Development of a Label-Free Immunosensor for Quantitative, Electrical Detection of Target Antigens

Abstract

Point-of-care immunoassays require rapid, sensitive, and user-friendly operation. The most common example is an at-home pregnancy test, which falls under a class of diagnostics called a lateral flow assay (LFA). However, LFAs present many disadvantages such as poor sensitivity and a difficulty for users to interpret test results accurately. The gold standard immunoassay, ELISA, exhibits excellent limits of detection (LOD) and limits of quantitation (LOQ). However, barriers to the implementation of ELISA must be considered, such as the consumption of large amounts of expensive reagents, the need for trained personnel to perform the assay, and the possibility of false positives or negatives due to improper washing or procedure. A clear demand exists for a label-free immunological sensor with high sensitivity and specificity, requiring only a small amount of sample and with fully automated operation. Previous work in our group has demonstrated the successful label-free detection of nucleic acids in a microfluidic device with an embedded 3D porous electrode by their hybridization to a bed of bioconjugated microbeads. An increase in conductivity, observed by a current-voltage curve, was attributed to the negative charge added to the beads upon hybridization of the target nucleic acids. In this presentation, we demonstrate label-free sensing of antibody targets by an analogous method despite their low net charge under the conditions employed. In this system, biotinylated goat anti-rabbit IgG immobilized on streptavidin-modified beads serves as the capture antibody. The analyte, Rabbit IgG, is introduced into the channel and incubated for 30 min. A current-voltage curve (CVC) is obtained before and after incubation to detect and quantify binding of Rabbit IgG to the bead surface. We evaluated the sensitivity of this method and found that the LOD is approximately 5 nM. Following validation of this method, we examined binding kinetics by obtaining CVCs at several time points during incubation. Next, to improve the LOD in this device, faradaic ion concentration polarization (fICP) was employed to electrokinetically enrich the negatively charged IgG over the bead bed test line. After an enrichment period of 30 min, the antigen was preconcentrated over 100-fold, which resulted in greater sensitivity. Finally, we characterize the impact of ionic strength on the sensor and evaluated its performance in a simulated biofluid, saliva, under several conditions: neat, spiked with Tris, and diluted. Ongoing research in our group will translate this method to biomarkers relevant to viral diagnostics. The authors gratefully acknowledge the Roy J. Carver Charitable Trust for the funding of this project.