Abstract
This study reports 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 immune sensing and surface regeneration mechanism for continuous detection. A method using gold-nanoparticles (GNPs) to enhance immune-sensing signal is proposed. It is suggested that an enlarged index mismatch and an elongated optical path by GNPs conjugated on recognition proteins will contribute most to signal enhancement in the interference fringe shift. Theoretical and experimental results show that the interference fringe shift is linearly related to both the amount and size of the GNPs binding on the sensor surface. The detected signal for 30 nm GNPs can reach a lowest detection limit of 18 pM (1010 particles/ml). Besides, surface regeneration of the FOI sensor was achieved through local pH level manipulation by means of a photoactive molecule, o-Nitrobenzaldehyde (o-NBA), which triggered the dissociation of immune complexes. Experimental results showed that the pH level of the o-NBA solution can be reduced from 7.0 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 shows a linear relationship to the concentration of anti-cytochrome C antibody solution and the detection limit approaches 10 ng/ml. This sensory mechanism has the potential to be applied for in-situ, in-vivo monitoring immune recognition signals in the future.