Development of an amperometric immunosensor based on flow injection analysis for the detection of red blood cells

An amperometric immunosensor, based on a non-competitive sandwich assay and flow injection analysis (FIA), was developed for the detection of human red blood cells (RBCs). A dual working electrode, on which specific IgM and nonspecific IgM were chemically immobilised to form sensing and blank electrodes, respectively, was employed to determine the binding of specific blood cells and non-specific adsorption in one determination. Horseradish peroxidase (HRP)-labelled antiblood group A IgM was used in the assay. Sensor preparation involved chemical immobilisation of the IgMs on glassy carbon electrodes using l-ethyl-3(3-dimethyl aminopropyl)carbodiimide (EDC) as a coupling reagent in the presence of N-hydroxysuccinimide (NHS). The interference contributions, such as the non-specific adsorption of the enzyme conjugate and the blood cells, were determined and removed. A quantitative relationship between the cell binding response and its concentration was obtained in the region 1 − 30 × 10⁸ cells ml⁻¹.