Rapid Slow Off-Rate Modified Aptamer (SOMAmer)-Based Detection of C-Reactive Protein Using Isotachophoresis and an Ionic Spacer.

We present an on-chip electrophoretic assay for rapid protein detection with a SOMAmer (Slow Off-Rate Modified Aptamer) reagent. We used isotachophoresis (ITP) coupled with an ionic spacer to both react and separate SOMAmer-protein complex from free SOMAmer reagent. ITP accelerates the reaction kinetics as the ionic spacer concurrently separates the reaction products. We developed a numerical and analytical model to describe ITP spacer assays, which involve low-mobility, nonfocusing targets that are recruited into the ITP zone by higher-mobility, ITP-focused probes. We demonstrated a proof-of-concept of this assay using C-reactive protein (CRP) in buffer, and achieved a 2 nM limit of detection (LOD) with a combined 20 min assay time (10 min off-chip preparation of reagents and 10 min on-chip run). Our findings suggest that this approach has potential as a simple and rapid alternative to other homogeneous immunoassays. We also explore the extension of this assay to a diluted serum sample spiked with CRP, where we observe decreased sensitivity (an LOD of 25 nM in 20-fold diluted serum). We describe the challenges in extending this assay to complex samples and achieving higher sensitivity and specificity for clinical applications.