Electrochemically confined effects on single enzyme detection with nanopipettes

Abstract

• Label-free detection of single enzymes using nanopipettes with varying orifices in near physiological solutions. • The ionic enhancement sensing strategy for single protein. • Nanopore confinement effect modulated translocation behavior of single enzymes. Dynamic conformational changes of enzymes during enzyme catalysis are crucial to the catalytic activities, however the subtle variations are usually buried in ensemble-averaged measurements. The nanopore sensing technique is a promising tool for single-molecule analysis by providing a nanoscale confined space to capture and insolate a single analyte from a bulk solution. Here, we employed nanopipettes with different diameters to analyze the translocation process of single glucose oxidase molecules in an electrolyte solution with low salt concentration. The unique confinement effect of pore including the volume exclusion and charge effect contributed to the current fluctuations, resulting in the current blockade and current enhancement protein traversing through the nanopipettes tip orifice with diameters of 67 nm and 34 nm, respectively. Our research shed light that the interaction between the enzyme and inner wall of nanopipette could significantly extend the duration time of analyte at the orifice, which may facilitate to capturing a single molecule and tracing the subtle conformational changes during enzyme catalysis without any labeling, providing new insights into single enzyme analysis.