Development Of An Atomic Force Microscopic Immunosensor For The Ferritin Antigen

The adsorption conditions for a range of different immunoreactants to both polystyrene and ultrasmooth silanised silicon surfaces are optimised. The conditions studied were concentration of protein, time and temperature of incubation, pH, molarity and ionic strength of the buffer. The anti-ferritin surface was selected as our model system. This immunosensor was used to develop and evaluate a millispot ELISA for ferritin. The millispot ferritin ELISA developed utilises a sample volume of 6pl and a sample incubation time of 10 minutes. Precision and accuracy studies show the assay to have a dynamic assay range of 7.8-lOOng/ml ferritin. The millispot ELISA detected a minimum ferritin concentration of 13ng/ml. An alternative method of immunecomplex detection was also studied. An Atomic force microscope was successfully applied to directly detect the presence of ferritin and alpha- fetoprotein antigens on the appropriate immunosensor surface, while further work is required to directly detect smaller proteins such as hCG. The detection of the ferritin protein was taken a step further with the development of a novel system to quantify the ferritin in the bulk solution via the Atomic Force Microscope (AFM). The number of binding events and surface roughness (Ra) over 5μm2 areas was determined for a range of different ferritin concentrations. Both parameters show a linear response to ferritin concentration for the concentration range (7.8 - 500ng/ml).