Development of a peptide-based lateral flow assay for the detection of the cancer biomarker Mdm2

Abstract

This study introduces peptide aptamers as a promising alternative to conventional antibodies for use as recognition units in lateral flow assays (LFAs). Two distinct strategies for immobilizing peptides onto nitrocellulose (NC) membranes were investigated: the first involved covalent coupling to bovine serum albumin (BSA) using EDC/sulfo-NHS chemistry, while the second utilized the well-known biotin/streptavidin complexation method. The former strategy only requires the addition of a lysine in the peptide sequence, while the latter exploits a commercially widely available chemical modification of these biomolecules. Both methods proved successful in immobilizing the capture probes onto the surface. Furthermore, we employed silver nanoplates functionalized with calixarenes as colorimetric reporters, that we have shown recently to exhibit excellent characteristics for LFAs, including high absorption coefficient, excellent stability, and strong contrast with the NC membrane. The peptide-based assay detected Mdm2, a well-established cancer biomarker, in low nanomolar range even in complex matrixes such as cell lysates. Notably, the utilization of peptide aptamers demonstrated superior performance and extended shelf life compared to polyclonal antibodies, underlining their potential as recognition units in LFAs. In addition to their ease of handling, peptide aptamer utilization also offers the prospect of substantial cost reductions compared to conventional antibody-based LFAs. This comprehensive approach enhances the utility of LFAs for the sensitive and cost-effective detection of target proteins using peptide aptamers as recognition units both on the colorimetric reporter and the membrane, opening doors to broader applications in biomarker analysis and diagnostic assays.