Scanning electrochemical microscopy (SECM) was applied to a dual enzyme immunoassay for the detection of pepsinogen 1 (PG1) and pepsinogen 2 (PG2). Sandwich-type immunocomplexes labeled with horseradish peroxidase (HRP) were constructed on microspots consisting of anti-PG1 IgG antibody and anti-PG2 IgG antibody. These microspots were fabricated on a hydrophobic glass substrate using a capillary microspotting technique. In the presence of H2O2 and ferrocenemethanol (FcOH; used as an electron mediator), the labeled HRP catalyzed the oxidation of FcOH by H2O2 to generate the oxidized form of FcOH (Fc+OH) at localized areas corresponding to microspots containing both immunocomplexes. The enzymatically generated Fc+OH was reduced and detected with a SECM probe (0.05 V versus Ag/AgCl), and the substrate surface was mapped to generate SECM images of the PG1 and PG2 spots. Relationships between the reduction current in the SECM images and the concentrations of PG1 and PG2 were obtained in the range 1.6-60.3 ng/ml protein. Dual imaging of PG1 and PG2 was achieved using microspots containing PG1 and PG2 immunocomplexes separated by a 200 μm physical barrier on the substrate. Pyramidal hole arrays with 100 μm × 100 μm openings on the silicon wafer were utilized to fabricate spots using antibodies on poly(dimethylsiloxane) (PDMS) membranes. Current responses obtained from microspots fabricated with pyramidal holes are significantly sharper compared to the responses obtained from spots fabricated using the capillary method.
- Dual imaging
- Enzyme immunoassay
- Scanning electrochemical microscopy