Development of a biomimetic enzyme-linked immunosorbent assay based on a molecularly imprinted polymer for the detection of cortisol in human saliva

Abstract

The development of reliable molecularly imprinted sorbent assays is ensured by in-depth knowledge of the binding between the tracer, conventionally based on a template analogue conjugated to an enzyme, and the imprinted polymer used as a recognition element. To this end, the binding properties of a cortisol-3-(O-carboxymethyl)oxime-horseradish peroxidase conjugate to cortisol-imprinted microparticles previously adsorbed at the bottom of microplates were assessed. The effect of different blocking agents as well as of different percentages of Tween 20 in the working buffer were investigated in order to minimise the non-specific binding of the enzyme tracer to the adsorbed imprinted microparticles. The capability of the enzyme tracer to bind the imprinted solid phase and to compete with free cortisol was assessed by measuring the apparent equilibrium dissociation constant, K_D (39.7 ± 13.5 pmol L⁻¹), and the apparent binding site concentration, B_max (21.7 ± 4.3 nmol L⁻¹), whereas the IC₅₀ value for the cortisol competition curve was found to be 5.32 ± 1.15 ng mL⁻¹. Moreover, binding selectivity measured for several cortisol-related steroids confirmed the experimental results previously published for cortisol-imprinted polymers. A competitive assay for the determination of cortisol in human saliva was developed with a limit of detection of 1.02 ng mL⁻¹, providing quantitative results comparable to those of a commercial ELISA.