In Situ Enzyme Activity Analysis in Single Living Cells Based on the EISA Reaction and Rotation or Translation...

Enzymes in living cells play crucial roles in various cellular physiological activities. However, few in situ single-particle spectroscopy methods were developed to quantitatively determine the enzyme activity within single living cells, in addition to some imaging methods. In this work, a novel single-particle spectroscopy method was proposed for the quantification of enzyme activity at different detection sites within single living cells by combining an enzyme-instructed self-assembly (EISA) reaction with a single-particle rotation or translation correlation spectroscopy (RTCS) technique, using caspase-3 as a model. In this measurement strategy, fluorescent peptide nanorods were self-assembled within living cells due to caspase-3-induced EISA reaction when peptides entered the cell and were cleaved by caspase-3. Then, RTCS curves of fluorescent nanorods were measured, and their rotational and translational diffusion times were extracted. Finally, based on the dependence of the ratiometric diffusion time (ratio of rotational to translational diffusion time) on caspase-3 concentration, the in situ quantitative analysis method of caspase-3 activity within single living cells was established under optimized experimental conditions. The distribution of the caspase-3 activity within single cells was determined, and the inhibition effect of the inhibitor on caspase-3 in living cells was measured. This single-particle spectroscopy method achieved the quantitative measurement of caspase-3 in a single cell and demonstrates significant potential for studying enzymatic functions and their inhibitor screening in native biological contexts.