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

• Published in: Analytical chemistry (Washington) 2015-07, Vol.87 (13), p.6736-6743
• Main Authors: Eid, Charbel, Palko, James W, Katilius, Evaldas, Santiago, Juan G
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Aptamers, Nucleotide - chemistry; Assaying; C-Reactive Protein - analysis; Dilution; Immunoassay; Ions; Isotachophoresis; Mathematical analysis; Mathematical models; Proteins; Reaction kinetics; Serums; Spacers
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.5b00886

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.