Simultaneous detection method for two cardiac disease protein biomarkers on a single chip modified with mixed aptamers using surface plasmon resonance

A simultaneous detection method for two cardiac disease protein biomarkers present in serum samples on a single planar gold chip using surface plasmon resonance (SPR) is described. The detection of N-terminal pro-brain natriuretic peptide (NT-proBNP) and tumor necrosis factor α (TNF-α), which are known as acute myocardial infarction (AMI) biomarkers, with predetermined clinically relevant concentrations was performed using mixed aptamers specific to each protein tethered on a single gold surface. After the binding of NT-proBNP and/or TNF-α to the mixed aptamers, an antibody specific to each target protein was injected to form a surface sandwich complex to improve selectivity. In order to adjust the dynamic ranges in the known clinically relevant concentration significantly different for NT-proBNP (0.13–0.24 nM) and TNF-α (0.5–3 pM), the surface density ratios of the corresponding pair of aptamer and antibody were first systematically determined, which were the 1:1 mixed aptamer chip with 40 nM anti-NT-proBNP and 100 nM anti-TNF-α. This allowed to establish the distinct dynamic ranges of 0.05–0.5 nM for NT-proBNP and 0.1–5 pM for TNF-α in a buffer, along with detection and quantification limits of 0.03 and 0.19 nM for NT-proBNP and 0.06 and 0.21 pM for TNF-α, respectively. The changes in refractive unit (RU) values observed when exposing both proteins at different concentrations alongside the corresponding fixed concentration of antibodies onto the 1:1 mixed aptamer chip were then correlated to the sum of RU values measured when using the injection of individual protein for evaluating each protein concentration. With a complete characterization of the simultaneous quantification of two protein concentrations in the buffer, the mixed aptamer chip was finally employed for direct measurements of NT-proBNP and TNF-α concentrations in undiluted serum samples from healthy controls and AMI patients. The results of simultaneous SPR measurements for the two proteins in the serum samples were further compared to the individual protein concentration results using an enzyme-linked immunosorbent assay.