Enzymes have catalytic turnovers. The field of nanozyme endeavors to engineer nanomaterials as enzyme mimics. However, a discrepancy in the definition of "nanozyme concentration" has led to an unrealistic calculation of nanozyme catalytic turnovers. To date, most of the reported works have considered either the atomic concentration or nanoparticle (NP) concentration as nanozyme concentration. These assumptions can lead to a significant under- or overestimation of the catalytic activity of nanozymes. In this article, we review some classic nanozymes including Fe3O4, CeO2, and gold nanoparticles (AuNPs) with a focus on the reported catalytic activities. We argue that only the surface atoms should be considered as nanozyme active sites, and then the turnover numbers and rates were recalculated based on the surface atoms. According to the calculations, the catalytic turnover of peroxidase Fe3O4 NPs is validated. AuNPs are self-limited when performing glucose-oxidase like activity, but they are also true catalysts. For CeO2 NPs, a self-limited behavior is observed for both oxidase- and phosphatase-like activities due to the adsorption of reaction products. Moreover, the catalytic activity of single-atom nanozymes is discussed. Finally, a few suggestions for future research are proposed.

Nanozyme Catalytic Turnover and Self-Limited Reactions.

Nanopipettes provide a promising confined space that enables advances in single-molecule analysis, and their unique conical tubular structure is also suitable for single-cell analysis. In this work, functionalized-nanopore-based single-entity electrochemistry (SEE) analysis tools were developed for the label-free monitoring of single-molecule glycoprotein-boronate affinity interaction for the first time, and immunoglobulin G (IgG, one of the important biomarkers for many diseases such as COVID-19 and cancers) was employed as the model glycoprotein. The principle of this method is based on a single glycoprotein molecule passing through 4-mercaptophenylboronic acid (4-MPBA)-modified nanopipettes under a bias voltage and in the meantime interacting with the boronate group from modified 4-MPBA. This translocation and affinity interaction process can generate distinguishable current blockade signals. Based on the statistical analysis of these signals, the equilibrium association constant (κa) of single-molecule glycoprotein-boronate affinity interaction was obtained. The results show that the κa of IgG in the confined nanopore at the single-molecule level is much larger than that measured in the open system at the ensemble level, which is possibly due to the enhanced multivalent synergistic binding in the restricted space. Moreover, the functionalized-nanopore-based SEE analysis tools were further applied for the label-free detection of IgG, and the results indicate that our method has potential application value for the detection of glycoproteins in real samples, which also paves way for the single-cell analysis of glycoproteins.

Nanopore-Based Single-Entity Electrochemistry for the Label-Free Monitoring of Single-Molecule Glycoprotein-Boronate Affinity Interaction and Its Sensing Application.

Single-particle collisions have made many achievements in basic research, but challenges still exist due to their low collision frequency and selectivity in complex samples. In this work, we developed an "on-off-on" strategy based on Pt nanoparticles (PtNPs) that catalyze N2H4 collision signals on the surface of carbon ultramicroelectrodes and established a new method for the detection of miRNA21 with high selectivity and sensitivity. PtNPs catalyze the reduction of N2H4 on the surface of carbon ultramicroelectrodes to generate a stepped collision signal, which is in the "on" state. The single-stranded DNA paired with miRNA21 is coupled with PtNPs to form the complex DNA/PtNPs. Because PtNPs are covered by DNA, the electrocatalytic collision of N2H4 oxidation is inhibited. At this time, the signal is in the "off" state. When miRNA21 is added, the strong complementary pairing between miRNA21 and DNA destroys the electrostatic adsorption of DNA/PtNP conjugates and restores the electrocatalytic performance of PtNPs, and the signal is in the "on" state again. Based on this, a new method for detecting miRNA21 was established. It provides a new way for small-molecule sensing and has a wide range of applications in electroanalysis, electrocatalysis, and biosensing.

Stochastic Collision Electrochemistry from Single Pt Nanoparticles: Electrocatalytic Amplification and MicroRNA Sensing.

Revealing the electrocatalytic features of single redox enzyme is significant to both fundamental biological processes and practical catalysis and sensing applications. Herein, we directly reveal the electrocatalytic current from a single enzyme inside the carbon nanopipettes via electrochemical collision strategies, based on the increased activity at nanoscale confinement. Besides the staircase current steps from surface blockage, discrete H2O2 oxidation and reduction current transients catalyzed by a single enzyme are also displayed and analyzed. The carbon nanopipette would increase the catalytic activities of enzymes and lead to a detectable current response, thus opening a new way to investigate the fundamental enzymatic mechanisms at the single enzyme level.

Nanoconfined Electrochemical Collision and Catalysis of Single Enzyme inside Carbon Nanopipettes.

Nanoparticles (NPs) are of considerable interest, owing to their enhanced catalytic activity and optical properties. Ionic liquids (ILs) are employed as a medium to prepare small (<20 nm in diameter) and relatively low dispersity (typically, ±1–2 nm) NPs. While ensemble techniques (e. g., UV/Vis) can provide a great deal of information, increasingly, single-entity electrochemistry (SEE) has been used to provide physical insight into individual NP catalysis and dynamics. Herein, Pt NP impact current spikes for particles prepared in the IL phase and based on electrocatalytic amplification (ECA) of the borohydride oxidation reaction (BOR) were recorded during i-t measurements. Additionally, direct oxidative LiBH4 nanocrystal (NC) impact signals were observed herein for the first time. By quantifying the charge transferred during the stochastic collision events, the radius of the NCs (rNC) was calculated (∼5.5 nm) and compares favorably with TEM images. Critically, small additions (1 % v/v) of a molecular, polar organic solvent (tetrahydrofuran, THF) disrupt the ILs supramolecular fluidic nature, which enhances NC impact frequency and the aggregation of Pt NPs. 

Single entity electrochemical detection of as‐prepared metallic and dielectric nanoparticle stochastic impacts in a phosphonium ionic liquid

Enzymes encapsulated in metal-organic frameworks (enzyme@MOFs), as a promising immobilized enzyme, were investigated for intrinsic catalytic activities at the single entity level via a stochastic collision electrochemical technique. Zeolitic imidazolate frameworks with amorphous (aZIF-8) and crystalline (ZIF-8) structures were chosen as model MOFs to encapsulate glucose oxidase (GOx). We carried out single enzyme@MOF nanoparticle (NP) collision experiments using the carbon ultramicroelectrode (CUME), which demonstrated that the catalytic activity of GOx@ZIF-8 NPs was much less than GOx@aZIF-8 NPs. Meanwhile, the kcat and TON per GOx in aZIF-8 NPs were obtained, revealing the intrinsic catalytic activity of GOx in aZIF-8 NPs at the single entity level. This strategy is the first approach for investigating enzyme@MOFs at a single entity level, which can not only broaden the horizons of single entity electrochemistry (SEE) but also provide further insights into research on electrochemistry, catalysis, and nanocomposites.

Jiahao Du

Papers

Intrinsic Catalytic Activities from Single Enzyme@Metal-Organic Frameworks by Using a Stochastic Collision Electrochemical Technique.