Fluorescent nanozyme-based biosensors: Classification, catalytic mechanisms, and emerging applications.

Fluorescent nanozymes have increasingly drawn attention as a unique type of nanozyme-like materials, whose value lies not in a single characteristic but in the combination of catalytic performance and fluorescence within one material system. Traditional nanozyme sensing systems are based on colorimetric detection methods that, although simple in operation, suffer from poor sensitivity and are easily influenced by coloured or turbid samples. The fluorescence approach addresses most of these issues, enabling sensitive detection, real-time monitoring of the signal, and suitability for microscale measurements-all of which play an important role in transferring research laboratory experiments into diagnostic tests at the point of care. In addition to sensitivity, fluorescent nanozymes still preserve such practical characteristics as high stability, long-term shelf life, cost-effective production, and tunability of surface chemistry for modulation of catalytic and fluorescent properties. In this review, recent progress in the field of fluorescent nanozyme-based biosensors has been analysed with an emphasis placed equally on accomplishments and limitations in the area. The classification of fluorescent nanozymes is explored based on different criteria, such as compositional, dimensional, catalytic and fluorescent types, as one single classification approach would be inadequate for describing the existing diversity of fluorescent nanozyme-based sensing platforms. A key aspect that needs to be considered is the catalytic reactions utilised to generate or enhance fluorescence signals, namely, peroxidase-like, oxidase-like and Fenton/Fenton-like mechanisms, with inherent mechanistic benefits and practical challenges associated with their utilisation that are not always addressed properly in experimental work. Potential applications of fluorescent nanozymes have also been discussed, particularly about performance that is relevant for each application domain, rather than just analytical capabilities. Concurrently, various issues are discussed in depth, including substrate specificity, that is often inadequate in most systems, insufficient understanding of catalysis mechanisms in many materials, fluorescence quenching due to biological matrices, and a lack of standardised testing protocols for nanozyme assays. The challenges mentioned above not only pose technical problems but also act as major hurdles for the further development of nanozyme technology to achieve translational validity. This review concludes by discussing strategies for developing highly selective and sensitive fluorescent nanozymes suitable for practical biosensing applications.