| Four different immobilization methods for antibody molecules onto a piezoelectric quartz crystal gold electrode surface were studied and compared. Anti-Vibrio parahaemolyticus antibody and its target antigen, Vibrio parahaemolyticus cells were employed as a model. An antigen concentration of 10,000 CFU in 10 {dollar}mu{dollar}L and a reaction time of 30 min in room temperature was used for the assay. The piezoelectric biosensor developed using Staphylococcal protein A as the coupling agent for immobilizing antibodies gave a frequency decrease of 63 Hz, compared to 43 Hz for polyethyleneimine-dimethyl pimelimidate crosslinking, 34 Hz for using polyethyleneimine-glutaraldehyde crosslinking, and 5 Hz for polyethyleneimine-dithiobis (succinimidyl propionate) crosslinking. Immobilization of antibodies onto the crystal surface using the Staphylococcal protein A as a coupling agent showed superior sensitivity, compared to the other three immobilization methods studied.; A continuous liquid-phase flow-injection analysis piezoelectric immuno-biosensor was developed to detect Vibrio parahaemolyticus. Staphylococcal protein A was used as the coupling agent for immobilization of the antibodies onto the gold electrode surface. The immuno-biosensor developed had a linear detection range of 10{dollar}sp4{dollar} to 10{dollar}sp7{dollar} CFU/mL with a response time of 20 min. The optimum injection volume of the immuno-sensor was 50 {dollar}mu{dollar}L. Specificity study of the immuno-biosensor using Salmonella typhimurium cells showed no apparent cross reactivity. The piezoelectric immuno-biosensor developed had a faster response time, excellent specificity, and competitive sensitivity compared to other rapid detection methods available for this pathogen.; Regeneration of piezoelectric quartz crystal immunobiosensor was studied. Four regeneration treatments were studied, 1% Triton X-100, glycine-HCl (pH 3.0), glycine-HCl with 50% ethylene glycol (pH 1.75), and 1% tripolyphosphate with 25% ethylene glycol. Two antibody-antigen models, anti-glucose oxidase antibody and glucose oxidase, and anti-Vibrio parahaemolyticus antibody and V. parahaemolyticus cells, were used. Glucose oxidase was successfully re-attached to the immunosensor and showed enzyme activity after treatments with all four regeneration procedures, except Triton X-100. For the V. parahaemolyticus biosensor, regeneration treatment by glycine-HCl with 25% ethylene glycol for 0.5 min was the most effective method among those studied. The immunosensor regenerated by this method retained 54% of the original signal. |
