Biospecific irreversible fishing coupled with atomic force microscopy for detection of extremely low-abundant proteins.

In the absence of an analog of PCR for proteins, the concentration detection limit (DL) becomes a real challenge. The problem may be solved by means of a combination of biospecific irreversible fishing with atomic force microscopy (AFM). AFM offers the ability to register individual molecules and their complexes, while biospecific fishing takes advantage of an affine interaction between analyte molecules spread over a large volume of biomaterial and ligand molecules immobilized on the chip surface. Fishing may be conducted in Kd-dependent reversible mode and in Kd-independent irreversible mode. In this study, the DLs of two previously applied proteomic approaches were determined and compared to the DL of a newly developed analytical method. The first approach, based on MS analysis of biomaterial after 2-DE or LC separation of proteins, attained a DL at the level of 10(-8)-10(-10) M. The second approach, based on the optical biosensor analysis of molecular interactions in the format of proteomic microarrays, had a DL of 10(-9)-10(-10) M. Our proposed method which combines biospecific fishing with AFM allowed us to attain DL values of 10(-11) M under reversible binding conditions and 10(-16) M under irreversible binding conditions.