Compared with their monometallic counterparts, bimetallic nanoparticles often show enhanced catalytic activity associated with the bimetallic interface. Direct quantitation of catalytic activity at the bimetallic interface is important for understanding the enhancement mechanism, but challenging experimentally. Here using single-molecule super-resolution catalysis imaging in correlation with electron microscopy, we report the first quantitative visualization of enhanced bimetallic activity within single bimetallic nanoparticles. We focus on heteronuclear bimetallic PdAu nanoparticles that present a well-defined Pd–Au bimetallic interface in catalyzing a photodriven fluorogenic disproportionation reaction. Our approach also enables a direct comparison between the bimetallic and monometallic regions within the same nanoparticle. Theoretical calculations further provide insights into the electronic nature of N–O bond activation of the reactant (resazurin) adsorbed on bimetallic sites. Subparticle activity correlation between bimetallic enhancement and monometallic activity suggests that the favorable locations to construct bimetallic sites are those monometallic sites with higher activity, leading to a strategy for making effective bimetallic nanocatalysts. The results highlight the power of super-resolution catalysis imaging in gaining insights that could help improve nanocatalysts.

Detecting and characterizing reaction intermediates is not only important and powerful for elucidating reaction mechanisms but also challenging in general because of the low populations of intermediates in a reaction mixture. Studying surface reaction intermediates in heterogeneous catalysis presents additional challenges, especially the ubiquitous structural heterogeneity among the catalyst particles and the accompanying polydispersion in reaction kinetics. Here we use single-molecule fluorescence microscopy to study two complementary types of Au nanocatalysts—mesoporous-silica-coated Au nanorods (i.e., Au@mSiO2 nanorods) and bare 5.3 nm pseudospherical Au nanoparticles—at the single-particle, single-turnover resolution in catalyzing the oxidative deacetylation of amplex red by H2O2, a synthetically relevant and increasingly important probe reaction. For both nanocatalysts, the distributions of the microscopic reaction time from a single catalyst particle clearly reveal a kinetic intermediate, which is hidden when the data are averaged over many particles or only the time-averaged turnover rates are examined for a single particle. This intermediate is further resolvable by single-turnover kinetics at the subparticle level. Detailed single-molecule kinetic analysis leads to a quantitative reaction mechanism and supports that the intermediate is likely a surface-adsorbed one-electron-oxidized amplex red radical. The quantitation of kinetic parameters further allows for the evaluation of the large reactivity inhomogeneity among the individual nanorods and pseudospherical nanoparticles, and for Au@mSiO2 nanorods, it uncovers their size-dependent reactivity in catalyzing the first one-electron oxidation of amplex red to the radical. Such single-particle, single-molecule kinetic studies are expected to be broadly useful for dissecting reaction kinetics and mechanisms.

Light-driven semiconductor-catalyzed oxidation reactions are of fundamental importance in photocatalysis and photoelectrocatalysis for removing organic contaminants in wastewater, solar energy conversion, and fine chemical synthesis. The underlying reaction mechanism is often unclear because it is difficult to measure directly and specifically the semiconductor-catalyzed reaction rates. For example, an organic molecule could be oxidized “directly” by photogenerated holes that are transported from the semiconductor interior to the semiconductor–electrolyte interface or “indirectly” by photogenerated intermediates (e.g., hydroxyl radical, superoxide anion, or hydrogen peroxide) that are produced at the semiconductor surface in aqueous solution. New experimental approaches that can distinguish these pathways are thus desirable. Here we introduce quantitative single-molecule, single-particle fluorescence imaging to measure the photoelectrocatalytic oxidation rate of a model organic substrate, amplex red, on the surface of individual rutile TiO2 nanorods. Our approach probes the oxidation product selectively before it becomes further degraded (which complicates bulk reaction kinetics measurements) while also avoiding interparticle charge transfer kinetics. By examining the reaction rate scaling relations versus light intensity at fixed potential and versus potential at fixed light intensity, together with the corresponding photocurrent scaling reactions, we demonstrate that amplex red oxidation on a TiO2-nanorod photoanode proceeds via an indirect mechanism.

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ70-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)—a Cu+-responsive MerR-family metalloregulator—modulates RNAP interactions with CueR’s cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a “biased sampling” instead of “dynamic equilibrium shifting” mechanism in regulating transcription initiation; it modulates RNAP’s binding–unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein’s displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

This review discusses the latest advances in using single-molecule microscopy of fluorogenic reactions to examine and understand the spatiotemporal catalytic behaviors of single metal nanoparticles of various shapes including pseudospheres, nanorods, and nanoplates. Real-time single-turnover kinetics reveal size-, catalysis-, and metal-dependent temporal activity fluctuations of single pseudospherical nanoparticles (<20 nm in diameter). These temporal catalytic dynamics can be related to nanoparticles' dynamic surface restructuring whose timescales and energetics can be quantified. Single-molecule super-resolution catalysis imaging further enables the direct quantification of catalytic activities at different surface sites (i.e., ends vs. sides, or corner, edge vs. facet regions) on single pseudo 1-D and 2-D nanocrystals, and uncovers linear and radial activity gradients within the same surface facets. These spatial activity patterns within single nanocrystals can be attributed to the inhomogeneous distributions of low-coordination surface sites, including corner, edge, and defect sites, among which the distribution of defect sites is correlated with the nanocrystals' morphology and growth mechanisms. A brief discussion is given on the extension of the single-molecule imaging approach to catalysis that does not involve fluorescent molecules.

Shape-controlled metal nanocrystals are a new generation of nanoscale catalysts. Depending on their shapes, these nanocrystals exhibit various surface facets, and the assignments of their surface facets have routinely been used to rationalize or predict their catalytic activity in a variety of chemical transformations. Recently we discovered that for 1-dimensional (1D) nanocrystals (Au nanorods), the catalytic activity is not constant along the same side facets of single nanorods but rather differs significantly and further shows a gradient along its length, which we attributed to an underlying gradient of surface defect density resulting from their linear decay in growth rate during synthesis (Nat. Nanotechnol.2012, 7, 237–241). Here we report that this behavior also extends to 2D nanocrystals, even for a different catalytic reaction. By using super-resolution fluorescence microscopy to map out the locations of catalytic events within individual triangular and hexagonal Au nanoplates in correlation with scanning electron microscopy, we find that the catalytic activity within the flat {111} surface facet of a Au nanoplate exhibits a 2D radial gradient from the center toward the edges. We propose that this activity gradient results from a growth-dependent surface defect distribution. We also quantify the site-specific activity at different regions within a nanoplate: The corner regions have the highest activity, followed by the edge regions and then the flat surface facets. These discoveries highlight the spatial complexity of catalytic activity at the nanoscale as well as the interplay amid nanocrystal growth, morphology, and surface defects in determining nanocatalyst properties.

High-throughput and quantitative screening of catalyst activity is crucial for guiding the work cycles of catalyst improvements and optimizations. For nanoparticle catalysts, their inherent heterogeneity makes it desirable to screen them at the single-particle level. Here, we report a single-molecule fluorescence microscopy approach that can screen the activity quantitatively of a large number of catalyst particles in parallel at the single-particle level and with subdiffraction spatial resolution. It can identify directly high activity catalyst particles and resolve subpopulations in mixtures of catalysts. It is readily scalable and broadly applicable to heterogeneous catalysts. Using ensemble measurements to establish activity correlations between different reactions, we further show that this approach can be extended to assess catalysts in reactions that do not involve fluorescent molecules. Coupled with high-throughput catalyst preparation and high-resolution structural/compositional analysis, this screening approach has promise in accelerating the development and discovery of new or better catalysts.Keywords: deacetylation; deoxygenation; heterogeneous catalysis; nitro-reduction; phenol oxidation; single-molecule imaging

Understanding how cells regulate and transport metal ions is an important goal in the field of bioinorganic chemistry, a frontier research area that resides at the interface of chemistry and biology. This Current Topic reviews recent advances from the authors’ group in using single-molecule fluorescence imaging techniques to identify the mechanisms of metal homeostatic proteins, including metalloregulators and metallochaperones. It emphasizes the novel mechanistic insights into how dynamic protein–DNA and protein–protein interactions offer efficient pathways via which MerR-family metalloregulators and copper chaperones can fulfill their functions. This work also summarizes other related single-molecule studies of bioinorganic systems and provides an outlook toward single-molecule imaging of metalloprotein functions in living cells.

Using single-molecule microscopy of fluorogenic reactions we studied Pt nanoparticle catalysis at single-particle, single-turnover resolution for two reactions: one an oxidative N-deacetylation and the other a reductive N-deoxygenation. These Pt nanoparticles show distinct catalytic kinetics in these two reactions: one following noncompetitive reactant adsorption and the other following competitive reactant adsorption. In both reactions, single nanoparticles exhibit temporal activity fluctuations attributable to dominantly spontaneous surface restructuring. Depending on the reaction sequence, single Pt nanoparticles may or may not show activity correlations in catalyzing both reactions, reflecting the structure insensitivity of the N-deacetylation reaction and the structure sensitivity of the N-deoxygenation reaction.

As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers Cu+ to the Wilson’s Disease Protein (WDP) via weak and dynamic protein–protein interactions. WDP contains six homologous metal binding domains (MBDs) connected by flexible linkers, and these MBDs all can receive Cu+ from Hah1. The functional roles of the MBD multiplicity in Cu+ trafficking are not well understood. Building on our previous study of the dynamic interactions between Hah1 and the isolated fourth MBD of WDP, here we study how Hah1 interacts with MBD34, a double-domain WDP construct, using single-molecule fluorescence resonance energy transfer (smFRET) combined with vesicle trapping. By alternating the positions of the smFRET donor and acceptor, we systematically probed Hah1–MBD3, Hah1–MBD4, and MBD3–MBD4 interaction dynamics within the multidomain system. We found that the two interconverting interaction geometries were conserved in both intermolecular Hah1–MBD and intramolecular MBD–MBD interactions. The Hah1–MBD interactions within MBD34 are stabilized by an order of magnitude relative to the isolated single-MBDs, and thermodynamic and kinetic evidence suggest that Hah1 can interact with both MBDs simultaneously. The enhanced interaction stability of Hah1 with the multi-MBD system, the dynamic intramolecular MBD–MBD interactions, and the ability of Hah1 to interact with multiple MBDs simultaneously suggest an efficient and versatile mechanism for the Hah1-to-WDP pathway to transport Cu+.

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu+-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

This tutorial review covers recent developments in using single-molecule fluorescence microscopy to study nanoscale catalysis. The single-molecule approach enables following catalytic and electrocatalytic reactions on nanocatalysts, including metal nanoparticles and carbon nanotubes, at single-reaction temporal resolution and nanometer spatial precision. Real-time, in situ, multiplexed measurements are readily achievable under ambient solution conditions. These studies provide unprecedented insights into catalytic mechanism, reactivity, selectivity, and dynamics in spite of the inhomogeneity and temporal variations of catalyst structures. Prospects, generality, and limitations of the single-molecule fluorescence approach for studying nanocatalysis are also discussed.

Covering: up to 2009

Nanoscale catalysts are important for electrocatalysis, especially in energy conversion as in photoelectrochemical cells and fuel cells. Understanding their reactivity is essential for improving their performances and designing new ones, but challenging due to their inherent structural heterogeneity. This article reviews recent developments in using single-molecule fluorescence microscopy to overcome this challenge and interrogate directly the individuality of nanoscale catalysts. Using electrocatalysis by single-walled carbon nanotubes (SWNTs) as an example, this article discusses how the single-molecule approach dissects the reaction kinetics at single-reaction resolution, unravels the reaction mechanism, and quantifies the reactivity and inhomogeneity of individual SWNT reactive sites, which are imaged to nanometre precision via super-resolution optical imaging. New scientific questions and opportunities are also discussed, as well as the related optical studies of single-molecule and single-nanoparticle electrochemistry.

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Nanoparticles can catalyze many important chemical transformations in organic synthesis, pollutant removal, and energy production. Characterizing their catalytic properties is essential for understanding the fundamental principles governing their activities, but is challenging in ensemble measurements due to their intrinsic heterogeneity from their structural dispersions, heterogeneous surface sites, and surface restructuring dynamics. To remove ensemble averaging, we recently developed a single-particle approach to study the redox catalysis of individual Au-nanoparticles in solution. By detecting the fluorescence of the catalytic product at the single-molecule level, we followed the catalytic turnovers of single Au-nanoparticles in real time at single-turnover resolution. Here we extend our single-nanoparticle studies to examine in detail the activity and heterogeneity of 6 nm spherical Au-nanoparticles. By analyzing the statistical properties of single-particle reaction waiting times across a range of substrate concentrations, we directly determine the distributions of kinetic parameters of individual Au-nanoparticles, including the rate constants and the equilibrium constants of substrate adsorption, and quantify their heterogeneity. Large activity heterogeneity is observed among the Au-nanoparticles in both the catalytic conversion reaction and the product dissociation reaction, which are typically hidden in ensemble-averaged measurements. Analyzing the temporal fluctuation of catalytic activity of individual Au-nanoparticles further reveals that these nanoparticles have two types of surface sites with different catalytic properties—one type-a with lower activity but higher substrate binding affinity, and the other type-b with higher activity but lower substrate binding affinity. Each Au-nanoparticle exhibits type-a behavior at low substrate concentrations and switches to type-b behavior at a higher substrate concentration, and the switching concentration varies largely from one nanoparticle to another. The heterogeneous and dynamic behavior of Au-nanoparticle catalysts highlight the intricate interplay between catalysis, structural dispersion, variable surface sites, and surface restructuring dynamics in nanocatalysis.

Recent experimental advances in single-molecule enzymology stimulated many efforts to develop single-molecule kinetic theories of enzyme catalysis, especially for the classic Michaelis−Menten mechanism. Our group recently studied redox catalysis by single metal nanoparticles at single-turnover resolution. Compared with enzymes, which are homogeneous catalysts and have well-defined active sites, nanoparticles are heterogeneous catalysts and have many different surface sites for catalysis. To provide a theoretical framework to understand nanoparticle catalysis at the single-molecule level, here we formulate in detail the single-molecule kinetic theory of a Langmuir−Hinshelwood mechanism for heterogeneous catalysis, which includes the multitude of surface sites on one nanoparticle. We consider two parallel product dissociation pathways that give complex single-molecule kinetics of the product dissociation reaction. We derive the probability density functions of the stochastic waiting times for both the product formation and the product dissociation reactions and describe their complex behaviors at different kinetic limiting conditions. We also obtain a single-molecule Langmuir−Hinshelwood equation that describes the saturation kinetics of the product formation rate over substrate concentrations and evaluate the randomness parameter of single-turnover waiting times. We further compare the single-molecule kinetics between the Langmuir−Hinshelwood mechanism for heterogeneous catalysis and the Michaelis−Menten mechanism for enzyme catalysis and formulate the modified single-molecule Michaelis−Menten kinetics with multiple product dissociation pathways. In the end, we suggest that the Langmuir−Hinshelwood mechanism is also applicable to describe the single-molecule kinetics of oligomeric enzymes that contain multiple catalytic sites. We expect that these theories will enable quantitative analysis of single-turnover kinetics of heterogeneous and enzyme catalysis and provide a theoretical foundation to understand the catalytic dynamics of nanoparticles and enzymes at the single-molecule level.

In recent years, single-molecule methods have enabled many innovative studies in the life sciences, which generated unprecedented insights into the workings of many macromolecular machineries. Single-molecule studies of bioinorganic systems have been limited, however, even though bioinorganic chemistry represents one of the frontiers in the life sciences. With the hope to stimulate more interest in applying existing and developing new single-molecule methods to address compelling bioinorganic problems, this review discusses a few single-molecule fluorescence approaches that have been or can be employed to study the functions and dynamics of metalloproteins. We focus on their principles, features and generality, possible further bioinorganic applications, and experimental challenges. The fluorescence quenching via energy transfer approach has been used to study the O2-binding of hemocyanin, the redox states of azurin, and the folding dynamics of cytochrome c at the single-molecule level. Possible future applications of this approach to single-molecule studies of metalloenzyme catalysis and metalloprotein folding are discussed. The fluorescence quenching via electron transfer approach can probe the subtle conformational dynamics of proteins, and its possible application to probe metalloprotein structural dynamics is discussed. More examples are presented in using single-molecule fluorescence resonance energy transfer to probe metallochaperone protein interactions and metalloregulator–DNA interactions on a single-molecule basis.A review on single-molecule fluorescence approaches and applications related to bioinorganic chemistry. Approaches discussed include fluorescence resonance energy transfer, fluorescence quenching via energy transfer and via electron transfer. Applications include studies of hemocyanin O2 binding, azurin redox, cytochrome c folding, protein conformational dynamics, metallochaperone–protein interactions, and metalloregulator–DNA interactions.

To maintain normal metal metabolism, bacteria use metal-sensing metalloregulators to control transcription of metal resistance genes. Depending on their metal-binding states, the MerR-family metalloregulators change their interactions with DNA to suppress or activate transcription. To understand their functions fundamentally, we study how CueR, a Cu1+-responsive MerR-family metalloregulator, interacts with DNA, using an engineered DNA Holliday junction (HJ) as a protein-DNA interaction reporter in single-molecule fluorescence resonance energy transfer measurements. By analyzing the single-molecule structural dynamics of the engineered HJ in the presence of various concentrations of both apo- and holo-CueR, we show how CueR interacts with the two conformers of the engineered HJ, forming variable protein-DNA complexes at different protein concentrations and changing the HJ structures. We also show how apo- and holo-CueR differ in their interactions with DNA, and discuss their similarities and differences with other MerR-family metalloregulators. The surprising finding that holo-CueR binds more strongly to DNA than to apo-CueR suggests functional differences among MerR-family metalloregulators, in particular in their mechanisms of switching off gene transcription after activation. The study also corroborates the general applicability of engineered HJs as single-molecule reporters for protein-DNA interactions, which are fundamental processes in gene replication, transcription, recombination, and regulation.

Nanoparticles can catalyze many important chemical transformations in organic synthesis, pollutant removal, and energy production. Characterizing their catalytic properties is essential for understanding the fundamental principles governing their activities, but is challenging in ensemble measurements due to their intrinsic heterogeneity from their structural dispersions, heterogeneous surface sites, and surface restructuring dynamics. To remove ensemble averaging, we recently developed a single-particle approach to study the redox catalysis of individual Au-nanoparticles in solution. By detecting the fluorescence of the catalytic product at the single-molecule level, we followed the catalytic turnovers of single Au-nanoparticles in real time at single-turnover resolution. Here we extend our single-nanoparticle studies to examine in detail the activity and heterogeneity of 6 nm spherical Au-nanoparticles. By analyzing the statistical properties of single-particle reaction waiting times across a range of substrate concentrations, we directly determine the distributions of kinetic parameters of individual Au-nanoparticles, including the rate constants and the equilibrium constants of substrate adsorption, and quantify their heterogeneity. Large activity heterogeneity is observed among the Au-nanoparticles in both the catalytic conversion reaction and the product dissociation reaction, which are typically hidden in ensemble-averaged measurements. Analyzing the temporal fluctuation of catalytic activity of individual Au-nanoparticles further reveals that these nanoparticles have two types of surface sites with different catalytic properties—one type-a with lower activity but higher substrate binding affinity, and the other type-b with higher activity but lower substrate binding affinity. Each Au-nanoparticle exhibits type-a behavior at low substrate concentrations and switches to type-b behavior at a higher substrate concentration, and the switching concentration varies largely from one nanoparticle to another. The heterogeneous and dynamic behavior of Au-nanoparticle catalysts highlight the intricate interplay between catalysis, structural dispersion, variable surface sites, and surface restructuring dynamics in nanocatalysis.

Nanoscale catalysts are important for electrocatalysis, especially in energy conversion as in photoelectrochemical cells and fuel cells. Understanding their reactivity is essential for improving their performances and designing new ones, but challenging due to their inherent structural heterogeneity. This article reviews recent developments in using single-molecule fluorescence microscopy to overcome this challenge and interrogate directly the individuality of nanoscale catalysts. Using electrocatalysis by single-walled carbon nanotubes (SWNTs) as an example, this article discusses how the single-molecule approach dissects the reaction kinetics at single-reaction resolution, unravels the reaction mechanism, and quantifies the reactivity and inhomogeneity of individual SWNT reactive sites, which are imaged to nanometre precision via super-resolution optical imaging. New scientific questions and opportunities are also discussed, as well as the related optical studies of single-molecule and single-nanoparticle electrochemistry.

This tutorial review covers recent developments in using single-molecule fluorescence microscopy to study nanoscale catalysis. The single-molecule approach enables following catalytic and electrocatalytic reactions on nanocatalysts, including metal nanoparticles and carbon nanotubes, at single-reaction temporal resolution and nanometer spatial precision. Real-time, in situ, multiplexed measurements are readily achievable under ambient solution conditions. These studies provide unprecedented insights into catalytic mechanism, reactivity, selectivity, and dynamics in spite of the inhomogeneity and temporal variations of catalyst structures. Prospects, generality, and limitations of the single-molecule fluorescence approach for studying nanocatalysis are also discussed.

This review discusses the latest advances in using single-molecule microscopy of fluorogenic reactions to examine and understand the spatiotemporal catalytic behaviors of single metal nanoparticles of various shapes including pseudospheres, nanorods, and nanoplates. Real-time single-turnover kinetics reveal size-, catalysis-, and metal-dependent temporal activity fluctuations of single pseudospherical nanoparticles (<20 nm in diameter). These temporal catalytic dynamics can be related to nanoparticles' dynamic surface restructuring whose timescales and energetics can be quantified. Single-molecule super-resolution catalysis imaging further enables the direct quantification of catalytic activities at different surface sites (i.e., ends vs. sides, or corner, edge vs. facet regions) on single pseudo 1-D and 2-D nanocrystals, and uncovers linear and radial activity gradients within the same surface facets. These spatial activity patterns within single nanocrystals can be attributed to the inhomogeneous distributions of low-coordination surface sites, including corner, edge, and defect sites, among which the distribution of defect sites is correlated with the nanocrystals' morphology and growth mechanisms. A brief discussion is given on the extension of the single-molecule imaging approach to catalysis that does not involve fluorescent molecules.

Detecting and characterizing reaction intermediates is not only important and powerful for elucidating reaction mechanisms but also challenging in general because of the low populations of intermediates in a reaction mixture. Studying surface reaction intermediates in heterogeneous catalysis presents additional challenges, especially the ubiquitous structural heterogeneity among the catalyst particles and the accompanying polydispersion in reaction kinetics. Here we use single-molecule fluorescence microscopy to study two complementary types of Au nanocatalysts—mesoporous-silica-coated Au nanorods (i.e., Au@mSiO2 nanorods) and bare 5.3 nm pseudospherical Au nanoparticles—at the single-particle, single-turnover resolution in catalyzing the oxidative deacetylation of amplex red by H2O2, a synthetically relevant and increasingly important probe reaction. For both nanocatalysts, the distributions of the microscopic reaction time from a single catalyst particle clearly reveal a kinetic intermediate, which is hidden when the data are averaged over many particles or only the time-averaged turnover rates are examined for a single particle. This intermediate is further resolvable by single-turnover kinetics at the subparticle level. Detailed single-molecule kinetic analysis leads to a quantitative reaction mechanism and supports that the intermediate is likely a surface-adsorbed one-electron-oxidized amplex red radical. The quantitation of kinetic parameters further allows for the evaluation of the large reactivity inhomogeneity among the individual nanorods and pseudospherical nanoparticles, and for Au@mSiO2 nanorods, it uncovers their size-dependent reactivity in catalyzing the first one-electron oxidation of amplex red to the radical. Such single-particle, single-molecule kinetic studies are expected to be broadly useful for dissecting reaction kinetics and mechanisms.