Robust Single-Molecule Enzyme Nanocapsules for Biosensing with Significantly Improved Biosensor Stability

The present study demonstrates the use of highly stable single-molecule enzyme nanocapsules (SMENs) instead of traditional native enzyme as biorecognition element in enzyme-based biosensors. The main purpose of this study is to resolve the major obstacle and challenge in the biosensor field, i.e., the poor stability of enzyme-based biosensors, including thermal stability, organic solvent tolerance, long-term operational stability, etc. Highly active and robust SMENs of glucose oxidase (GOx, as a model enzyme) were synthesized (nGOx) using an in situ polymerization strategy in an aqueous environment. The particle-size distribution, transmission electron microscopic (TEM) images, and UV-vis spectral characterization revealed the formation of a thin polymer layer around each enzyme molecule. The polymer shell effectively stabilized the GOx enzyme core while enabling rapid substrate transportation, resulting in a new class of biocatalytic nanocapsules. Multiple covalent attachments between a thin polymer layer and an enzyme molecule strengthened the encapsulated GOx molecule. Encapsulation created a favorable microenvironment to avoid any structural dissociation at high temperature and helped to retain essential water during the organic solvent operation. The present work reports a study implementing nGOx SMENs as highly stable nano(bio)sensors for point-of-care diagnostic applications. Prepared nGOx SMENs manifested significantly improved thermal stability (even at 65 degrees C) and organic solvent tolerance without any compromise in biocatalytic activity. For example, the native GOx-based biosensor lost its catalytic activity for glucose after 4 h of incubation at high temperature (65 degrees C), while the nGOx/N-CNTs-Chi/GCE nano(bio)sensor maintained similar to 56% of its original catalytic activity for glucose oxidation. The proposed SMENs-based nano(bio)sensors with robust stability in variable working environment could promote the development and applications of biosensors in point-of care diagnostics, biomedical detection, wearable devices, implantable equipment, and biofuel cells.