Generalizing single-atom-catalysis fluorescence correlation spectroscopy (SAC-FCS) beyond two-site-class resolution and a single fluorogenic probe chemistry

Single-atom-catalysis fluorescence correlation spectroscopy (SAC-FCS) was introduced in 2023 as the first autocorrelation-based single-molecule method to identify and kinetically resolve unequal active sites in iron-nitrogen single-atom catalysts, but it resolved only two kinetically distinct site classes, relied on a simplified two-site renewal model, and depended on one fluorogenic probe chemistry (o-phenylenediamine oxidation to 2,3-diaminophenazine) under narrow measurement conditions. The open problem, stated by the method's authors as their improvement agenda, is to generalize the method: resolve three or more kinetically distinct single-atom site classes, extend the autocorrelation framework beyond the simplified two-site reaction and catalyst models, and demonstrate more universal probe chemistries beyond the original fluorogenic pair. Contemporaneous reviews of single-molecule fluorescence imaging independently identify single-atom catalysts, and fluorogenic probes for electrocatalysis products, as an underexplored frontier, but they propose activity mapping rather than autocorrelation-based multi-site kinetic resolution and do not extend SAC-FCS itself; transfer to other transformations such as electrocatalysis therefore remains motivating context rather than part of the source's stated agenda. No published work is known to extend the method as of 2026, leaving this generalization frontier open.

Background

Single-atom catalysts (SACs) are heterogeneous catalysts in which isolated metal atoms are anchored to a support — for example, iron atoms coordinated by four, five, or six nitrogens in a carbon matrix (denoted FeN4, FeN5, FeN6 coordination). Because different coordination environments coexist in the same sample and can differ enormously in catalytic activity, the genuinely active sites are concealed by conventional ensemble-averaged measurements, which mix contributions from all site types. Single-molecule fluorescence microscopy addresses this by watching individual catalytic turnovers: a non-fluorescent substrate molecule is converted into a fluorescent product at the catalyst surface, and each emitted fluorescence burst marks one completed reaction event at one site. Fluorescence correlation spectroscopy (FCS) adds a statistical layer: it computes the autocorrelation of the fluorescence-intensity fluctuations — how likely a photon is to be followed by another after a lag time $t$ — and fits this decay with exponential terms whose time constants and amplitudes reveal kinetically distinct catalytic site populations and their rate constants, using renewal theory (the statistical description of random waiting times between events) and Michaelis-Menten-type stochastic kinetics (the standard substrate-binding and product-forming scheme with rate constants $k_1$ , $k_{-1}$ , $k_2$ ). In 2022, Liu et al. demonstrated single-molecule fluorescence at single-turnover resolution on individual platinum single atoms, establishing feasibility but analyzing only one site type. In 2023, Huang et al. developed SAC-FCS: immobilizing iron phthalocyanine (FePc, a model with well-defined FeN4 sites) and practical FeN $_x$ /C single-atom catalysts on microscope slides, they used the oxidase-like oxidation of o-phenylenediamine (OPD, non-fluorescent) to fluorescent 2,3-diaminophenazine (DAP) with O $_2$ , recorded photon arrival times with a time resolution far finer than the microsecond-order spacing between successive catalytic turnovers in the measurement area, and fit the autocorrelation to decompose the site population. They resolved exactly two kinetically distinct site classes in FeN $_x$ /C — an active FeN5 class (~26% of sites, rate constants up to 182- and 1463-fold higher than the other sites in FeN $_x$ /C and the FeN4 site in FePc) and a slower FeN4/6 class — and showed these single-site kinetics differ by tens of times from ensemble averages. The method, however, was a bounded proof of concept: the autocorrelation derivation assumed two kinetic classes with stochastic renewal behavior and one specific fluorescent-product redox scheme; the measurement required ~1 nM substrate and a narrow fluorescence-intensity window ( $\sim 5\times 10^{4}$ counts/s, avoiding triplet-state artifacts); and the analysis was tuned to the OPD-to-DAP probe. The authors state the limitation explicitly: 'the SAC-FCS method has limitations, such as the relative low resolution and sensitivity to different categories of single-atom sites, as well as simplified reaction and catalyst models; the accuracy can be improved by utilizing more universal probe molecules and optimizing the autocorrelation analysis.' Independently, a contemporaneous review of single-molecule fluorescence imaging in catalysis (Shen, Rackers and Sadtler, 2023) concluded that correlating single-molecule activity maps with the chemical nature of active sites 'remains a critical challenge' and named single-atom catalysts — and fluorogenic probes for electrocatalysis products such as CO, nitrite, and ammonia — as the underexplored frontier.

Problem Statement

Generalize single-atom-catalysis fluorescence correlation spectroscopy (SAC-FCS) from its demonstrated proof of concept — two kinetically distinct site classes on FeN $_x$ /C single-atom catalysts, a simplified two-site stochastic-renewal autocorrelation model, and the single OPD-to-DAP fluorogenic probe under oxidase-like conditions — into a general quantitative method for identifying and resolving heterogeneous active sites in single-atom catalysts. Concretely, as stated in the source's limitation agenda: (1) resolve three or more kinetically distinct single-atom site categories, with their populations and rate constants, on single-atom catalysts — a practical catalyst such as FeN $_x$ /C and/or model catalysts of known composition such as phthalocyanine mixtures — where resolution means that the classes are kinetically distinguished in the autocorrelation and corroborated by independent structure-sensitive evidence on the same samples, rather than being an artifact of an over-parameterized multi-exponential fit; (2) extend the autocorrelation derivation beyond the two-site-class renewal and single-fluorescent-product redox scheme, yielding generalized autocorrelation expressions for less simplified reaction and catalyst networks together with an optimized analysis whose site-class populations and rate constants remain identifiable from finite photon-arrival statistics; and (3) demonstrate the method with more universal fluorogenic probe molecules beyond o-phenylenediamine oxidation. These components serve one objective — making single-molecule, ensemble-free counting and kinetic characterization of unequal single-atom active sites a general tool — and a resolution would jointly demonstrate site-class resolution, model generality, and probe scope beyond the original restrictions.

References

Liang Huang, Qiong Liu, Weiwei Wu, Ge Gao, Xiliang Zheng, Jin Wang, Shaojun Dong (2023). Identifying the active sites in unequal iron-nitrogen single-atom catalysts. Nature Communications 14:5594. DOI: 10.1038/s41467-023-41311-9. https://doi.org/10.1038/s41467-023-41311-9
Xiaodong Liu, Xin Ge, Jing Cao, Yi Xiao, Yan Wang, Wei Zhang, Ping Song, Weilin Xu (2022). Revealing the catalytic kinetics and dynamics of individual Pt atoms at the single-molecule level. Proceedings of the National Academy of Sciences USA 119(14):e2114639119. DOI: 10.1073/pnas.2114639119. https://doi.org/10.1073/pnas.2114639119
Meikun Shen, William H. Rackers, Bryce Sadtler (2023). Getting the Most Out of Fluorogenic Probes: Challenges and Opportunities in Using Single-Molecule Fluorescence to Image Electro- and Photocatalysis. Chemical & Biomedical Imaging 1(8):692-715. DOI: 10.1021/cbmi.3c00075. https://doi.org/10.1021/cbmi.3c00075
Eslam M. Hamed, Mostafa M. Elsaady, Sam F. Y. Li (2025). Single-Atom Nanozymes in Analytical Chemistry: Opportunities and Challenges. Analytical Chemistry 97(48):26313-26325. DOI: 10.1021/acs.analchem.5c05006. https://doi.org/10.1021/acs.analchem.5c05006
Koki Sekioka, Nazanin Mosleh, Richard Hailstone, Xiangcheng Sun (2026). Single-Molecule Fluorescence Reveals Catalytic Heterogeneity and Active Species in Copper Nanocatalyzed Click Reactions. ChemRxiv preprint (posted 2026-07-08). DOI: 10.26434/chemrxiv.15005790/v1. https://doi.org/10.26434/chemrxiv.15005790/v1