Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices

Analysis of proteins released from living single cells is strongly required in the fields of biology and medicine to elucidate the mechanism of gene expression, cell-cell communication and cytopathology. However, as living single-cell analysis involves fL sample volumes with ultra-small amounts of analyte, comprehensive integration of entire chemical processing for single cells and proteins into spaces smaller than single cells (pL) would be indispensable to prevent dispersion-associated analyte loss. In this study, we proposed and developed a living single-cell protein analysis device based on micro/nanofluidics and demonstrated analysis of cytokines released from living single B cells by enzyme-linked immunosorbent assay. Based on our integration method and technologies including top-down nanofabrication, surface modifications and pressure-driven flow control, we designed and prepared the device where pL-microfluidic- and fL-nanofluidic channels are hierarchically allocated for cellular and molecular processing, respectively, and succeeded in micro/nanofluidic control for manipulating single cells and molecules. 13-unit operations for pL-cellular processing including single-cell trapping and stimulation and fL-molecular processing including fL-volumetry, antigen-antibody reactions and detection were entirely integrated into a microchip. The results suggest analytical performances for countable interleukin (IL)-6 molecules at the limit of detection of 5.27 molecules and that stimulated single B cells secrete 3.41 IL-6 molecules per min. The device is a novel tool for single-cell targeted proteomics, and the methodology of device integration is applicable to other single-cell analyses such as single-cell shotgun proteomics. This study thus provides a general approach and technical breakthroughs that will facilitate further advances in micro/nanofluidics, single-cell life science research, and other fields.