Single-Cell Recognition and Separation via Matched Reciprocating-Flow Interfaces and Peptide Recognition Dynamics.

Single-cell capture and separation in microfluidics requires simultaneous optimization of distinct cascade processes: 1) precise interaction regulation enabling rapid point-to-point recognition, and 2) face-to-face interfacial contact, which determines binding stability and duration. Since these two processes occur on different length scales, constructing an engineered affinity interface that couples both remains highly challenging. Herein, we report a dynamic microfluidic single-cell capture platform that integrates peptide-based rapid recognition interfaces with kinetically matched fluid-field regulation. A series of targeting peptides toward PD-L1 (programmed cell death ligand-1) with antiparallel β-sheet structure were rationally engineered, leading to an optimized sequence (EAF) characterized by flexible recognition termini and electrostatically dominated binding. At the molecular level, this promotes rapid and specific point-to-point recognition. Upon self-assembly, EAF forms nanofibers densely displaying recognition motifs, thereby enhancing multipoint specific interactions with the cell membrane. By coupling this with a reciprocating-flow microfluidic design, locally perturbed fluidic fields are generated to enhance cell-surface contact, thereby achieving stable binding. This synergistic kinetic modulation facilitates rapid target recognition and stable interfacial binding, resulting in efficient and sustained single-cell capture. Collectively, this work demonstrates a generalizable strategy for engineering dynamic affinity interfaces that align molecular recognition kinetics with microfluidic control, offering a versatile platform for single-cell isolation.