Label-free detection of C-reactive protein using an electrochemical DNA immunoassay

Abstract

A label-free electrochemical immunoassay that combines DNA-directed immobilization (DDI) with electrochemical impedance spectroscopy (EIS) on microwire sensors is reported for the detection of C-reactive protein (CRP). CRP is an acute-phase protein that is strongly correlated with systemic inflammation. Since inflammation plays a role in pathogenesis of cardiovascular diseases, CRP can be used to predict the likelihood of coronary events. To demonstrate the new chemistry, 25-μm Au electrodes were modified with single strand DNA (ssDNA) and exposed to a solution containing complementary ssDNA conjugated to monoclonal anti-CRP. The charge-transfer resistance of the [Fe(CN)6]3−/4− redox couple was used to determine the CRP concentration after binding. A stepwise increase in the charge transfer resistance was observed using EIS for each modification step, ssDNA, ssDNA-anti-CRP hybridization and the final CRP capture. Cyclic voltammetry (CV) was used to verify the EIS results, and showed an increase in peak potential splitting in a similar stepwise manner for each modification step. Finally, fluorescence microscopy was used to confirm the DNA hybridization and CRP binding. Standard addition of CRP revealed that EIS could be used to detect CRP at clinically relevant levels in serum samples. This new form of electrochemical DNA immunoassay (eDI) has significant potential as a simple, label-free sensor for proteins in microfluidic devices. Keywords: Electrochemical impedance spectroscopy (EIS), C-reactive protein, Gold microwire electrode, DNA-directed immobilization, Label-free detection