Detection of single protein molecules at interfaces after antibody-antigen binding

The fluorescence-based detection of single dye labeled protein molecules at interfaces is presented. Glass substrates with covalent immobilized antibodies serve for capturing matching antigens from volume concentrations between 10^-12 and 10^-17 mol/l. The unspecific binding at the interface has been reduced to a level down to 0.1% of the maximum signal level. At concentrations lower than 10^-13 mol/l we observe single antibody-antigen complexes at the surface. We developed a scanning method for counting single antibody- antigen complexes. The counting results are used for calibrating the volume concentration dependency. AT the present stage, the detection limit of this molecule counting process is of the order of 10^-17 mol/l, and the dynamic range detectable antigen concentration is more than 8 orders of magnitude, without reaching a limiting value. The instrumental set-up is similar to that of a confocal microscope. A diode laser is used as an excitation source. As an first application in early-stage-diagnostics, we investigated the detection of a single cardiac-actin molecule in human plasma.