Inside biological cells there are very small numbers of molecules undergoing non-equilibrium processes. We present the role of fluctuations in the dynamics of small number systems in connection to nano-diffusion experiment. We study the usual two-state processes which can be relevant to single molecule experiments in different biological systems. We use the principle of maximum caliber to model such problems. Finally, we discuss heterogeneous kinetics of proteins, the origin of cooperative transition and the folding in the micro-second timescale, with an attempt to understand the ultimate speed-limit in protein folding.