Isotachophoresis-Based Surface Immunoassay

• Published in: Analytical chemistry (Washington) Vol. 89; no. 14; pp. 7373 - 7381
• Main Authors: Paratore, Federico, Zeidman Kalman, Tal, Rosenfeld, Tally, Kaigala, Govind V, Bercovici, Moran
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.07.2017
• Subjects: Abundance; Analytical chemistry; Antibodies; Antibodies - immunology; Chemistry; Counterflow; Design standards; Fluorescence; Green fluorescent protein; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - immunology; Immunoassay; Immunoassays; Isotachophoresis; Kinetics; Min protein; Optimization; Proteins; Surface Properties
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.7b00725

In the absence of amplification methods for proteins, the immune-detection of low-abundance proteins using antibodies is fundamentally limited by binding kinetic rates. Here, we present a new class of surface-based immunoassays in which protein–antibody reaction is accelerated by isotachophoresis (ITP). We demonstrate the use of ITP to preconcentrate and deliver target proteins to a surface decorated with specific antibodies, where effective utilization of the focused sample is achieved by modulating the driving electric field (stop-and-diffuse ITP mode) or applying a counter flow that opposes the ITP motion (counterflow ITP mode). Using enhanced green fluorescent protein (EGFP) as a model protein, we carry out an experimental optimization of the ITP-based immunoassay and demonstrate a 1300-fold improvement in limit of detection compared to a standard immunoassay, in a 6 min protein–antibody reaction. We discuss the design of buffer chemistries for other protein systems and, in concert with experiments, provide full analytical solutions for the two operation modes, elucidating the interplay between reaction, diffusion, and accumulation time scales and enabling the prediction and design of future immunoassays.