Research Of The Detection For Food Allergen Protein Based On Mast Cell Sensors

Food allergy has become a potential hazard to food safety in recent yearswith its high frequency and severe clinical outcomes, and it has become an hot research area in the field of public health and food safety. However, there are no effective methods for allergy detection since the the traditional methods are time-consuming and not suitable for rapid identification. Therefore, there is an imperative need to establish a rapid, sensitive ands afe method for food allergen detection. The cell-based sensing technology using living cells from immune system as sensing component to build sensors has been widely used in environmental monitoring, drug screening, safety evaluation, and medical diagnosis. In this study, RBL-2H3 mast cell, as a recognition component, was used to detect food allergens by combining with electrochemical sensing technology, fluorescent probe, magnetic nanoparticles and microfluidic chip technology.Firstly, a novel allergic ICR mice model for milk and shrimp allergen evaluation and identification was developed based on cell histaminerelease in vitro. The ICR mice were immunized with the purified shrimp and milk protein once a week. The spefic Ig E of food allergens was measured by indirect ELISA at day 7, 14 and 21.And the peritoneal mast cells were collected at day 14, and stimulated by allergens.The release rate of tryptase was measured by ultraviolet spectrophotometry assay.The levels of Ig E in allergic serum reached the peak at14 th day after challengingwith a value of 1/25600 in shrimp group and 1/12800 in milk group.Tthe tryptase release rate was 38.5%±2.62%in milk group and 47.1±2.2% in shrimp group compared with a control of 5.7±3.2%. The successful ICR mice model set the foundation for development of cell-based sensing technology.Then Cell-based electrochemical biosensor was developed first to quantify major shrimp allergen tropomyosin Pen a 1. RBL-2H3 mast cells, encapsulated in type I collagen, were immobilized on a gold electrode that is modified by self-assembled L-cysteine/gold nanoparticlesto monitor Ig E-mediated mast cell sensitization and activation. The correlation between the concentration of DNP-BSA and impedance was found to be linear within the range of 1×10-3 ng/m L to 10 ng/m L(y=1.33x+7.851, R2=0.998).To demonstrate the utility of this mast cell-based biosensor for quantification of shrimp allergen. RBL cells were activated by the shrimp allergen Pen a 1and was quantitatively recorded by EIS measurements. The Ret value of impedance increased along the concentration of tropomyosinin in a range of 0.5 μg/m L to 2.5 μg/m L with a detection limit of 0.15 μg/m L. The electrochemical result was confirmed by β-hexosaminidase assayand SEM analysis.After that, we developed a fluorescence mast cell based sensor to detect and identify the major fish allergen paralbumin. A CD63-EGFP plasmid containing the gene of fluorescent protein was first constructed, and then RBL cells were transfected through a highly efficient, lipid-mediated, DNA-transfection procedure. Stable fluorescent transfectant mast cells were obtained through G418 screening and confocal laser scanning microscopy assay. Both the allergenic DNP-BSA and fish paralbumin could stimulate the expression of fluorescent protein in the mast cellsindicating that the fluorescent mast cells can be used as a biosensor to to detect allergens through a fluorescent assay Experimental results on fish PV allergen showed that fluorescence intensity increased along PV concentrations from 1 ng/m L to 100 ng/m L, and the detection limit is 0.35 ng/m L(RSD 4.5%). This highly sensitive biosensor has been evaluated by three different kind fish samples.Next a RBL-2H3 cell sensor was constructed by combining with fluorescent magnetic beads. Fluorescein isothiocyanate(FITC) was fused inside the Si O2 layer of Si O2 shell-coated Fe3O4 nanoparticles. The synthesized fluorescent magnetic beads were then transfected into mast cells through a highly efficient, lipid-mediated magnetofection procedure. Magnetic glassy carbon electrode(MGCE), which possesses excellent reproducibility and regeneration qualities, was then employed to adsorb the CMFNP-transfected RBL-2H3 cells activated by an allergen for electrochemical assay. The correlation between the concentration of DNP-BSA and impedance was found to be linear within the range of 1×10-3 ng/m L to 1×10-1 ng/m L(y= -1.22x+1.028, R2=0.996) with a detection limit of 3.3×10-4 ng/m L. Anti-Pen a1 Ig E and AntiPV Ig E-activated cells were employed to quantify both shrimp allergen tropomyosin(Pen a 1) and fish allergen parvalbumin(PV), and the result shows that a limit of 0.03 μg/m L(shrimp Pen a 1) and 0.16 ng/m L(fish PV), respectively, could be successfully detected.Finally, an ANA-1 macrophages and RBL-2H3 mast cells co-culture system on integrated microfluidic chip was constructed, and signaling molecule and metabolites detected by ELISA and electrochemical impedance was used as the base to quantify food allergens. Results show that high levels of DNP-BSA caused the production of TNF-α、IL-6、INF-γ in co-culture system in which 10-1 ng/m L of DNP-BSA created the most efficient effect within 12 h. This system can be used to detect the physiological activity of cells in real time. Based on the electrochemical parameter of impedance, the cells grew well individually, but in the coculture system, and macrophages can promote the proliferation of the mast cells. When the cells in co-culture system were exposed to DNP-BSA with different concentrations, the impedance value of the mast cells decreased significantly. The result was comparable to the ELSIA assay, which confirmed that the cell co-culture system could be used for the detection of food allergens.In conclusion, RBL-2H3 mast cells were selected as a novel recognition element of cellbased sensor for food allergen detection and evaluation. RBL-2H3 mast cells-based biosensor combining with electrochemistry, fluorescence transfection, magnetic beads and microfluidic chip could be effectively used to detect and evaluate food allergens. This offer a new technical support for the promotion and application in food safty detection and prediction.