Modular Cell-Surface-to-Solution CRISPR-Cas12a Platform for Wash-Free Quantification of Cell−Surface Proteins on Living Cells

Abstract Cell−surface proteins are important biomarkers for cell phenotyping, disease diagnosis, therapeutic evaluation, and drug screening. However, their quantitative analysis on intact living cells remains challenging because conventional methods often require cell lysis, fixation, repeated washing, extensive sample processing, or bulky instrumentation. Herein, we developed a modular and reconfigurable cell-surface-to-solution CRISPR-Cas12a platform for wash-free quantitative analysis of cell−surface proteins on intact living cells. Aptamer recognition of extracellularly accessible targets deblocks a DNA activator, thereby activating Cas12a trans-cleavage in solution and converting localized cell−surface recognition into amplified fluorescence while maintaining a low background in the absence of targets. Using EpCAM as a model target, we engineered a split dual-aptamer architecture that enabled cooperative target recognition, efficient activator deblocking, and low-background signal amplification. The platform achieved sensitive EpCAM detection with a limit of detection of 32 fM and enabled live-cell analysis down to 2 cells/μL. It distinguished cell populations with different surface EpCAM levels and quantitatively monitored epithelial−mesenchymal transition-associated changes in cell−surface EpCAM abundance. Coordinated redesign of the target-responsive nucleic acid components extended the platform to PTK7 detection and the analysis of drug-induced cell−surface PTK7 remodeling. Coupling the system to a lateral flow readout enabled portable visual detection. This work establishes a modular and reconfigurable CRISPR-Cas12a sensing strategy for wash-free amplified quantification of cell−surface proteins on living cells, providing a convenient tool for investigating their dynamic remodeling during physiological and pathological processes and therapeutic responses, as well as for drug screening.