An integrated microfluidic platform for sensitive and rapid detection of biological toxins

TLDR

The authors aim to create a portable, rapid (<20 min) microfluidic immunoassay device that detects biotoxins in bodily fluids with minimal sample volume (<10 µL) for point‑of‑care use. The device integrates a microfluidic chip with miniaturized electronics, optics, fluid‑handling components, and software, performing electrophoretic immunoassays that preconcentrate analytes via a photopolymerized nanoporous membrane, separate complexes in polymeric gels, and quantify toxins by laser‑induced fluorescence of bound/unbound antibody ratios. The platform achieved limits of detection of 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin without preconcentration, and reduced SEB LOD to <10 pM with a 10‑min on‑chip preconcentration, demonstrating its suitability for rapid, quantitative detection of multiple protein biomarkers.

Abstract

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 µL) and have appreciable sensitivity and dynamic range (µM–pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of ∼10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody–analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is <10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.