Enzyme immunosensing for C-reactive protein with scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) has been applied to enzyme immunoassay for the detection of C-reactive protein (CRP). The immunocomplexes of the sandwich type with horseradish peroxidase (HRP) labeling were constructed at the microspots of an anti-CRP IgG antibody fabricated on a hydrophobic glass substrate by capillary microspoting. In the presence of ferrocenemethanol (FcOH) as an electron mediator and hydrogen peroxide as a substrate of HRP, the oxidized form of FcOH (Fc⁺ OH) was generated at localized areas corresponding to the microspot of immunocomplexes by an enzymatic reaction of captured antibodies with HRP label. The reduction current of Fc⁺ OH was detected with a microelectrode at. 0.05 V vs. Ag/AgCl and mapped by scanning the microelectrode to view SECM images of the spots for CRP. An amperometric determination of CRP was also performed using the microelectrode positioned at 10 μm above the microspots. Relationships between the reduction current, in SECM images and the concentration of CRP, were obtained in the range of 0.1 ng/ml to 100 ng/ml. A system for multi-samples measurements has been developed using amperometric determination and antibody array chips.