A multiplexed point-of-care assay for C-reactive protein and N-terminal pro-brain natriuretic peptide

Several new plasma protein biomarkers have been associated with increased risk of cardiovascular events. It would be of great value if sets of these markers could be measured in a multiplexed format at point-of-care settings. A major challenge is the extremely wide concentration range in which different plasma biomarkers are present. Two promising biomarkers for cardiac risk prediction are C-reactive protein (CRP) and N-terminal pro-brain natriuretic peptide (NTproBNP). The concentrations of these markers can differ by more than six orders of magnitude. Here we present a chip-based multiplexed assay for CRP and NTproBNP. The high-concentration analyte, CRP, is analyzed in a competitive format, whereas the low-concentration analyte, NTproBNP, is analyzed in a sandwich format. This allows concurrent measurement of the two analytes in a single multiplexed assay. The dynamic ranges for the two assays were optimized to match the relevant serum concentration ranges; thus, no dilutions were needed. Both assays exhibit good precision (5–15% in the clinically relevant concentration ranges), and the limit of detection for the NTproBNP assay was 5 ng/L. Patient plasma samples were used for comparison with clinical methods, resulting in coefficients of determination (R2) of 0.9762 and 0.9606 for NTproBNP and CRP, respectively.