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Development of an optical immunoassay based on peroxidase-mimicking Prussian blue nanoparticles and a label-free electrochemical immunosensor for accurate and sensitive quantification of milk species adulteration

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Abstract

In contrast to reported enzyme-based immunoassays, an enzyme-free immunoassay (optical and electrochemical) is presented here for the first time that can be used as point-of-need detection bioplatforms of bovine IgG as goat milk adulterant. In the first format, Prussian blue nanoparticles (PBNPs) were used as antibody catalytic labels in a competitive colorimetric microplate immunoassay. Absorbance measurement was performed photometrically at 450 nm. After in-depth optimization, excellent sensitivity was achieved (0.01% cow/goat volume ratio), which is 100 times lower than the limit allowed by the European legislation (EL) (1% v/v), thanks to the high catalytic activity of PBNPs compared with natural peroxidase. Moreover, the antibody-PBNPs bioconjugates showed excellent stability over 4 weeks (> 94% of the initial response) confirming the successful anchoring of the antibodies to the surface of the PBNPs. On the other hand, a label-free voltammetric immunoassay for the detection of bovine IgG was developed. The sensing principle was based on the hindrance of charge transfer between ferri-ferrocyanide redox couple and the screen-printed gold electrodes modified with bovine IgG antibody. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step modification of the electrode surface. Under optimal conditions, this single-step electrochemical analysis achieved a high sensitivity of 0.1% (cow/goat) when monitoring the ferrocyanide oxidation at + 0.092 V (vs. Ag/AgCl) using differential pulse voltammetry (DPV). The selectivity of the developed immunoassays was evaluated for different species of milk of similar composition, and both immunoassays exhibited a selective response only to bovine IgG. Unlike conventional immunoassays, the developed enzyme-free immunoassays have many attractive features for the detection of milk adulteration, whether they are used in quality control laboratories for routine milk analysis (optical immunoassay) or at on-site checkpoints (electrochemical immunoassay) using wireless electrochemical detectors. The sensors provide high sensitivity (≤ 0.1%), excellent precision (RSD < 6%), low cost (no enzyme is required) and ease of operation, including handling of milk samples.

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  1. Laboratory of Process Engineering and Environment, Faculty of Sciences and Techniques, Hassan II University of Casablanca, P.A. 146, Mohammedia, Morocco

    Narjiss Seddaoui, Raouia Attaallah & Aziz Amine

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  1. Narjiss Seddaoui
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Narjiss Seddaoui contributed to conceptualization, methodology, validation, investigation, writing—original draft preparation, visualization and writing—review and editing. Raouia Attaallah contributed to conceptualization, investigation and writing—review and editing. Aziz Amine contributed to conceptualization, methodology, validation, writing—review and editing, and supervision.

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Correspondence to Aziz Amine.

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Seddaoui, N., Attaallah, R. & Amine, A. Development of an optical immunoassay based on peroxidase-mimicking Prussian blue nanoparticles and a label-free electrochemical immunosensor for accurate and sensitive quantification of milk species adulteration. Microchim Acta 189, 209 (2022). https://doi.org/10.1007/s00604-022-05302-9

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