Chimeric antigen receptor (CAR) T-cell therapy has become a key treatment for blood cancers thanks to its strong capacity to identify and destroy malignant cells. Evaluating the performance of these immune cells at the individual level is essential for optimizing diagnostic and therapeutic outcomes. There is a pressing need for a versatile, rapid, high-throughput, and comprehensive system to assess immune cell efficacy. In this work, the killing ability, expansion capacity, and long-term survival of TIM3/CD28-engineered CD19 CAR-T cells were compared against standard CD19 CAR-T cells using advanced, high-throughput graphene oxide quantum dot (GOQD)-based single-cell microfluidic devices. Comprehensive profiles of secreted factors, therapeutic performance across varying effector-to-target ratios, spatial distribution effects on immune activity, and cellular subpopulation characterization were analyzed to clarify the enhanced immunotherapy potential of the modified cells. TIM3/CD28-modified CD19 CAR-T cells demonstrated superior tumor-killing activity and sustained strong therapeutic responses even under low effector-to-target ratios and at greater distances. The TIM3/CD28 modification also improved localized targeting precision. Notably, these engineered cells displayed more pronounced and stable Th1/Th2-like persistent and highly cytotoxic subpopulations, supporting more tailored therapeutic strategies. In summary, this approach offers a broadly applicable method for assessing existing CAR-T designs and testing novel CAR-based treatments moving forward.
