Simultaneous counting of two subsets of leukocytes using fluorescent silicananoparticles in a sheathless microchip flow cytometer

Abstract

A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45⁺CD4⁺ cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45⁺CD4⁻ cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R² = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.