A single-molecule flow analyzer based on laser induced fluorescence and hydrodynamic focusing was developed in our lab. There were three key aspects of this design for achieving single molecule detection sensitivity: (1) a detector with a high photon-detection efficiency(>65% for avalanche photo-diode detector); (2) a small probe volume obtained by sheath-protected, hydrodynamically focused sample stream to reduce background; and (3) a slow flow to provide extended dwell time(milliseconds) in the probe volume so that more fluorescence cycling can occur. The single molecule detection sensitivity was demonstrated by the successful detection of single phycoerythrin molecules. The house-constructed single-molecule flow analyzer was applied to DNA fragment sizing with linear correlation between DNA fragment length and mean burst size.
