The investigation of Protein A and Salmonella antibody adsorption onto biosensor surfaces by atomic force microscopy.
The investigation of Protein A and antibody adsorption on surfaces in a biological environment is an important and fundamental step for increasing biosensor sensitivity and specificity. The atomic force microscope (AFM) is a powerful tool that is frequently used to characterize surfaces coated with a variety of molecules. We used AFM in conjunction with scanning electron microscopy to characterize the attachment of protein A and its subsequent binding to the antibody and Salmonella bacteria using a gold quartz crystal. The rms roughness of the base gold surface was determined to be approximately 1.30 nm. The average step height change between the solid gold and protein A layer was approximately 3.0 +/- 1.0 nm, while the average step height of the protein A with attached antibody was approximately 6.0 +/- 1.0 nm. We found that the antibodies did not completely cover the protein A layer, instead the attachment follows an island model. Salt crystals and water trapped under the protein A layer were also observed. The uneven adsorption of antibodies onto the biosensor surface might have led to a decrease in the sensitivity of the biosensor. The presence of salt crystals and water under the protein A layer may deteriorate the sensor specificity. In this report, we have discussed the application and characterization of protein A bound to antibodies which can be used to detect bacterial and viral pathogens.
Lee
Kyoung G
KG
Department of Chemical Engineering, University of Louisville, Louisville, Kentucky 40292, USA.
Pillai
Shreekumar R
SR
Singh
Shree R
SR
Willing
Gerold A
GA
D000327:Adsorption; D000906:Antibodies / Q000737:chemistry*; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D012475:Salmonella / Q000276:immunology* / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013499:Surface Properties
D000906:Antibodies; D013205:Staphylococcal Protein A