Resonant Energy Transfer Detection for Low Volume Immunoassay in Environmental Applications

Abstract

Environmental monitoring activities aim at the assessment of the environmental situation and its change due to anthropogenic influence or natural factors. Ideally the environmental picture derived from monitoring activities should be tight in space and time. On the other hand economic factors limit the number of samples which can be processed and analysed. Rapid and cost effective pre-screening techniques are required to reduce the number of samples, which are subject to costly instrumental analysis. Immunoassays are already recognised as a mass screening technique. Immunoassays with reduced sample volume and high sample density may become an answer to the demand for cost effective high throughput screening requirements. Approaches to low volume assays may be adapted from fields like molecular biology, where limited sample amounts or high reagent costs drive similar developments.

This project targets the development of a nanotitreplate immunoassay for environmental analytes. In this paper we focus on the general concept and on results from the immunoanalytical system. The assay volumes foreseen are 100 nl to 500 nl. Microstructured assay compartments (nanotitre plates) are readily available by a variety of techniques (e.g. isotropic or anisotropic etching of silicon, injection moulding, laser manufacturing). Sample and reagent handling will be done by piezomicrodrop techniques, allowing the accurate dispensing of volumes below 1 nl. The small assay volumes foreseen preclude typical sequential ELISA protocols, where washing steps are mandatory. Therefore a wash free proximity type assay is under development. The detection is based on fluorescence Resonant Energy Transfer (RET). A donor-acceptor pair with two “red” (long wavelength excitable) fluorescent dyes is used. This reduces fluorescence background and allows the use of semiconductor laser light sources.

A model assay for atrazine was set up by using an antibody labelled with donor molecules and a conjugate of bovine serum albumin with hapten and acceptor. Initial calibrations gave a test midpoint of 6 ppb.