Is One Enough?

Photostable Single-Molecule Nanoparticle Optical Biosensors for Real-Time Sensing of Single Cytokine Molecules and Their Binding Reactions

“…Similar interesting phenomena were observed in our previous study, in which we directly observed such a complete binding reaction process as single nanoparticle biosensors diffused to the surface of cells, searched for receptors on the surface of the cells, attempted its binding with the receptors several times, and finally bound with the receptors . We did not observe immediate disassociation of ligand−receptor complex (Off-state), which could be attributable to the large affinity of the ligand−receptor complex ( K B = 4.3 × 10 7 M −1 ) that led to a long association time (On-rate), as predicted by simulations …”

“…The result suggests that either single TNFα molecules did not disassociate from MAB on the surface of nanoparticles or the disassociated single TNFα molecules might be trapped among the surface functional groups (MMUA-PVP) of nanoparticles (Scheme ) and therefore were unable to depart from the surface of nanoparticles. The large binding constant ( K B = 1.7 × 10 6 M −1 ) of TNFα-MAB could hinder their disassociation (Off-state), leading to a long association time (On-rate) (>1.6 × 10 6 s = 444 h), as predicted by recent simulations . Similar interesting phenomena were observed in our previous study, in which we directly observed such a complete binding reaction process as single nanoparticle biosensors diffused to the surface of cells, searched for receptors on the surface of the cells, attempted its binding with the receptors several times, and finally bound with the receptors .…”

“…Notably, we did not observe a digital response (bound/unbound or On/Off) of single molecule reaction of TNFα with MAB, that was supposed to be uniquely associated with single molecule reaction, as predicted and reported using fluorescence detection. , Even though a single molecule reaction is supposed to occur at a single step as described by quantum mechanics, a complete reaction dynamic process takes several steps, because both molecules need to diffuse together, interact (collide several times) to search for the optimum bind sites, and finally bind. Studies of single molecule reactions thus far reported primarily used fluorescence probes which were limited by the photodecomposition and blinking of fluorescence probes, and hence it was impossible to observe such a complete single-molecule reaction process.…”

Real-Time Imaging and Tuning Subcellular Structures and Membrane Transport Kinetics of Single Live Cells at Nanosecond Regime

“…Recent studies show that 10-14 measurements of individual events (e.g., single molecules) are sufficient sample sizes to extrapolate ensemble properties with accuracy of analytical measurements at single-entity level. 39 Thus, the sample sizes presented in this study are adequate for the study of bulk cells at the single-cell resolution. Unlike ensemble measurements, single-cell imaging allows us to unmask the heterogeneous nature of individual cells in bulk solution and probe intracellular molecules and structures of individual cells with both spatial and temporal resolution.…”

“…Recent studies show that 10−14 measurements of individual events (e.g., single molecules) are sufficient sample sizes to extrapolate ensemble properties with accuracy of analytical measurements at single-entity level . Thus, the sample sizes presented in this study are adequate for the study of bulk cells at the single-cell resolution.…”