This paper proposes a novel perfusion-based micro opto-fluidic system (PMOFS) as a reusable immunosensor for in-situ and continuous protein detection. The PMOFS includes a fiber optic interferometry (FOI) sensor housed in a micro-opto-fluidic chip covered with a microdialysis membrane. It features a surface regeneration mechanism for continuous detection. Gold nanoparticles (GNPs) labeled anti-rabbit IgG were used to enhance the immune conjugation signal by the elongated optical path from GNPs conjugation. Surface regeneration of the sensor was achieved through local pH level manipulation by means of a photoactive molecule, o-Nitrobenzaldehyde (o-NBA), which triggered the elution of immune complexes. Experimental results showed that the pH level of the o-NBA solution can be reduced from 7 to 3.5 within 20 seconds under UV irradiation, sufficient for an effective elution process. The o-NBA molecules, contained within poly(ethylene glycol) diacrylate (PEG) complexes, were trapped within the sensing compartment by the microdialysis membrane and would not leak into the outside environment. The pH variation was also limited in the neighborhood of the sensor surface, resulting in a self-contained sensing system. In-situ immune detection and surface regeneration of the sensing probe has been successfully carried out for two identical cycles by the same sensing probe, and the cycle time can be less than 8 minutes, which is so far the fastest method for continuous monitoring on protein/peptide molecules. In addition, the interference fringe shift of the sensor is linearly related to the concentration of anti-cytochrome C antibody solution and the detection limit approaches 10 ng/ml.
