Electrochemical Magnetic Immunoassay for the Determination of the Fish Allergen β-Parvalbumin

Abstract

β-parvalbumin (β-PV) is the primary fish allergen responsible for most allergic reactions in individuals sensitive to fish. To ensure food safety, a sandwich-based magnetic immunoassay was developed using maleimide-functionalized magnetic beads (NH-MBs). Specific anti-β-PV antibodies were immobilized on these MBs, and a screen-printed carbon electrode was employed as the electrochemical transducer. A linear concentration range from 10 to 1000 ng/mL, a limit of detection of 1.8 ng/mL, and a limit of quantification of 7.1 ng/mL were achieved. Nineteen commercial food samples were analyzed to assess the potential of the sensor for routine use in food quality control. Important factors such as protein source and food processing (e.g., boiling, grilling, and frying) and preservation (e.g., in oil, and vacuum) were evaluated. The validated results confer the usefulness of the assay for food quality control.

Keywords: chronoamperometry; electrochemical biosensor; fish allergen; food safety; immunomagnetic assay; maleimide-modified magnetic-beads; seafood; β-parvalbumin.

Figure 1

Schematic representation of the magnetic sandwich immunoassay: (i) NH-MB biomodification with CAb, and addition of (ii) β-PV, (iii) DAb-HRP, and (iv) TMB-H₂O₂, with exemplification of the analytical signal recorded by chronoamperometry.

Figure 2

Immunosensor optimization in absence (B, 0 ng/mL) and presence of β-PV (S, 2000 ng/mL), and the corresponding S/B ratio: (A) effect of the amount of NH-MB suspension; (B) blocking with or without BSA before β-PV and DAb-HRP incubation; (C) concentration of CAb (µg/mL) and assay format (A, simultaneous incubation of β-PV and DAb-HRP; and B, step-by-step assay); and (D) amount of BSA added to the DAb-HRP solution.

Figure 3

Electrode surface characterization: (A,B) scanning electron microscopy micrographs (scale bar: 10 and 2 µm, respectively; magnification: 10,000 and 50,000×, respectively) and (C) energy-dispersive X-ray spectroscopy spectra of the transducer surface containing the NH-MBs.

Figure 4

Analytical characterization of the immunoassay: (A) chronoamperograms of increasing concentrations of β-PV (10, 25, 100, 250, and 1000 ng/mL); (B) calibration curve obtained for the immunoassay; (C) interference study in the presence of different possible interferents (Ara h 1, Api g 1, and TTX) at concentrations of 1000 ng/mL; (D) comparison of curves of β-PV in buffer and a food extract; and (E) stability study over 43 days (dashed line, average of the measurements; solid lines, average ± 2 s (s = standard deviation).

Figure 5

(A) Effect of β-PV extraction buffer on the signal using a fresh salmon sample: B-1—10 mM PBS with 5 mM EDTA, pH 7.4; B-2—10 mM PBS with 137 mM NaCl, pH 7.4; B-3—100 mM Tris buffer, pH 7.4, and B-4—16 mM Na₂CO₃ with 128 mM NaCl, pH 9.6. (B) Comparison of the results obtained by the immunoassay and the conventional ELISA for the following foods: i—egg, ground celery, peanut butter, rice, soy lecithin, liquid protein yoghurt, broccoli and cauliflower soup, roast chicken, sardine pâté, canned fish surimi, squid, canned tuna, and tuna burger; ii—fish surimi; iii—fresh salmon; and iv—tuna pâté.