An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays

An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA = 0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (Kd = 1.98 μM) and CRP molecules (Kd = 2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (Kd = 8.58 μM). The dynamic detection range for CRP was determined to be 1–8 μM (0.58–4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.

► The aptamer can competitively bind its target protein or a complementary strand (cRNA) in co-mixtures. ► An RNA aptamer wrapped CNT activated capacitor array was used for detecting C-reactive protein (CRP) by label-free method. ► The ratio and concentration of the CRP:cRNA will determine the affinity separation of CNT wrapped aptamer, which enable determining the presence and concentration of target protein.