A high-sensitivity and clogging-free microfluidic impedance flow cytometer based on three-dimensional hydrodynamic focusing.

Abstract

Microfluidic impedance flow cytometry has functioned as an enabling instrument in single-cell analysis, which, however, suffers from the limiting tradeoff between high sensitivity and clogging-free operation. In order to address this issue, this study presented a microfluidic impedance flow cytometer based on three-dimensional (3D) hydrodynamic focusing, in which the crossflow of conductive sample fluids and insulating sheath fluids was leveraged to centralize and restrict electric field lines to the sample fluid, thereby achieving high impedance sensitivity of single cells without the concern of channel blockage. Different from conventional impedance flow cytometry, in this study, impedance amplitude dips (rather than pulse singles) generated by single microparticles traveling through the 3D hydrodynamic focusing region were experimentally validated using microbeads. Based on the home-developed microfluidic impedance flow cytometer, high-sensitivity and clogging-free impedance profiles of three leukemia cell lines (K562, Jurkat, and HL-60) and four types of purified leukocytes (neutrophil, eosinophil, monocyte, and lymphocyte) were quantified as -8.01 ± 2.96%, -4.53 ± 1.09%, -6.36 ± 1.54%; -8.11 ± 0.84%, -7.23 ± 1.06%, -9.05 ± 2.00% and -5.68 ± 1.24%, respectively. When a recurrent neural network was adopted for cell-type classification, high classification accuracies of 93.9% for three leukemia cell lines and 87.8% for four types of purified leukocytes were achieved. This study presented a promising impedance flow cytometer that combines high sensitivity with sustainable working capabilities, potentially overcoming the limitations of conventional microfluidic impedance flow cytometry and significantly advancing its commercial development.