Abstract

The high prevalence of cardiovascular diseases demands a reliable and sensitive risk assessment technique. In order to develop a fast and label-free immunosensor for C-reactive protein, a risk factor for this condition, anti-CRP antibodies were physically adsorbed to the hydrogen -terminated surface of nanocrystalline diamond. An Enzyme-Linked ImmunoSorbent Assay reference technique showed that this was a suitable substrate for antibody-antigen recognition reactions. Electrochemical Impedance Spectroscopy was used to electronically detect CRP recognition. The specificity of the immunosensor was demonstrated by incubation with CRP and plasminogen as reference molecule. A different impedance behavior was observed in real-time after CRP addition as compared to plasminogen addition: the impedance increased only during CRP incubation. Fitting the data showed that this corresponded with a decrease in capacitance of the molecular layer due to its increased thickness by specific CRP recognition. Sensitivity experiments in real-time showed a clear discrimination between 1 μM, 100 nM, and 10 nM of CRP after 10 min at 100 Hz. Since, 10 nM of CRP was still clearly distinguishable from buffer solution, our CRP-directed immunosensor prototype reaches a sensitivity that is within the physiologically relevant concentration range of this biomarker in healthy controls and CVD patients. Moreover, this prototype displayed real-time discriminating power between spiked and unspiked serum, and thus also shows its applicability in this biological matrix. © 2011 Elsevier B.V. All rights reserved.