A paper-based fluorescence resonance energy transfer (FRET) immunodevice for cotinine detection in human saliva

Abstract

We report a paper-based fluorescence resonance energy transfer (FRET) immunodevice for cotinine detection in human saliva. This system is composed of Cy5-labeled silica nanoparticles (Cy5-SiNPs) as the FRET donor, and gold nanorods (GNRs) as the acceptor. The GNRs used as an acceptor showed maximum absorption at 673 nm, which corresponded with the emission of the Cy5-SiNPs. In the absence of cotinine, FRET occurred between the donor and acceptor, followed by the quenching of fluorescence of Cy5-SiNPs because of the specific recognition between the anti-cotinine antibody conjugated-Cy5-labeled SiNPs (donor conjugates) and GNR-cotinine-HRP (acceptor conjugates). When cotinine in human saliva was added, the competitive reaction between cotinine in the sample and the GNR-cotinine-HRP conjugates decreased FRET between donor and acceptor conjugates, consequently facilitating a stronger fluorescence of the anti-cotinine antibody conjugated-Cy5-labeled SiNPs than that observed in absence of cotinine. This FRET principle was integrated with the advantages of low-cost and easy fabrication of a paper-based device, which consisted of two pieces of wax-printed papers and a transfer pad. The donor and acceptor conjugates were immobilized and dried on the first and the second paper layers, respectively. The components of the developed immunodevice were stacked in order of donor conjugates-immobilized layer, acceptor conjugates-immobilized layer, and a transfer pad. In this fabricated immunodevice, upon injection of the human saliva samples containing cotinine, the fluorescence signal increased with increasing concentration of cotinine within the range of 1–1000 ng mL-1 within 30 min without the need for multiple steps. Thus, we believe that the proposed paper-based immunodevice has a significant potential for clinical monitoring and for on-site diagnostics of other trace analytes in a homogeneous manner.