Study on molecularly imprinted nanoparticle modified microplates for pseudo-ELISA assays.

Nanosized Molecularly Imprinted Polymers (nanoMIPs) are designed artificial nanoreceptors with a predetermined selectivity and specificity for a given analyte, lately proposed as a replacement to antibodies in immunoassays. The nanoMIP-plate preparation based on nanoparticle adsorption was studied with the aim to rationally identify and discuss the critical points in the nanoMIP-assay development, in an example based on the iron homeostasis biomarker hepcidin and hepcidin-specific nanoMIPs (K = 9nM). Plates were prepared by deposition and drying of nanoMIP (0.5-4µg/well), or by nanoMIPs co-depositions (proteins, PVA). Rehydration (> 1h) of dry nanoMIP-plates showed the reconstitution of the imprinted binding sites. NanoMIP-plate mechanical stresses (several washings; pipetting) caused nanoMIP desorption (~90%). After 10 washes the quantity of nanoMIP was 0.2µg/well, the imprinted binding sites were ~270 fmol/well, their accessibility the 92%. Co-depositions resulted in higher amount of adsorbed nanomaterial (1.2µg/well), but low accessibility of the imprinted binding sites (2-47%). Tested in a competitive sequential assay, using as competitor horseradish peroxidase conjugate to hepcidin, the nanoMIP-plate permitted to determine hepcidin in serum samples, yet with a narrow dynamic range of response (0.9-10nM). Critical points in the assay were: the instability of the nanoMIP adsorption, which lead to the progressive loss of binding sites/well, and the affinity of the nanoMIP for the analyte (K = 9nM), which corresponds to kinetics dissociation constants on the time-scale of the washing lengths (minutes), thus compatible with the release of the bound hepcidin during the washings. The found limits set the conditions to develop a successful nanoMIP-assay: (i) stable microplate derivatization; (ii) maximized number of imprinted binding sites/well; (iii) nanoMIP/analyte equilibrium not perturbed on the time scale of the minutes (i.e. K ~ pM).