized GO and AuNPs as amplification agents. The immunomagnetic beads used here have sensitive magnetic responsiveness, good colloidal stability and abundant binding sites, which could be used to effectively capture and separate PV by an external magnet. Hence the operation time of the assay could be reduced (L. Wang & Gan, 2009), which is considered to be crucial for food safety (Xu et al., 2015). GO is a two-dimensional carbon nanomaterial (Mccoy, Turpin, Teo, & Tabor, 2019) with versatile properties such as excellent thermal, mechanical, and optical properties (Mccoy et al., 2019). GO has shown great potential in combining with biomolecules (Tian et al., 2019). Various organic small molecules, macromolecules, biomacromolecules and functional materials containing active groups could be covalently bonded to GO by amidation or esterification reactions of active carboxyl groups on the surface of GO (X. Wang et al., 2012). Therefore, GO was employed as a nanocarrier for the mAb. AuNPs have attracted great interest due to their unique structural, electronic, magnetic, optical, catalytic (Adriano, Federico, & Arben, 2010; Xu, Gao, Kuang, Liz-Marzan, & Xu, 2018; Xu et al., 2013) and immunological properties (A. Dykman & Khlebtsov, 2016) and feasibility for surface modification with molecular probes. Hence, the secondary antibody labeled with HRP was modified onto AuNPs through hydrophobic, hydrophilic, and electrostatic interactions. The resulted GO-mAb and HRP-Ab-AuNPs were used to enhance the optical signal in the proposed ELISA for PV (Fig. 1). The experimental results demonstrated that this method had a good linear response in the range of 1–50 ng/mL and exhibited low detection limit (4.29 ng/mL). This proposed method is expected to provide new insights to the minute quantity of allergen detection and food safety evaluation.

2.2. Preparation of purified fish PV Extraction of the fish PV was carried out via Guo's (Guo, Kubota, & Shiomi, 2012) method. Alaska pollack used to extract the allergenic protein was purchased as frozen fillet from local supermarket. The white fish muscle samples were stored at −20 °C before used and all procedures were performed at 4 °C. The fish fillet (50 g) was homogenized with five volumes of 50 mM phosphate buffer (pH 7.4), then stirred for 30 min. The mixture was kept at 4 °C overnight and subjected to centrifugation at 8000g for 15 min. The supernatant was heated in a boiling water bath for 30 min, after which was cooled to room temperature. After centrifugation at 4 °C for 15 min at 8000 g, the obtained supernatant was precipitated by 60% saturation ammonium sulfate. Two hours later, the solution was centrifuged at the same condition and the supernatant was precipitated for a second time by 100% saturation ammonium sulfate for 2 h. Then the solution was centrifuged and the resulted precipitate was suspended in 10 mM Tri-HCl (pH 7.5). The crude protein was dialyzed against the same buffer extensively. The dialyzed protein solution was purified by the AKTA protein purification system with DEAE-Sepharose FF ion-exchange column. The purified PV was stored at −20 °C. 2.3. Capture of PV by the amino magnetic beads Capture of PV by the amino magnetic beads (MB) was conducted according to the methods reported by Chen and Hadadi (Chen et al., 2018; Hadadi & Habibi, 2019). Glutaraldehyde was used as coupling agent. Briefly, 0.5 mL (1 mg/mL) MB was added to 0.5 mL glutaraldehyde (25%) solution, and the mixture was placed in a 400 rpm orbital shaker at R.T. for 1 h. The resulted MB was washed by phosphate buffer (0.1 mol/L, pH = 7.4) for three times to remove excess glutaraldehyde. Then PV was added into the above MB solution and the mixture was stirred at 400 rpm for 4 h. Finally, the PV-MB solution was washed by PBS three times. The ability of MB in capturing proteins was studied by analyzing the remained proteins in supernatant by BCA

2. Materials and methods 2.1. Material and reagent Graphene oxide was purchased from Nanjing XF Nano Materials

Fig. 1. Schematic representation of the detection procedure. 2

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Finally, the reaction was stopped by addition of 50 μL 2 M H2SO4, and the absorbance at OD 450 nm was measured.

method. Briefly, 0.25, 0.5, 1 μg proteins were added to 0.05 mg MB respectively, which was activated by glutaraldehyde in advance. The mixture was stirred at 400 rpm for 4 h. Then the BCA protein quantitative method was applied to measure the protein content in the supernatant.

2.7. Fish sample preparation and analysis Four different species of fish bought from local supermarket were subjected to GO and AuNPs dual amplification and immunomagnetic beads-derived ELISA. The total fish protein extraction was performed using a commercial kit and all the procedure was carried out according to the instructions. Firstly, fish muscle was grounded into powder by liquid nitrogen and mixed with the buffer (1 mL of lysis buffer, 1 μL of protease inhibitor, 10 μL of phosphodiesterase, 5 μL of 100 mM PMSF) to a final concentration of 0.1 g/mL. Then the total protein mixture was manually shaken 30–50 times and was centrifuged at 12000 rpm for 5 min. The supernatant was collected for PV content assay. The total protein of different fish was analyzed by SDS-PAGE using AlphaView SA 3.4.0 software.

2.4. Preparation of the monoclonal antibody modified graphene oxide (GOmAb) GO-mAb was synthesized according to the previous work (Dan et al., 2011; Hongjun et al., 2012). Firstly, GO was strongly sonicated and dispersed in deionized water to reach a concentration of 1 mg/mL. To convert ester, hydroxyl, and epoxide groups to carboxylic groups, 1 mg/mL GO solution was added with 50 mg NaOH and 50 mg ClCH2COONa. Then the mixture was sonicated for 2 h and neutralized with dilute HCl. The carboxylic GO was washed three times and dispersed in deionized water. After 48 h dialysis, the final product GOCOOH was suspended in 1 mL solution of pH 6.0 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing 400 mM EDC and 200 mM NHS to activate for 30 min. The mixture was centrifuged at 13000 rpm for 5 min and the supernatant was discarded to remove the excess EDC and NHS. This process was repeated three times. The precipitate was subsequently dispersed in 1 mL PBS buffer (pH 7.4) and sonicated for 5 min. Next, the monoclonal antibody (mAb) was added to the solution with a concentration of 30 μg/mg and the mixture was stirred overnight at 4 °C. The resulted GO-mAb was centrifugated and washed three times and re-dispersed in 1 mL of PBS (PH 7.4) containing 1% BSA. The GOmAb was stored at 4 °C before use.

3. Results and discussion 3.1. Preparation of PV-MB The purified fish PV was successfully prepared for the further experiments, and Fig. S1 shows the SDS-PAGE of purified PV. PV was covalently immobilized onto MB by glutaraldehyde, as the aldehyde groups on the both ends of glutaraldehyde could react with the amino groups of magnetic beads and mAb (Fig. 2A). From the FTIR spectra in Fig. S2, it could be seen that the PV-MB showed a new stretching vibration of amide Ⅱ at 1550 cm−1. This was due to the Schiff base (-CH]N-) formation after the reaction between -CHO and -NH2. The differential FTIR spectra of MB and PV-MB clearly showed the C]N stretching vibration peak at 1635 cm−1. At the same time, the bending vibration peak of -CH2 was strengthened. Compared to the spectrum of PV, the C–N and C]O stretching vibration at 1134 cm−1 and1400 cm−1 could be found in PV-MB. From the above results, it could be found that PV-MB was successfully prepared.

2.5. Preparation of the Goat anti-mouse IgG2a (HRP) modified AuNPs (HRP-Ab-AuNPs) AuNPs were prepared by reduction of HAuCl4 by sodium citrate (Elahi, Kamali, & Baghersad, 2018; Y.; Wang, Rao, Zhou, Zheng, & Fu, 2019). The Goat anti-Mouse IgG2a that labeled with HRP was used as the second antibody and stored at 4 °C before use. The HRP-Ab-AuNPs was synthesized by direct adsorption and direct conjugation (Ciaurriz, Fernández, Tellechea, Moran, & Asensio, 2017; Tripathi & Driskell, 2018). This strategy relies on hydrophobic, hydrophilic, and electrostatic interactions between HRP-Ab and the surface of AuNPs (Meissner, Prause, Bharti, & Findenegg, 2015). Firstly, 3 μL K2CO3 was added to the AuNPs (20 nm, 2 mL) solutions to adjust pH to 8.5. Then 16 μL 0.5 mg/mL HRP-Ab was added and incubated for 1 h at R.T. to allow for direct adsorption. Following the incubation, 5% BSA was added to the solution and shaken for 10 min. Then the sample was centrifuged at 14,000 g for 20 min to remove unbound HRP-Ab. The HRP-Ab-AuNPs in pellet was resuspended in 200 μL of 10 mM borate buffer (containing 5% sucrose, 2% glycerol, 0.5% BSA, and 0.01% Tween, pH 8.0), and stored at 4 °C before further use.

3.2. Preparation of GO-mAb The GO-mAb was prepared in a simple and convenient route as shown in Fig. 2B. The TEM images of the unmodified GO is shown in Fig. 3A. GO exhibited as a single layer state or presented as very thin layers, which showed typical wrinkle paper-like structures. Many dark spots could be observed in Fig. 3B on the GO sheets for GO-mAb. The dark spots were suggested to be the conjugated mAb on GO sheets. The hydrodynamic size of GO was measured to be 274.05 ± 4.31 nm (Fig. 3D). To convert the ester, hydroxyl, and epoxide groups into carboxyl groups, GO was mixed with ClCH2COONa under strong basic conditions. Fig. 3C shows the FTIR spectra of GO, GO-COOH and GOmAb. Compared to GO, it could be seen that the epoxide C–O–C stretching vibrational absorption peaks (1250 and 1050 cm−1) of GOCOOH were obviously weakened. Conjugation of mAb to GO-COOH was achieved through the reaction between the NH2 groups of the mAb and the COOH of GO (Dan et al., 2011). The new strong bands at 1690 and 1550 cm−1 of GO-mAb were related to N–H in-plane stretching of the amide I and II. The GO-mAb was further characterized by UV–vis spectrometry. Fig. 3E showed the characteristic absorption peaks of GO at 230 nm and 300 nm. Then a blue-shift (Fig. 3F) was observed for GO after modification by mAb, indicating successful conjugation of mAb onto GO (Shen et al., 2010).

2.6. Immunoassay procedure for detection of PV The GO-mAb and HRP-Ab-AuNPs were used to amplify the detection signal in the immunomagnetic beads-derived ELISA of PV. Initially, PV solutions with concentration being 1, 5, 10, 20, 30, 40, 50 ng/mL were prepared as working solution and coupled with the activated MB. After stirring for 4 h, 1% BSA was added to block the active sites of the MB. The supernatant was removed by magnet. And then the precipitate was washed three times with phosphate buffer (10 mM, pH = 7.4). Subsequently, 100 μL GO-mAb (3 μg/mL) was reacted with PV-MB with thoroughly stirring at R.T. for 1 h. Following the removal of the excessed GO-mAb by washing three times with buffer, 100 μL of HRP-AbAuNPs (2 μg/mL) was added to the PV-MB and GO-mAb complex and was incubated at R.T. with shaking for 1 h. After the complexes were separated with magnet and washed three times, 100 μL TMB was added and the resulted solution were incubated for 15 min at 37 °C in the dark.

3.3. Preparation of HRP-Ab-AuNPs AuNPs were synthesized according to the method reported by Wang et al. (Y. Wang et al., 2019) (Fig. 2C). TEM images and the DLS results of the unmodified AuNPs are shown in Fig. 4A and Fig. 4B. The AuNPs 3

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Fig. 2. (A) Synthesis of PV-MB. (C) Synthesis of HRP-Ab-AuNPs. (B) Schematic principle of preparation of GO-mAb.

and tended to reach a steady value above the concentration of 30 μg/ mg of mAb, which was probably due to the fact that the surface of GO was close to saturation. Therefore, the optimal feed ratio of mAb and GO was chosen to be 30 μg/mg. Next, the concentration of GO-mAb used in the immunoassay was optimized. 100 μL GO-mAb with the mAb concentration ranged from 0 to 6 μg/mL was added to the same amount of PV-MB. It could be seen that the OD 450 nm changed significantly along with the concentration of the GO-mAb. When the concentration of GO-mAb was above 3 μg/ mL, the OD 450 nm reached a plateau (Fig. 5B). Hence, 3 μg/mL was chosen to be the optimal GO-mAb ratio for further experiments. The optimal concentration of the HRP-Ab-AuNPs was also a critical factor that needed to be studied. 100 μL HRP-Ab-AuNPs with the HRPAb concentration ranged from 0 to 5 μg/mL were added to PV-MB and GO-mAb to conduct the immunoassay. Fig. 5C shows the results of the relationship between the concentration of the HRP-Ab-AuNPs and the OD 450 nm in the range of 0–5 μg/mL. The OD 450 nm increased with elevated concentration of the HRP-Ab-AuNPs and reached a plateau above 2 μg/mL. Thus, 2 μg/mL of HRP-Ab-AuNPs was chosen to be the

particles were homogeneously distributed with a hydrodynamic size of 23.41 ± 0.23 nm. Fig. 4C demonstrates the UV–vis spectra of the AuNPs and HRP-Ab-AuNPs. It could be seen that the absorption peak changed from 516 nm to 525 nm after conjugation of HRP-Ab onto AuNPs. The red-shift in absorption peaks revealed that the HRP-Ab was successfully conjugated onto AuNPs. 3.4. Optimization of detection conditions In order to acquire an optimal analytical performance, different detection conditions were explored. The ratio of the mAb to GO was very crucial because the abundance of primary antibodies on amplification agents could directly affect the sensitivity of the immunoassay (X. Fu, Wang, Liu, Liu, & Chen, 2019). GO-mAbs with different feed ratios of mAb to GO were prepared. Then the above GO-mAbs of different feed ratios were added with the same amount of HRP-Ab and TMB and allowed to react at the same condition. The reaction was stopped by adding H2SO4, after which the absorbance at OD 450 nm was collected. Fig. 5A shows that the OD 450 nm gradually increased

Fig. 3. (A) TEM image of the GO. (B) TEM image of the GO-mAb. (C) FTIR spectra of GO, GO-COOH and GO-mAb. (D) Size distribution of GO. (E) UV–vis spectra of GO. (E) UV–vis spectra of GO-mAb. 4

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Fig. 4. (A) TEM image of AuNPs. (B) Size distribution of AuNPs. (C) UV–vis spectra of a) AuNPs and b) HRP-Ab-AuNPs.

Fig. 5. Optimization of the ELISA detection conditions (A) Ratio of mAb and GO. (B) Concentration of GO-mAb. (C) Concentration of HRP-Ab-AuNPs. Fig. 6. (A) Calibration curve for the dual amplification ELISA detection of different concentration of PV. (B) Calibration curve for the different detections of different concentration of PV a) dual amplification ELISA b) HRP-Ab-AuNPs based ELISA c) GO-mAb based ELISA d) conventional ELISA. (C) Specificity of the ELISA detection of OVA, BSA, TM, AK, COL, and PV (S/N presents the ratio of sample and negative absorbance at OD 450 nm). Statistical significance was determined by t-test. ***p < 0.001.

Table 1 The precision of the spiked PV containing different amounts of PV.

Intra-day

Inter-day

Table 3 Amounts of PV in four fishes detected by dual amplification ELISA.

Spiked concentration (ng/ ml)

n

Measured concentration (ng/ml)

CV (%)

Fish species

n

Measured total protein Conc. (mg/g)

Measured PV Conc. (mg/g) ± SDa

10 20 30 10 20 30

3 3 3 3 3 3

10.21 ± 0.96 22.45 ± 0.67 28.20 ± 2.40 8.68 ± 0.25 22.49 ± 3.74 32.31 ± 6.25

9.61 3.67 5.50 5.83 8.60 7.74

Ctenopharyngodon idellus Aristichthys nobilis Oreochroms mossambcus Gadus macrocephalus

3 3 3 3

24.17 20.48 23.48 27.45

0.88 0.77 0.85 1.21

± ± ± ±

0.10 0.13 0.08 0.24

optimal concentration for the proposed ELISA. Table 2 Recovery rates of spiked PV in three samples containing different amounts of PV. Sample No.

Spiked concentration (ng/ml)

Measured concentration (ng/ml)

Recovery (%)

1

10 20 30 10 20 30 10 20 30

10.61 22.32 29.35 11.47 17.96 34.61 11.37 18.96 33.23

106.10 111.60 97.84 114.67 89.82 115.37 113.73 94.83 110.78

2

3

± ± ± ± ± ± ± ± ±

1.83 0.23 4.96 0.80 0.41 3.37 1.18 5.22 4.04

3.5. Method validation of detection of PV Detection of PV by the GO-mAb and HRP-Ab-AuNPs-based ELISA was conducted under the optimized conditions as discussed above. The corresponding linear range, limit of detection (LOD), limit of quantification (LOQ), cross-reactivity (specificity), precision and accuracy (recovery) were carefully validated according to the method reported before (Zhou, Wang, Qian, Zhang, & Fu, 2019). 3.5.1. Linearity, LOD and LOQ PV standards in nine different concentration (1, 5, 10, 20, 30, 40, 50 ng/mL) were immobilized onto magnetic beads and subjected to the linear range determination. The corresponding calibration plot versus the target PV concentration is displayed in Fig. 6A. There was a linear relationship between OD 450 nm and the concentration of PV in the range of 1–50 ng/mL. The dose-response curve was analyzed to be 5

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immunoassay are ensured by the CV (%) of intra-day and inter-day as 3.67%–9.61% and 5.83%–8.60%. The fish sample detection further confirmed the practical applicability of the method. These results suggested that the proposed ELISA was a reliable and sensitive method for the detection of PV.

y = 0.0631x + 0.4321, where y was the value of OD 450 nm and x was the concentration of PV (ng/mL). The correlation coefficient (R2) was found to be 0.981. The calculated LOD and LOQ were 4.29 ng/mL (S/ N = 3) and 14.15 ng/mL (S/N = 10), respectively. Compared with the detection methods merely using GO-mAb or HRP-Ab-AuNPs as well as the conventional ELISA (Fig. 6B), the proposed ELISA showed an improved signal density and a lower detection limit. That is, the signal of OD 450 nm of GO and AuNPs dual amplificated ELISA was five times higher than that of the conventional ELISA, and about twice times higher than that of the singly amplified ELISA. Therefore, the merits of the dual amplification by GO-mAb and HRP-Ab-AuNPs for ELISA of PV could be clearly seen.

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. Acknowledgments

3.5.2. Cross-reactivity In order to explore the specificity of this method to detect PV in fish, bovine serum albumin (BSA), ovalbumin (OVA), tropomyosin (TM), arginine kinase (AK) and collagen (COL) were used for the further experiments. As shown in Fig. 6C, with the same detection process, the signal observed for these non-specific proteins was significantly low compared to PV. The results demonstrated that this method possessed excellent specificity and reliability toward PV.

This study was financially supported by the Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars of China [grant number LR19C200001], and the National Natural Science Foundation of China [grant number 31871735]. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106989.

3.5.3. Precision and accuracy The precision and accuracy of the detection were also assessed. The intra-day and inter-day for the signal of OD 450 nm by different concentrations (10, 20, 30 ng/mL) of PV were investigated for three times. According to Table 1, the CV% of intra-day was 3.67%–9.61% and inter-day was 5.83%–8.60%, indicating the reliability of this method. The recovery test was performed by blank samples mixed with PV standards at the concentrations of 10, 20, 30 ng/mL. The blank samples referred to total protein (50 ng per sample) extracted from fish samples with PV being removed in advance and the related SDS-PAGE was shown in Fig. S3. As Fig. S4 showed, the MB (0.05 mg) was able to capture almost all proteins in solution when the amount of proteins was below 0.5 μg. Herein, at least for 30 ng/mL spiked samples, the MB (0.0125 mg) could capture all proteins (~0.06 μg) in the samples. In fact, incorporating primary antibody into the MB could predictably improve the sensitivity of the methods. However, considering the extra costs and the already enough protein capture capability, the nonspecific MB used here was suggested to be suitable for subsequent recovery test. The results in Table 2 showed the recovery ranged from 89.82% to 115.37%. Together with the non-spiked sample (0 ng/ml) as a negative control shown in Fig. S5, it's suggested that this method had a good accuracy and specificity.

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3.6. Analysis of fish samples To demonstrate the applicability of the developed GO and AuNPsbased dual amplification ELISA method, four different kinds of fish samples were tested. The SDS-PAGE electropherogram of the total protein extracted from four different fish was shown in Fig. S6. PV was extensively existed in fish, and the concentrations of PV in fish samples were back-calculated from the calibration curves and shown in Table 3. The results provided the information about the PV content in crude fish samples, and demonstrated that this GO and AuNPs-based dual amplification and beads-derived ELISA method have potential application in PV detection. 4. Conclusion In this study, we demonstrated an immunomagnetic beads-derived ELISA of PV with enhanced signal by taking advantage of the GO-mAb and HRP-Ab-AuNPs. The proposed ELISA had a linear detection range for PV over 1–50 ng/mL and the detection limit was as low as 4.29 ng/ mL. The recovery rates of spiked PV ranged from 89.82% to 115.37%. Moreover, the good repeatability and reproducibility of this 6

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