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.

A wide range of medical conditions can be diagnosed through sequence-specific analysis of nucleic acids. However, a major challenge remains in detecting a specific target in samples containing a high concentration of mismatching sequences. A single-step kinetic homogenous (free solution) assay is presented in which free sequence-specific probes are continuously separated from probe-target hybrids during electrophoretic sample focusing, allowing monitoring of dissociation kinetics. Under these conditions, the different kinetics of targets versus mismatches result in distinct patterns of the signal (for example, linear increase for target versus exponential decay for mismatch), allowing the detection of desired sequences even in the presence of high background nucleic acid content. Additionally, an analytical model provides insight into the underlying dynamics, and allows design of assays based on this mechanism.

A wide range of medical conditions can be diagnosed through sequence‐specific analysis of nucleic acids. However, a major challenge remains in detecting a specific target in samples containing a high concentration of mismatching sequences. A single‐step kinetic homogenous (free solution) assay is presented in which free sequence‐specific probes are continuously separated from probe–target hybrids during electrophoretic sample focusing, allowing monitoring of dissociation kinetics. Under these conditions, the different kinetics of targets versus mismatches result in distinct patterns of the signal (for example, linear increase for target versus exponential decay for mismatch), allowing the detection of desired sequences even in the presence of high background nucleic acid content. Additionally, an analytical model provides insight into the underlying dynamics, and allows design of assays based on this mechanism.