Simulations for guiding the delivery and trapping of single biomolecules in a nanofluidic device

Abstract

A microfluidic device has been developed wherein single molecules in solution are electrokinetically transported along a nanochannel. The nanochannel is irradiated by two adjacently focused laser beams so that the timing of fluorescence photons induced by each beam indicates the position of a molecule along the nanochannel. This is then used to actively control the electrokinetic flow, so that the molecule may be held within the confocal volume for a prolonged time and then rapidly replaced following photobleaching or completion of the single-molecule measurement. Here we focus on Monte Carlo computer simulations of the physical processes that occur during the delivery and trapping. The simulations help in understanding the constraints imposed by experimental limitations, such as the latency of feedback, the maximum achievable speed of electrokinetic flow, and photophysical processes such as triplet crossing and photobleaching. They also aid in evaluating the effects of shot noise and photon timing error and in predicting optimum experimental operating parameters. Studies indicate that the 6 μs latency of feedback in our experiments is well below that required for stable trapping (~100 μs); for small freely diffusing molecules, a limited flow speed of ~2 μm/ms can result in ~10-20 % of molecules escaping before they photobleach; there is an optimum laser power of ~30-40 μW that provides a sufficient rate of fluorescence photons for trapping while reducing loss due to photobleaching; an increase in the spacing between the beams or increase in relative power of the down-stream beam increases the trapping time.