Simultaneous quantification of intracellular ATG4B and CTSB activities using a dual-channel nanopipette to assess autophagic flux

Simultaneous detection of key autophagic proteases—autophagy-related 4B (ATG4B) and cathepsin B (CTSB)—in living cells remains challenging, yet it is essential for understanding their dual regulatory roles in tumor progression and immune modulation, such as major histocompatibility complex I (MHC-I)-mediated antigen presentation. To address this gap, we developed a dual-channel peptide-functionalized glass nanopipette for simultaneous quantification of intracellular ATG4B and CTSB activities. Each channel is chemically modified with a protease-specific peptide substrate; upon specific cleavage by the target protease, the ionic current through the corresponding channel is altered, enabling label-free detection. The nanopipette exhibits exceptional selectivity, with current responses to ATG4B and CTSB over three-fold higher than those induced by common biological interferents (e.g., cations, amino acids, and redox-active molecules), and demonstrates acceptable stability for cellular assays. In cell studies, it successfully distinguished autophagy levels between normal hepatocytes and hepatocellular carcinoma cells, and further captured rapamycin-induced autophagy enhancement. Given the established correlation between protease activity and MHC-I expression, this method enables direct assessment of autophagy and indirect evaluation of immunogenicity for immunotherapy prediction.