Molecularly Imprinted Polymers as an alternative to biological receptors in toxicology

Consumer and environmental safety decisions are based on exposure and hazard

data interpreted using risk assessment approaches. The adverse outcome pathway

(AOP) conceptual framework has been presented as a logical sequence of

events occurring across multiple levels of biological organisation. This aims to increase

the mechanistic understanding of the key events occurring following exposure from a

chemical to develop the scientific rationale underpinning a risk assessment. The AOP

approach brings many challenges including the identification of Molecular Initiating

Events (MIEs), the initial interactions between a molecule and a biomolecule or biosystem

that can be causally linked to an adverse outcome. One of the major types of MIEs

is receptor ligand interaction. Although studying these interactions has been possible

for years, the in vitro assays currently available are expensive and time-consuming,

mainly because they rely on the natural receptor for recognition. Natural receptors are

expensive, molecularly unstable, and difficult to produce and store. Therefore the need

for the development of more suitable binding assays is important.

Among the existing alternatives to using a natural receptor as a recognition material

Molecularly Imprinted Polymers (MIPs) offers promising technology. However, designing

MIPs which closely represent a natural receptor is a significant challenge. Natural

receptors bind to a variety of molecules with differing affinities, leading to a range

of responses which are dependant upon the molecular interactions and 3D properties

involved. Reproducing this level of complexity with single MIPs, which are inherently

highly specific, represents a real challenge.

For this purpose the choice of natural receptor to focus on, template to use for imprinting,

validation dataset of ligand to compare the selectivity of MIPs and natural receptor

and monomer to use in MIPs synthesis was crucial. Therefore, computational tools and

molecular modelling were used initially to make the most sensible choice possible. Several

MIPs were produced using solid-phase synthesis and tested using surface plasmon

resonance and fluorescence polarisation. However, as these techniques did not enable

suitable analyses of the MIPs, a novel assay based on fluorescent MIPs and magnetic

template was designed. This assay demonstrated promising results and the performance

of the fluorescent MIPs were studied and compared to those of the natural receptor. The

MIPs binding pattern proved to be too different from the natural receptor to be used as

an analogue. However, the results obtained and the successful design of this assay paved

the way toward the design of more suitable analogues based on molecular imprinting.