Immunological identification of fibrinogen in dual-component protein films by AFM imaging

The success of long-term blood-contacting implanted devices is largely dependent upon the interaction of the blood components with the device biomaterial surface. The ability to study these interactions has been hindered by a lack of methods to measure single-molecule interactions in complex multi-protein environments similar to the environment found in vivo. In this paper, we demonstrate the use of atomic force microscopy (AFM) in conjunction with gold nanolabels to detect the protein fibrinogen under aqueous conditions without the topographical clues usually necessary for high resolution visualization. BSA was patterned onto both muscovite mica and plasma-treated polydimethylsiloxane (PDMS) substrates and these test substrates were subsequently backfilled with fibrinogen to yield a featureless protein layer. The fibrinogen in this dual-protein layer was detected using high resolution AFM imaging following infusion of anti-fibrinogen conjugated with nanogold particles. This AFM immuno-detection technique will potentially be applicable to complex multi-component protein films adsorbed on clinically relevant polymers used in medical devices.