Improving immunoassays based on the analysis of scanning force microscopy images

Scanning force microscopy has been demonstrated to be an effective binding event detection step for immunoassays. In its simplest form—analysing small area images—the detection limit of the scanning force microscopic immunoassay (SFMIA) has been shown to be comparable to existing techniques. In the present work, we have examined how the performance of image analysis-based SFMIA can be improved. Firstly, we have used a surface analysis parameter that increases linearly with the concentration of binding events. This parameter—the surface area ratio—is the ratio of the surface area after antigen binding to the surface area of the original biospecific surface. With this parameter, SFMIA images can be rapidly analysed and converted into assay units. Secondly, we have demonstrated that by using silicon wafer supports that carry fiducial marks we can relocate to very high accuracy onto the biospecific surfaces and identify the changes due to antigen binding. By relocating in this manner the signal to noise ratio of the technique is enhanced. Thirdly, from simulations we have determined the SFM tip size and image area that optimizes the immunoassay sensitivity.