Surface plasmon resonance immunosensor for the detection of bacterial cells was first reported in 1998 with high detection limit as much as 107 cfu/ml. Since then, many efforts have been made aiming to lower the detection limit and improve the sensitivity of detection. The aim of this study was to compare the effect of four most frequently used immobilization strategies, including direct physical adsorption (physisorption), covalent immobilization via self-assembled monolayer (SAM) formation, bioaffinity immobilization using protein G-mediated immobilization and using mixed SAM of alkane thiols on signal strength of detection of Vibrio cholerae using these modified surfaces. The most widely used strategy, covalent binding of antibodies to sensor chip via SAM formation, gave the highest immobilization density and mixed SAM of 20/80 (v/v) of 11-mercaptoundecanoic acid (11-MUA)/9-mercapto-1-nonanol resulted in the least surface coverage in antibody immobilization step. To optimize surface density in covalent immobilization, four different concentrations (12.5, 25, 50, and 100 μg/ml) of anti-OmpW were immobilized on 11-MUA modified gold chips and maximum interaction response was achieved at 25 μg/ml. The interaction response signals for detection of V. cholerae using immobilized anti-OmpW were in this order: Oriented immobilization using protein G/antibody complex > mixed SAM of 11-MUA and 9-mercapto-1-nonanol > homogenous 11-MUA SAM > direct physical adsorption. In order to evaluate interaction studies in real sample condition, waste water samples that were artificially spiked with V. cholerae were tested and the authors concluded that for real samples, it is better to setup experiment with low surface coverage such as mixed SAM to overcome nonspecific adsorption.

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See supplementary material at http://dx.doi.org/10.1116/1.4971270 for the stabilization of the 11-MUA modified sensor chip before antibody immobilization.

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