Ultrasensitive and label-free probing of binding affinity using recognition imaging.

Reliable quantification of binding affinity is important in biotechnology and pharmacology, and increasingly coupled with a demand for ultra-sensitivity, nano-scale resolution, and minute sample amounts. Standard techniques are not able to meet these criteria. This study provides a new platform based on atomic force microscopy (AFM) derived recognition imaging to determine affinity by visualizing single molecular bindings on nano-sized dendrons. Using DNA hybridization as a demonstrator, an AFM sensor adorned with a cognate binding strand senses and localizes target DNAs at nanometer resolution. To overcome the limitations of speed and resolution, the AFM cantilever is sinusoidally oscillated close to resonance conditions at small amplitudes. The equilibrium dissociation constant of capturing DNA duplexes was obtained, yielding KD = 2.4×10-10 M. Our label-free single-molecular biochemical analysis approach evidences the utility of recognition imaging and analysis in quantifying bio-molecular interactions of just a few hundred molecules.