TEMPO-based immuno-lateral flow quantitative detection of dengue NS1 protein

Abstract

The development of a rapid, affordable, and sensitive diagnostic kit for point-of-care is important in most healthcare settings. In this follow-up paper to our previous work on quantification of dengue NS1 protein via impedimetric measurement, our present technology aims to provide quantification by utilizing proprietary stabilized and improved electroactive immunonanoparticles that bind to the target biomarker and subsequently move along toward the biofunctionalized screen-printed gold electrodes (SPGE) to generate an amperometric signal. The SPGE functions simultaneously as a signal transducer and a solid-state support for a sandwich ELISA-like immunoassay. The successful immunocomplex formation is then recorded electrochemically using a potentiostat, whereby the signal was contributed by the presence of a more hydrophilic redox label than ferrocene, namely radical TEMPO (TEMPO), on the formulated nanoparticles. In this paper, a bifunctional ligand, thiolated polyethylene glycol (PEG-thiol) polymer, was used to stabilize 20 nm gold colloidal nanoparticles (AuNPs) in the formulation. PEG was incorporated to not only prevent the salt-mediated AuNPs aggregations, but also provide an anchor for antibody and redox species conjugation. To-date, we have successfully miniaturized a 3D-printed prototype device able to sensitively detect and quantify dengue NS1 protein with only 0.6 μL human clinical serum samples diluted in a volume ratio of 1:100 (PBS diluent) in less than 30 min with a simple cyclic voltammetry analysis. The positive and negative samples were also tested with ELISA, a gold standard validation method, which means that the development of the prototype is a promising start as a point-of-care diagnostic technology.