Development of ic-ELISA and lateral-flow immunochromatographic strip for detection of vitamin B₂ in an energy drink and vitamin tablets

ABSTRACT

Vitamin B₂ (riboflavin) is a water-soluble vitamin that has important roles in human health. In this study, we developed a sensitive and specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip for the rapid detection of vitamin B₂. Following routine fusion and selection, the optimum monoclonal antibody against vitamin B₂ was obtained. The 50% inhibitory concentration and limit of detection of ic-ELISA were 8.18 and 1.80 ng/mL, respectively. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. The results revealed that our developed methods are suitable for the on-site detection and mass screening of vitamin B₂ in food and pharmaceutical products.

KEYWORDS:

• Vitamin B₂
• monoclonal antibody
• ic-ELISA
• lateral-flow immunochromatographic assay strip

Introduction

Vitamin B₂, or riboflavin, is one of the most widely distributed B vitamins, which participates in several cellular processes. Vitamin B₂ plays vital roles in biological systems such as cellular respiration and metabolism of fats, carbohydrates, and proteins (Basaranoglu et al., 2017; Henriques, Olsen, Bross, & Gomes, 2010; Lewicka et al., 2017; Powers, 2003). Vitamin B₂ is present in a wide variety of animal and plant foods, such as liver, dairy products, and leafy vegetables. Low intake of dairy products, excessive alcohol consumption, and certain diseases may contribute to vitamin B₂ deficiency, characterized by anemia, angular cheilitis, cataracts, seborrheic dermatitis, and growth retardation (Kennedy, 2016; MacMillan et al., 2017; Mazur-Bialy & Pocheć, 2017; Mazur-Bialy, Pochec, & Plytycz, 2015; Powers et al., 2011; Qi, Kniazeva, & Han, 2017; Thomas & Mirowski, 2010). Therefore, vitamin B₂ supplementation is particularly important for individuals with vitamin B₂ deficiency. In the US, the recommended daily allowance for vitamin B₂ is 1.7 mg/day for pregnant women (Fankhanel & Gassmann, 1998), 0.3–0.4 mg/day for infants, and 0.6–1.8 mg/day for adults. To reduce the risk of vitamin B₂ deficiency, vitamin B₂ is commonly added to several foods. Therefore, a highly sensitive and specific vitamin B₂ analytical method is critical for assessing the nutritional quality and safety of foods.

Currently, the available methods to determine vitamin B₂ include microbiological assays (Barton-Wright & Booth, 1943; Hyma, 1945), high-performance liquid chromatography (HPLC); (Hampel, York, & Allen, 2012; Marti-Andres, Escuder-Gilabert, Martin-Biosca, Sagrado, & Medina-Hernandez, 2015; Schmidt, Schreiner, & Mayer, 2017), electrochemical methods (Kowalczyk, Sadowska, Krasnodebska-Ostrega, & Nowicka, 2017), and fluorescence spectroscopy (Chen, Li, Cui, Yu, & Zhai, 2015; Gliszczyńska-Świglo & Rybicka, 2015; Privitera & Lozano, 2017). Microbiological assays, which are based on Lactobacillus rhamnosus, are highly sensitive. A commercial kit (VitaFast® Vitamin B₂, R-Biopharm Co., Ltd., Darmstadt, Germany) has recently been developed for vitamin B₂ determination with a limit of detection (LOD) of 1.8 µg vitamin B₂/100 g (mL). However, the incubation time is lengthy (44–48 h), and the operation process is time-consuming. HPLC allows the quantification of vitamin B₂ in foods and biological samples. However, due to the complexity of food matrices, HPLC requires tedious sample pre-treatment steps, sophisticated equipment, and trained personnel (Geng et al., 2017; Nurit, Lyan, Piquet, Branlard, & Pujos-Guillot, 2015). These determination methods cannot be adapted for high-throughput screening or on-site detection. Electrochemical methods and fluorescence spectroscopy have been used to determine vitamin B₂; however, they are non-reproducible, have poor sensitivity for complex food matrices, and are not suitable for on-site testing. Biosensors based on electrochemistry methods and fluorescence spectroscopy have been developed to improve detection sensitivity (Farzin & Shamsipur, 2017; Puangjan, Chaiyasith, Taweeporngitgul, & Keawtep, 2017; Sá, da Silva, Jost, & Spinelli, 2015); however, biosensors require complex sample preparation steps and expensive instruments.

The enzyme-linked immunosorbent assay (ELISA) based on antibody–antigen interactions has received considerable attention due to its high sensitivity, specificity, and throughput (Berlina, Zherdev, Xu, Eremin, & Dzantiev, 2017; Guo et al., 2015; Kuang, Liu, et al., 2013). An indirect competitive ELISA (ic-ELISA) based on polyclonal antibodies against vitamin B₂ has been used for the determination of vitamin B₂ in foods and pharmaceuticals (Wang et al., 2013). Even though this method is sensitive and specific, polyclonal antibodies have great limitations in commercial applications because they cannot be reused and have poor reproducibility. Therefore, a highly sensitive and specific monoclonal (mAb)-based ic-ELISA for vitamin B₂ determination is required. The lateral-flow immunochromatographic assay (ICA) strip, which is based on antigen–antibody interactions, have been widely used in the toxins, heavy metals, and chemicals analysis (Kong, Xie, Liu, Song, Kuang, Cui, et al., 2017; Kuang, Xing, et al., 2013; Peng et al., 2017). The lateral-flow ICA strip is simple, rapid (results can be obtained with the naked eye within 10 min), effective, and suitable for semi-quantitative detection, qualitative detection, and mass sample screenings.

In this study, we produced a mAb against vitamin B₂. Highly sensitive and specific mAb-based ic-ELISA and lateral-flow ICA strip were developed for the determination of vitamin B₂ in food and pharmaceutical products.

Materials and methods

Reagents and materials

Vitamin B₂, N,N′-carbonyldiimidazole (CDI), bovine serum albumin (BSA), ovalbumin (OVA), Freund’s complete adjuvant, Freund’s incomplete adjuvant, and enzyme immunoassay-grade horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin were purchased from Sigma-Aldrich (Shanghai, China). Gelatin and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Other reagents, which were of analytical grade, were acquired from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Energy drink and compound vitamin B tablet were purchased from a local supermarket. Nitrocellulose (NC) high-flow-plus membranes (Pura-bind RP) were supplied by Whatman-Xinhua Filter Paper Co. (Hangzhou, China), and polynylchloride (PVC) backing materials, absorbance pad, and sample pad were obtained from Goldbio Tech Co. (Shanghai, China).

Solutions

The buffers used in this study included (1) PBS buffer (0.01 M phosphate-buffered saline, pH 7.4), (2) coating buffer (0.01 M sodium carbonate buffer, pH 9.6), (3) blocking buffer (0.2% w/v gelatin in coating buffer), (4) washing buffer (PBS buffer containing 0.05% v/v Tween-20), (5) antibody dilution buffer (PBS buffer containing 0.1% w/v gelatin and 0.05% v/v Tween-20), (6) stop buffer (2 M sulfuric acid), and (7) substrate solution (2 mL of 0.06% w/v TMB in glycol mixed with 10 mL of 0.1 M citrate phosphate buffer, pH 5.0 containing 1.8 µL of 30% hydrogen peroxide).

Antigen preparation

Vitamin B₂ antigens were synthesized by conjugating vitamin B₂ with carrier proteins (BSA or OVA). We prepared vitamin B₂-BSA-1, vitamin B₂-BSA-2, and vitamin B₂-BSA-3 at reaction ratios 50:1, 100:1, and 150:1, respectively. Similarly, we prepared vitamin B₂-OVA-1, vitamin B₂-OVA-2, and vitamin B₂-OVA-3 at reaction ratios 50:1, 100:1, and 150:1, respectively. Briefly, BSA/OVA were dissolved in 0.1 M sodium carbonate–bicarbonate buffer (CB, pH 9.6) at 5 mg/mL. Vitamin B₂ (8.42 mg) and CDI (18.15 mg) were dissolved in 1 mL of DMF, heated to 40°C, and stirred in the dark for 1 h. Subsequently, the reaction mixture was slowly added to 2 mL of carrier protein solution and stirred overnight at room temperature. The conjugate was dialyzed against 0.01 M PBS for 3 days. Antigens were characterized by UV-Vis spectroscopy.

MAb preparation

Vitamin B₂-BSA-1, vitamin B₂-BSA-2, and vitamin B₂-BSA-3 were used as immunogens. Female BALB/c mice (8–10 weeks of age) were immunized by continuous multipoint subcutaneous injections with the immunogens to generate polyclonal antibodies against vitamin B₂. The mouse with the highest serum antibody titer and lowest 50% inhibitory concentration (IC₅₀) was sacrificed, and its spleen was fused with Sp2/0 murine myeloma cells (Kong, Liu, Song, Kuang, & Xu, 2017). The target cells were selected by ic-ELISA and obtained by the limiting dilution method. MAbs were purified by the caprylic acid-ammonium sulfate precipitation method.

Development of ic-ELISA

In this experiment, 96-well microplates were coated with coating antigen diluted in coating buffer (100 µL/well) and incubated at 37°C for 2 h. Subsequently, the wells were washed three times with washing buffer. After washing the plates, 200 µL blocking buffer was added to each well and incubated at 37°C for 2 h. After washing, 50 µL anti-vitamin B₂ mAb and 50 µL vitamin B₂ standard were added to each well and incubated for 0.5 h at 37°C. After washing, 100 µL HRP-labeled goat anti-mouse IgG was added to each well, and the plates were incubated for 0.5 h at 37°C. After washing the plates three times, 100 µL substrate solution was added to each well and incubated for 15 min at 37°C in the dark. The reaction was stopped with the addition of 2 M sulfuric acid (50 µL/well). Absorbance was measured at 450 nm (Ding, Liu, Song, Kuang, & Xu, 2017). IC₅₀ and LOD were calculated by a standard curve generated by plotting optical density at 450 nm (OD₄₅₀) on the y-axis against vitamin B₂ concentration on the x-axis.

Cross-reactivity

MAb specificity was evaluated by measuring cross-reactivity (CR). Vitamins B₁, B₃, B₅, B₆, and B₁₂, biotin, and folic acid were analyzed by ic-ELISA. CR was calculated using the following equation: $\text{CR}\left(\text{\%}\right)=\left(\frac{\text{I}{\text{C}}_{50}\text{of}\text{vitamin}{\text{B}}_2}{\text{I}{\text{C}}_{50}\text{of}\text{cross - reacting}\text{compound}}\right)\times \text{100}\text{\%}.$

Preparation of the gold nanoparticle (GNP)-labeled mAb

GNPs and GNP-labeled mAb were synthesized in our laboratory. For the synthesis of GNP-labeled mAb, the pH value of a GNP solution was adjusted to 8.0 with 0.1 M K₂CO₃. Subsequently, 0.2 mg anti-vitamin B₂ mAb was slowly added to 10 mL GNP solution and maintained at room temperature for 1 h. BSA (0.5% w/v, 1 mL) was added dropwise. Following a 2-h incubation, the solution was centrifuged (7000g for 30 min at 4°C). Unconjugated gold nanoparticle and anti-vitamin B₂ mAb in the supernatant were discarded. The resulting precipitate was washed three times with 0.02 M PBS (containing 5% sucrose, 1% BSA, and 0.5% polyethylene glycol 6000, pH 7.4), dissolved in 5 mL of 0.02 M PBS (containing 0.02% NaN3), and stored at 4°C (Kong, Xie, Liu, Song, Kuang, & Xu, 2017).

Preparation of the lateral-flow ICA strip

The lateral-flow ICA strip was developed in our laboratory as previously reported (Xing et al., 2015). PVC backing card, NC membrane, sample pad, and absorption pad were assembled in layers. The NC membrane was pasted onto the center of the PVC backing card. The sample and absorption pads were glued to the bottom and upper section of the PVC backing card, respectively, with a 2-mm overlap of the NC membrane. Using a membrane dispenser, goat anti-mouse IgG and coating antigen were sprayed onto the NC membrane at 1 µL/cm to form the control line and test line (C line and T line, respectively). After drying at 37°C, the card was cut into 3-mm wide individual test strips and stored in a desiccator for further experiments.

Principle of the lateral-flow ICA strip

The detection principle of the lateral-flow ICA strip is based on a competitive interaction between vitamin B₂ present in the sample and the coating antigen sprayed on the T line for GNP-labeled mAb. Sample solution (100 µL) was mixed with 50 µL of GNP-labeled mAb in the wells of a microtiter plate and allowed to react at room temperature for 5 min. The ends of the strips with sample pad were inserted into the mixture. By capillary action, the mixture migrates from the sample pad to the NC membrane and reacts with the coating antigen on the T line and the goat anti-mouse IgG on the C line, finally reaching the absorption pad. The results are visualized by the naked eye within 5 min. In vitamin B₂-negative samples, red T and C lines are obtained because GNP-labeled mAbs are captured by the coating antigen. In vitamin B₂-positive samples, only a red C line is obtained. Vitamin B₂ present in the sample combines with GNP-labeled mAbs; therefore, less GNP-labeled mAbs are available to combine with the coating antigen on the T line, resulting in a weak red color on the T line. With increasing vitamin B₂ concentrations, the T line color intensity decreases. When the concentration of vitamin B₂ in the sample exceeds a certain value, no color appears on the T line. This critical concentration is defined as the cut-off value of the strip (Isanga et al., 2017). GNP-labeled mAbs or vitamin B₂-GNP-labeled mAbs continue to migrate and react with the goat-anti-mouse IgG antibody, resulting in a deep red color on the C line. The sensitivity of the lateral-flow ICA strip is determined by the detection of a series of vitamin B₂ standards. In this study, each test was repeated seven times.

Sample analysis

An energy drink and compound vitamin B tablet were analyzed. Following pH adjustment to 7–7.5, the energy drink (vitamin B₂ concentration: 4 mg/100 mL) was passed through a 0.2-µm filter membrane. The filtrate was diluted to a suitable concentration for analysis. Two pieces of compound vitamin B tablets (vitamin B₂ concentration: 1.25 mg/piece) were dissolved in pure water to generate a stock solution. The solution was centrifuged at 6500g for 25 min at 4°C. The lipid layer was removed, and the supernatant was collected. The solution was diluted to a suitable concentration for analysis.

Results and discussion

UV spectroscopic characterization

Vitamin B₂-BSA and vitamin B₂-OVA conjugates were used as immunogen and coating antigen, respectively. Vitamin B₂-BSA and vitamin B₂-OVA conjugates were characterized by UV absorption spectroscopy (Figure 1). The carrier proteins (BSA and OVA) had a characteristic absorption peak at 278 nm. Vitamin B₂-BSA and vitamin B₂-OVA conjugates had absorption peaks at 364 and 445 nm, and vitamin B₂ had absorption peaks at 263, 364, and 445 nm. A significant blue shift was observed between the antigens and carrier proteins, probably due to vitamin B₂ coupling at 263 nm. These results confirmed that the antigens were successfully conjugated to the carrier proteins.

Figure 1. The UV-Vis spectra of different antigens. (A) Antigens with BSA as carrier protein and (B) antigens with OVA as carrier protein.

Development and characterization of ic-ELISA

Vitamin B₂-BSA was used as immunogen in mice immunization, and vitamin B₂-OVA was used as coating antigen in ic-ELISA. The mouse with the highest serum antibody titer and lowest IC₅₀ was selected for cell fusion. Following cell infusion, the hybridoma cell line 3H8 was obtained, and vitamin B₂ antibody was purified from ascites by the caprylic acid-ammonium sulfate precipitation method.

The standard curve (Figure 2) of mAb 3H8 against vitamin B₂ concentration (y = 0.195 + 1.347/[1 + (x/8.180) × 1.691]) had a linear regression correlation coefficient (R²) of 0.991. The IC₅₀ value was 8.18 ng/mL, and the LOD defined as IC₁₀ value, was 1.80 ng/mL with a linear range of 3.60–18.56 ng/mL. The results revealed that our developed method was sensitive for the detection of vitamin B₂. MAb specificity was evaluated by ic-ELISA using other B vitamins (Table 1). The results showed that vitamin B₂ mAb did not significantly react with other B vitamins (CR values <0.1%). Therefore, the developed ic-ELISA was specific to vitamin B₂.

Figure 2. The standard curve of developed Ic-ELISA method.

Table 1. The CR value of related analytes by the Ic-ELISA method.

Analytes	IC50 (ng/mL)	Cross-reactivity (%)
Vitamin B2	8.18	100
Vitamin B1 (Thiamine)	>10,000	<0.1
Vitamin B3 (Nicotinic acid)	>10,000	<0.1
Vitamin B5 (Pantothenic acid)	>10,000	<0.1
Vitamin B6 (Pyridoxine)	>10,000	<0.1
Vitamin B6 (Pyridoxamine)	>10,000	<0.1
Vitamin B6 (Pyridoxal)	>10,000	<0.1
Vitamin B7 (Biotin)	>10,000	<0.1
Vitamin B9 (Folic acid)	>10,000	<0.1
Vitamin B12	>10,000	<0.1

Optimization and characterization of the lateral-flow ICA strip

The analytical performance and sensitivity of the lateral-flow ICA strip may be affected by a series of parameters, including coating antigen and suspension buffer. To determine the appropriate reaction ratio of vitamin B₂-OVA, three kinds of coating antigens (reaction ratios 50:1, 100:1, and 150:1) were sprayed onto the NC membrane. The lateral-flow ICA strip was used to analyze a vitamin B₂-negative sample (0 ng/mL) and a vitamin B₂-positive sample (50 ng/mL). The results are presented in Figure 3(A). No color on T line was obtained with the vitamin B₂-positive sample at any reaction ratio. A reaction ratio of 50:1 contributed to the deepest color on the T line with the vitamin B₂-negative sample. Therefore, the coating antigen with reaction ratio 50:1 was used for subsequent experiments.

Figure 3. The optimization of the lateral-flow ICA strip. (A) Strip with three kinds of coating antigens at 0.5 mg/mL: (1) coating antigen with reaction ratio 50:1; (2) coating antigen with reaction ratio 100:1; (3) coating antigen with reaction ratio 150:1. (B) Optimization of suspension solution for sample pad: (1) basic suspension buffer; (2) 1% PVP; (3) 1% PEG; (4) 1% PVA; (5) 1% BSA; (6) 1% Casein; (7) 1% sucrose; (8) 1% trehalose; (9) 1% sorbitol; (10) 1% mannitol; (11) 1% brij 35; (12) 1% triton X-100; N, vitamin B₂-negative sample (0 ng/mL); P, vitamin B₂-positive sample (50 ng/mL). (C) Optimization of two different surfactants in basic suspension buffer: (1) 1% brij 35; (2) 1% triton X-100; N, vitamin B₂-negative sample (0 ng/mL); P, vitamin B₂-positive sample (25 ng/mL).

The composition of the suspension buffer affects the flow rate, background color, sharpness, and intensity of the T line. The basic suspension buffer contained 20 mM Tris (pH 8.2), 0.1% PEG, 0.1% Tween, 5% sucrose, 5% trehalose, and 0.2% BSA. Different surfactants were subsequently added. The vitamin B₂-negative sample and the vitamin B₂-positive sample were analyzed (Figure 3(B)). When the basic suspension buffer containing 1% PEG, 1% PVA, or 1% casein were used, we obtained no color on the T line with the vitamin B₂-negative sample. The basic suspension buffer and suspension buffer containing 1% PVP, 1% BSA, 1% sucrose, 1% trehalose, 1% sorbitol, or 1% mannitol contributed to a weak T color with the vitamin B₂-positive sample. The basic suspension buffer containing brij 35 or triton X-100 contributed to a deep red T line color with the vitamin B2-negative sample. Therefore, subsequent experiments were performed using suspension buffer that contained brij 35 and triton X-100.

Figure 3(C) shows that when the vitamin B₂ concentration was decreased from 50 to 25 ng/mL, no red line on T line could be observed for both two kinds the suspension buffer. However, for vitamin B₂-negative sample, the suspension buffer containing brij 35 contributed to a weak red T line color, while the basic suspension buffer containing triton X-100 contributed to a distinct red T line color. Therefore, the optimum conditions of the developed lateral-flow ICA strip consisted of a reaction ratio of 50:1 and a suspension buffer containing 1% triton X-100.

PBS samples spiked with different concentrations of vitamin B₂ (0, 2.5, 5, 10, 25, and 50 ng/mL) were analyzed by the lateral-flow ICA strip (Figure 4). The cut-off value for vitamin B₂ was 50 ng/mL.

Figure 4. Vitamin B₂ detection by lateral-flow ICA strip. The PBS samples: (1) 0 ng/mL; (2) 2.5 ng/mL; (3) 5 ng/mL; (4) 10 ng/mL; (5) 25 ng/mL; and (6) 50 ng/mL.

Sample analysis

An energy drink and compound vitamin B₂ tablet were analyzed. The energy drink and compound vitamin B₂ tablet samples were analyzed at 6400-, 3200-, 1600-, 800-, 400-, 200-, and 100-fold dilution (Figure 5). Ultrapure water was used as the negative control. For the energy drink sample, a lighter color was observed on the T line with the 1600-fold diluted sample, and the T line disappeared with the 800-fold diluted sample (50 ng/mL). For the compound vitamin B tablet, a lighter color was observed on the T line with the 3200-fold diluted sample, and the T line disappeared with the 1600-fold diluted sample (78.13 ng/mL). The results obtained from the energy drink sample were more sensitive compared with the results obtained from the compound vitamin B tablet sample, which is probably due to substrate conditions and matrix interference effects. More sensitive results could be obtained by repeated measurements of different dilution ratios. The results revealed that our developed lateral-flow ICA strip assay was sensitive, accurate, and suitable for the detection and screening of vitamin B₂ in real samples.

Figure 5. The sample analysis with lateral-flow ICA strip. (A) Energy drink sample: (1) ultrapure water; (2) 6400 times dilution; (3) 3200 times dilution; (4) 1600 times dilution; (5) 800 times dilution; (6) 400 times dilution; (7) 200 times dilution; (8) 100 times dilution. (B) Compound vitamin B tablet sample: (1) ultrapure water; (2) 6400 times dilution; (3) 3200 times dilution; (4) 1600 times dilution; (5) 800 times dilution; (6) 400 times dilution; (7) 200 times dilution; (8) 100 times dilution.

Conclusions

In this study, we developed a highly sensitive and specific mAb-based ic-ELISA and lateral-flow ICA strip for the detection and screening of vitamin B₂ in food and pharmaceutical products. The IC₅₀ and LOD of ic-ELISA was 8.18 and 1.80 ng/mL, respectively, with a linear dynamic range of 3.60–18.56 ng/mL. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. These methods were effective for the analysis of both simple and complex matrices. Therefore, the developed mAb-based ic-ELISA and lateral-flow ICA strip are effective for on-site detection and mass sample screening of food and pharmaceutical products.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is financially supported National Key R & D Program (2016YFF0202300) and grants from Natural Science Foundation of Jiangsu Province and MOF (BE2016307, BK20150145, BX20151038, BK20140003, BE2014672, CMB21S1614, 201513006), and Taishan Industry Leading Talent Special Funds.

Notes on contributors

Lu Zeng

Lu Zeng got her bachelor degree from Zhejiang Chinese Medical University, Hangzhou, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interests are immunoassay applications in food.

Wei Jiang

Wei Jiang got her bachelor degree from Yangzhou University, Yangzhou, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interest includes immunoassay development for food safety.

Liqiang Liu

Liqiang Liu got his Ph. D in Food science in 2014 from Jiangnan University, Wuxi, China and then became a faculty in the college of Food science and technology of Jiangnan University. His research interests are immunochromatographic strip design and application.

Shanshan Song

Shanshan Song got her Master degree in Food science in 2012 from Jiangnan University, Wuxi, China and then became a research assistant in the college of Food science and technology of Jiangnan University. Her research interests are monoclonal antibody development.

Hua Kuang

Hua Kuang got her Ph. D from China Agricultural University in 2009 and then began to work as a faculty in the college of Food science and technology of Jiangnan University. She is currently a full professor in food safety. Her research interest is biosensor development.