While single-molecule detection in flowing sample streams has been reported by a number of groups, the instrumentation can be somewhat prohibitive for many applications due to the complexity and extensive expertise required to operate such a device. In this paper we report on the construction of a single-molecule detection device that is rugged, compact, inexpensive, and easily operated by individuals not well trained in optics and laser operations. The single-molecule detection apparatus consists of a semiconductor diode laser operating in a continuous-wave (CW) mode and a single photon avalanche diode transducer for converting the detected photons into transistor–transistor logic (TTL) pulses for displaying the data. In addition, the sampling volume is produced by a single-component lens, to create a volume on the order of 1 pL, allowing the sampling of microliter volumes of material on reasonable time scales. The device is targeted for operation in the near-IR region (700–1000 nm), where matrix interferences are minimal. Our data will demonstrate the detection of single molecules for the near-IR dyes IR-132 and IR-125, in methanol solvents in flowing sample streams at sampling rates of 100–250 samples/s. Detection efficiencies for the investigated near-IR dyes were found to be 98% for IR-132 and 50% for IR-125. Previous attempts in our laboratory to detect single molecules of IR-125 using time-gated detection were unsuccessful because of the short upper-state lifetime of this fluorophore (τf = 472 ps).
Applications of capillary electrophoresis and laser-induced fluorescence detection to the analysis of trace species: From single cells to single molecules