The peroxidase‐like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2O2 into .OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2O2. We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2O2 limitations under physiological conditions. As a proof of concept, aptamer‐functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule “APGH”, which can be activated by bacteria‐secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2O2 for the in‐situ generation of .OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 μm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

Peroxidase‐mimicking nanozymes such as Fe3O4 nanoparticles are promising substitutes for natural enzymes like horseradish peroxidase. However, most such nanozymes work efficiently only in acidic conditions. In this work, the influence of various liposomes on nanozyme activity was studied. By introducing negatively charged liposomes, peroxidase‐mimicking nanozymes achieved oxidation of 3,3′,5,5′‐tetramethylbenzidine (TMB) in neutral and even alkaline conditions, although the activity towards anionic 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) was inhibited. The Fe3O4 nanoparticles adsorbed on the liposomes without disrupting membrane integrity as confirmed by fluorescence quenching, dye leakage assays, and cryo‐electron microscopy. Stabilization of the blue‐colored oxidized products of TMB by electrostatic interactions was believed to be the reason for the enhanced activity. This work has introduced lipids to nanozyme research, and it also has practically important applications for using nanozymes at neutral pH, such as the detection of hydrogen peroxide and glucose.