Graduation Year
2013
Document Type
Dissertation
Degree
Ph.D.
Degree Granting Department
Chemistry
Major Professor
Rudy Schlaf
Keywords
Antibody, Biosensors, Inmmobilization, Physisorption, Spray
Abstract
The immobilization of antibodies on silica surfaces has been a wide and common practice via cross-linking with the formation of covalent bonds between surface and antibody. The formation of antibody thin films on solid surfaces using pneumatic spray (PS) as the deposition technique and the analysis of the surface morphology of these films were investigated during this study. The pneumatic spray method was compared with the covalent bonding method Avidin-Biotin Bridge (ABB). The intensities and capture efficiency tests showed similar results for both techniques with a lower signal-to-noise ratio (SNR) for the PS deposited films. Specificity tests suggested that the bio-sensitivity of the antibody films that were pneumatically sprayed maintained their capture abilities after the immobilization process. Analysis obtained from the attenuated total reflectance Fourier transform infrared ATR-FTIR support these results indicating that the antibodies retained their native structure and chemical stability thorough the induced physisorption process. The pneumatic spray films also preserve mechanical stability by adhering to the surface after the rinsing procedures.
Capture efficiency was tested for both immobilization techniques, the results of which were similar. The pneumatic spray technique was also tested using a diverse range of deposition times. It was shown that a 2 minute deposition time was sufficient to produce a film with similar capture efficiency to the avidin-biotin bridge technique. The surface density obtained for the 2 minute deposition was 9.05 ng/mm2, which is higher than the range of 2.2 to 4.74 ng/mm2 reported for the avidin-biotin bridge technique[1-3]. The contact angle measurements for the pneumatic spray films showed a higher hydrophobicity compared with the avidin-biotin bridge films. This is due to the higher surface roughness obtained for the avidin-biotin bridge films, a higher surface density for the PS and the random orientation of the antibodies in the pneumatic spray films. A study of shelf life showed that the pneumatic spray technique produces stable films that can be used for as long 100 days (study performed only up to 100 days) with similar capture efficiency to those prepared in the same day.
To further understand the improvement in capture efficiency of the pneumatic spray films, the surface morphology was investigated to determine its influence in the cell adhesion process. The surface was characterized by several different techniques: ellipsometry to determine the thickness of the films, atomic force microscopy (AFM) to calculate the surface roughness, optical microscopy to identify particle formation during antibody immobilization process, fluorescent microscopy and sandwich fluorescent immunoassay to observe the immobilization patterns of antibodies and antigens on the surface, contact angle measurements to analyze the wettability of the antibody films and X-ray photoelectron spectroscopy (XPS) to confirm the presence of antibody on both deposition methods and to propose a growth model for the pneumatic spray deposition technique.
A possible explanation for the similar results of capture efficiency for both techniques can be attributed to three main factors. First, the antibodies retained their native structure thorough the induced physisorption process allowing then to capture antigen normally. Second, the lack of orientation of the antibodies in the pneumatic films was compensated by high surface density thereby offering more binding sites to capture antigens. Third, hydrophobic surfaces are favorable to cell adhesion, therefore the high hydrophobicity of the pneumatic spray films increases the capture efficiency. It is important to mention that the time that it takes to produce the immunoassay surfaces was reduced dramatically from more than twenty four hours for the avidin-biotin bridge films to only a few minutes for the pneumatic spray films. In addition, pneumatic spray films significantly reduce the amount of materials and chemicals used in the deposition process. These factors make the pneumatic spray technique an excellent technique for the immobilization of antibodies on glass slides for commercial bio-sensor devices.
Scholar Commons Citation
Figueroa, Jhon J., "Immobilization and Characterization of Physisorbed Antibody Films Using Pneumatic Spray as Deposition Technique" (2013). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/4889
