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Single-cell assays have broad applications in cellular studies, tissue engineering, fundamental studies of cell-cell interactions, and understanding of cell-to-cell variations. Most existing methods for micron-sized cell patterning are still based on lithography-based microfabrication process. Thus, exploiting new mask-free strategies while maintaining high-precision single-cell patterning is still a great challenge. Here, we presented a facile, low-cost, and mask-free approach for constructing high-resolution patterning on sticky superhydrophobic (SH) substrates based on inkjet printing with ordinary precision. In this work, the SH surface with both high contact angle and relatively high contact angle hysteresis can not only obtain high-resolution spots but also avoid droplets bouncing behavior. We improved the feature size of printed protein spots as small as 4 μm, which is much smaller than protein spots used for single-cell trapping. Moreover, with the assistance of a narrow microchannel, the inkjet-printing patterned chip with fibronectin ink allows for fast and high-efficiency trapping of multiple single-cell arrays. Using this method, single-cell occupancy could reach approximately 81% within 30 min on subcellular-sized patterning chip, and there was no significant effect on cell viability. As a proof of concept, this chip has been applied to study the real-time apoptosis of single cells and demonstrated the potential in cells' heterogeneity analysis.

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