Development of an Ultrasensitive Dual-Channel Flow Cytometer for the Individual Analysis of Nanosized Particles and Biomolecules

A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO2 nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R2 > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was ∼90% on average for particle concentrations ranging from 1.62 × 105 to 3.93 × 107 particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.