Gating or threshold selection is very important in analyzing data from a microflow cytometer, which is especially critical in analyzing weak signals from particles/cells with small sizes. It has been reported that using the amplitude gating alone may result in false positive events in analyzing data with a poor signal-to-noise ratio. Transit time (τ) can be set as a gating threshold along with side-scattered light or fluorescent light signals in the detection of particles/cells using a microflow cytometer. In this study, transit time of microspheres was studied systematically when the microspheres passed through a laser beam in a microflow cytometer and side-scattered light was detected. A clear linear relationship between the inverse of the average transit time and total flow rate was found. Transit time was used as another gate (other than the amplitude of side-scattering signals) to distinguish real scattering signals from noise. It was shown that the relative difference of the measured microsphere concentration can be reduced significantly from the range of 3.43%–8.77% to the range of 8.42%–111.76% by employing both amplitude and transit time as gates in analysis of collected scattering data. By using optimized transit time and amplitude gate thresholds, a good correlation with the traditional hemocytometer-based particle counting was achieved (R² > 0.94). The obtained results suggest that the transit time could be used as another gate together with the amplitude gate to improve measurement accuracy of particle/cell concentration for microfluidic devices.

The eutrophication of aquatic ecosystems caused by rapid human urbanization has led to an increased production of potentially hazardous bacterial populations, known as blooms. One of the most notorious forms of these aquatic blooms are cyanobacteria, which in sufficiently large quantities can pose a hazard to human health through ingestion or prolonged exposure. Currently, one of the greatest difficulties in regulating and monitoring these potential hazards is the early detection of cyanobacterial blooms, in real time. Therefore, this paper presents an integrated microflow cytometry platform for label-free phycocyanin fluorescence detection, which can be used for the rapid quantification of low-level cyanobacteria and provide early warning alerts for potential harmful cyanobacterial blooms. An automated cyanobacterial concentration and recovery system (ACCRS) was developed and optimized to reduce the assay volume, from 1000 mL to 1 mL, to act as a pre-concentrator and subsequently enhance the detection limit. The microflow cytometry platform utilizes an on-chip laser-facilitated detection to measure the in vivo fluorescence emitted from each individual cyanobacterial cell, as opposed to measuring overall fluorescence of the whole sample, potentially decreasing the detection limit. By applying transit time and amplitude thresholds, the proposed cyanobacteria detection method was verified by the traditional cell counting technique using a hemocytometer with an R² value of 0.993. It was shown that the limit of quantification of this microflow cytometry platform can be as low as 5 cells/mL for Microcystis aeruginosa, 400-fold lower than the Alert Level 1 (2000 cells/mL) set by the World Health Organization (WHO). Furthermore, the decreased detection limit may facilitate the future characterization of cyanobacterial bloom formation to better provide authorities with ample time to take the appropriate actions to mitigate human risk from these potentially hazardous blooms.