Evaluation of antibody immobilization methods for piezoelectric biosensor application

Abstract

The immobilization of anti- Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature–time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4±3.8% on the gold surface which then allowed an immobilization of 42.1±2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6±0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to μg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only ∼8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S.typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI–GA immobilization. The frequency decreases with S.typhimurium concentration of ∼1.5×10 9 CFU/ml were 50±2 Hz and 44±3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.