Magnetic beads-based electrochemical immunosensor for monitoring allergenic food proteins.

Analytical biochemistry (2015-05-13)
Michaela Čadková, Radovan Metelka, Lucie Holubová, Daniel Horák, Veronika Dvořáková, Zuzana Bílková, Lucie Korecká
RESUMEN

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222nM and a detection limit of 5nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.

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Hydrogen peroxide solution, 30 % (w/w) in H2O, contains stabilizer
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N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, commercial grade, powder
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1,2-Dichloroethane, anhydrous, 99.8%
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MES, low moisture content, ≥99% (titration)
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N-Hydroxysulfosuccinimide sodium salt, ≥98% (HPLC)
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N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, crystalline
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N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, BioXtra
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N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, ≥99.0% (AT)
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Hydrogen peroxide solution, 34.5-36.5%
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o-Phenylenediamine, flaked, 99.5%
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o-Phenylenediamine, Peroxidase substrate, ≥98.0%, powder
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MES solution, BioUltra, for molecular biology, 0.5 M in H2O
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Thionin acetate salt, for microscopy (Bact., Bot., Hist.)
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o-Phenylenediamine, tablet, 20 mg substrate per tablet
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Thionin acetate salt, certified by the Biological Stain Commission, Dye content ≥85 %
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o-Phenylenediamine, sublimed, ≥99%
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Hydrogen peroxide solution, contains potassium stannate as inhibitor, 30-32 wt. % in water, semiconductor grade, 99.999% trace metals basis