Label-free and High-throughput Flow Cytometry for Single-cell Surface Protein Biosensing Using Multi-stage Constriction Microchannels

Flow cytometry (FCM) can measure various characteristics of single cells in a fast and efficient way. However, FCM relies on fluorescent labeling, which may alter cell behaviors. This may raise concerns about safety and efficacy of the cell therapies using the sorted labeled cells. There is apparently a need for innovative label-free solutions that overcome the constraints of traditional FCM and enable a new paradigm in cell analysis. The recent advancements in microfluidics enable quantitative measurements of cell properties which have been difficult for the traditional cell research techniques. For instance, the biochemical properties of floating cancer cells with molecular labels can be measured by multiple microfluidic techniques with improved biosensing performance. There are also label-free microfluidic techniques based on cell-solid or cell-flow interactions, such as the deformability microcytometry for quantifying biomechanical properties of tumor cells, which have been proven to reflect their metastatic tendency and cancer status. Nevertheless, there is still great demand for the microfluidic label-free and high-throughput FCM techniques for cell phenotyping, classification and screening, and the associated clinical applications. Our aim is to develop a label-free, high-throughput, and fully automated electrokinetic flow micro-cytometry system for multiparametric characterization in biophysical and biochemical domains. It is combined with chip antibody coupling and sensing microelectrodes, to detect the properties of single cells, including cell size, cell deformability, dielectric constant, and cell surface antigen expressions, to perform cell typing and cell subpopulation identification in a label-free manner. The automated system elimination of microscopic imaging and fluorescence sensing such that the unit cost can be largely reduced, inducing its value toward commercialization as a new generation of FCM. The core element for the success of this research is a representative theoretical model to promisingly convert the measured cell signals to the cell properties. We have a strong background and have performed solid preliminary study for this research, which includes a theoretical model describing the cell size and elasticity and the measured signals. We plan to further develop such a model to further consider the interaction between cell surface proteins and antibody-coated channel walls for achieving the cell surface protein quantification. This system can overcome the existing flow cytometry paradigm and can be applied to many fields where conventional FCM cannot be used, such as label-free detection and sorting of rare cells (such as circulating tumor cells, hematopoietic stem cells), and various stages of cell therapy drug manufacturing (such as CAR-T therapy).