Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay.

Abstract

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary 'antisense' strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a 'fit-for-purpose' model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.