Immunogold–silver staining-on-a-chip biosensor based on cross-flow chromatography

Immunogold–silver staining (IGSS) was adopted in cross-flow chromatographic analysis in which immunological reactions and silver intensification were sequentially conducted in the vertical and horizontal directions, respectively. Factors controlling the performance, except the silver substrate solution, were optimized to increase the signal-to-background ratio in measurements of cardiac troponin I as a model analyte. In generating the signal, the size of colloidal gold catalyst was critical; the smallest size (5-nm diameter) in the selected range yielded the highest colorimetric signal. To maintain the low background, two processes, blocking the remaining surfaces of membrane after antibody immobilization and washing the residual tracer after immunological reaction, were necessary. Self-nucleation of silver ions also caused a background signal and was controlled to some degree by decreasing the hydrodynamic force that arose when the substrate solution was supplied in the horizontal direction. Finally, a new chip (IGSS-on-a-chip; IOC) that allowed for convenient, efficient IGSS was produced by injection molding of plastic. This method enhanced the detection capability by 51-fold compared to the conventional rapid test kit using 30 nm-sized colloidal gold as the tracer. The IOC biosensor results also showed that silver intensification yield via cross flow after immunological reaction was 19% higher than that by traditional incubation.