Multiplexed Aptamer-Lattice Nanodevice for One-Step Phenotyping and Functional Quality Control of CAR-T Cells.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized cancer treatment but continues to face substantial quality control challenges due to cellular heterogeneity. Here, we report a multiplexed nanodevice, the Aptamer Lattice-Powered Hybrid Architecture (ALPHA), for one-step phenotypic and functional analysis of CAR-T cells. By integrating aptamers targeting CD3, CD25, and CD122 into a programmable DNA lattice scaffold, ALPHA enables rapid, wash-free, and orthogonal fluorescence activation. Within 30 min, the platform distinguishes resting from activated T cells and generates an Activation Index that strongly correlates with cytokine secretion and cytotoxic function. To directly monitor cytotoxicity, an integrated ATP-responsive aptamer detects extracellular ATP released from lysed targets, enabling real-time assessment of target cell killing. ALPHA also captured donor-dependent functional variability in CAR-T samples, consistent with conventional functional readouts. Overall, ALPHA represents a versatile and scalable tool for comprehensive quality control of engineered immune cells during manufacturing.