Site Engineering of Single-Atom Nanozymes: Designing Across Intrinsic Activity, Density, and Accessibility for Biomedical Applications

Conspectus Nanozymes have garnered substantial interest due to their intrinsic enzyme-like catalytic activities and their capacity to circumvent several inherent drawbacks of natural enzymes, including laborious purification, high production costs, stringent reaction conditions, and poor operational stability. These merits render nanozymes promising candidates for a wide array of applications, ranging from colorimetric analysis and the prevention of drug-resistant bacterial infections to tumor therapy. Despite the rapid expansion of the field and the development of thousands of nanozyme systems, only a limited subset exhibits catalytic efficacies on par with those of their natural counterparts. This performance gap has significantly restricted their broader practical deployment. The rational design of highly active nanozymes necessitates a comprehensive understanding of active-site engineering, encompassing three interrelated pillars: the structure–activity relationship of the catalytic site, site density, and site accessibility. In this context, single-atom nanozymes (SAzymes) offer an ideal platform to tackle these challenges, benefiting from maximal atom-utilization efficacy, atomically dispersed and structurally homogeneous active centers, and readily tunable electronic configurations. Considerable progress has been made in elucidating structure–activity relationships at the single-site level through coordination engineering, dimensionality regulation, functional-group modification, spin-state modulation, and related strategies. However, site density and site accessibility, which are equally important for determining overall catalytic output, have received comparatively less attention. In this Account, we summarize our recent endeavors, alongside representative studies from the literature, toward the rational design and construction of high-performance SAzymes and their applications across diverse fields. We begin with a brief overview of nanozyme development, followed by a focused discussion on site engineering strategies for the de novo synthesis of SAzymes. Emphasis is placed on three key optimization axes: enhancing the intrinsic activity of individual active sites, increasing site density, and improving the accessibility of catalytic centers. These three parameters thus address three fundamental questions: how active each site is, how many sites are present, and how many of them can participate in the reaction. We then discuss strategies for standardizing the quantification of accessible active-site density in SAzyme systems, focusing on two representative techniques: low-temperature CO adsorption combined with temperature-programmed desorption and nitrite adsorption followed by electrochemical reductive stripping. We critically compare their respective strengths, limitations, and application scopes. We then summarize recent progress in site engineering of SAzymes for biomedical applications, including colorimetric sensing, antibacterial therapy against drug-resistant pathogens, tumor therapy, and anti-inflammatory treatment. Finally, we highlight future research directions, including enhancement of catalytic activity and specificity, development of nonredox enzyme-mimetic systems, design of biodegradable supports, and the integration of emerging data-driven methodologies. We believe that optimizing the structure–activity relationship, site density, and site accessibility together is the way to close the performance gap between SAzymes and natural enzymes and to make them truly useful in practice.