Abstract
The array-based biosensors contain several advantages including quick screening, massive parallel analysis, easy operation, good sensitivity and selectivity. The immobilization of biological compounds onto the solid surfaces is one of the bottlenecks in the development array-based biosensor array. In this study, a sol-gel technique with additives and optimized parameters that is suitable for the immobilization of antibody was developed to fabricate the array-based immunosensor for the determination of hepatitis. Tetramethylorthosilicate (TMOS) was selected as the sol solution. Glycerol (6 %) was added into the sol-gel solution to effectively extend the gelation time of sol-gel. The cracking condition in the gelation process could be improved by adding 0.6 % polyvinyl alcohol (PVA). When the ratio of sol/solution was higher than 20 %, leakage of biomolecules was not significant. In addition, protein was immobilized within sol-gel network in the presence of polyvinyl acetate (PVAc) on the glass slide. The optimized array biosensor was used for fluorescein-immunoassay to detect antigens or antibodies. The mouse IgG was entrapped within sol-gel, and anti-mouse IgG with FITC was allowed to diffuse through the pores of the micro-patterned gel structure. The antigen-antibody was observed after 3 hr incubation. The detection limits of direct fluorescein-immunoassay were 5 ng/ml and 10 ng/m, respectively for Mouse-IgG and Sheep-IgG systems. In the sandwich immunoassay of HBV antigen/antibody reaction, a detection limit of 30 ng/ml can be achieved. For multi-protein measurement experiment, results showed that the antibody could recognize or identify the specific corresponding receptor in one experiment even in sandwich immunoassay. In addition, the activity of the mouse-IgG could be retained for more than two weeks at 4oC. This storage period was longer than that using the normally covalent immobilization technique (less than 7 days). The relative standard deviation (RSD) of the array-based immunosensor was less than 5% (n = 7). Results obtained in this study clearly show that the developed sol-gel based array immunosensor has good potentials to perform the simultaneous and parallel analysis for the determination of multi-samples in the presence of multianalytes.