Design and synthesis of single-nanoparticle optical biosensors for imaging and characterization of single receptor molecules on single living cells

At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver nanoparticle biosensors (AgMMUA-IgG) by functionalizing 11.6 +/not superset of 3.5-nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (1:3 mole ratio) and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21-0.37 molecule/mu m(2)), and measure their binding affinity and kinetics at concentrations below their dissociation constant on single living cells in real time over hours. The dynamic range of detection is 0-50 molecules per cell. We also found that the binding rate (2-27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k(1) = (9.0 +/not superset of 2.6) x 10(3) M-1 s(-1), k(-1) = (3.0 +/not superset of 0.4) x 10(-4) s(-1), and K-B = (4.3 +/not superset of 1.1) x 10(7) M-1, respectively.