Engineering Single-Atom Nanozymes for Catalytic Biomedical Applications

Nanomaterials with enzyme-mimicking properties, coined as nanozymes, are a promising alternative to natural enzymes owing to their remarkable advantages, such as high stability, easy preparation, and favorable catalytic performance. Recently, with the rapid development of nanotechnology and characterization techniques, single atom nanozymes (SAzymes) with atomically dispersed active sites, well-defined electronic and geometric structures, tunable coordination environment, and maximum metal atom utilization are developed and exploited. With superior catalytic performance and selectivity, SAzymes have made impressive progress in biomedical applications and are expected to bridge the gap between artificial nanozymes and natural enzymes. Herein, the recent advances in SAzyme preparation methods, catalytic mechanisms, and biomedical applications are systematically summarized. Their biomedical applications in cancer therapy, oxidative stress cytoprotection, antibacterial therapy, and biosensing are discussed in depth. Furthermore, to appreciate these advances, the main challenges, and prospects for the future development of SAzymes are also outlined and highlighted in this review.     

Activity Regulating Strategies of Nanozymes for Biomedical Applications

On accounts of the advantages of inherent high stability, ease of preparation and superior catalytic activities, nanozymes have attracted tremendous potential in diverse biomedical applications as alternatives to natural enzymes. Optimizing the activity of nanozymes is significant for widening and boosting the applications into practical level. As the research of the catalytic activity regulation strategies of nanozymes is boosting, it is essential to timely review, summarize, and analyze the advances in structure–activity relationships for further inspiring ingenious research into this prosperous area. Herein, the activity regulation methods of nanozymes in the recent 5 years are systematically summarized, including size and morphology, doping, vacancy, surface modification, and hybridization, followed by a discussion of the latest biomedical applications consisting of biosensing, antibacterial, and tumor therapy. Finally, the challenges and opportunities in this rapidly developing field is presented for inspiring more and more research into this infant yet promising area.     

Structure and activity of nanozymes: Inspirations for de novo design of nanozymes

Nanozymes, nanomaterials with enzyme-like activities, are becoming powerful competitors and potential substitutes for natural enzymes because of their excellent performance, including design from scratch, controllable activity, and environmental resistance. In recent years, various nanozymes have been discovered or designed, and gradually applied to molecular detection, biomedical treatment and environmental management. Nevertheless, nanozymes are often regarded as fascinating and confusing black boxes as their catalytic mechanisms remain largely indistinct. Interestingly, recent researches have shed light into these black boxes. It appears that the enzymatic activities of nanozymes are closely related to their size, surface lattice, surface modification and composition, etc. Some regular structure–activity relationships have been elucidated in recent reports. In this review, we systematically summarized the studies on the structure–activity relationship of nanozymes in recent years, aiming to illustrate the catalytic mechanism of nanozymes and clarify the key factors regulating their behavior, so as to provide ideas and inspiration for the de novo design of nanozymes.     

Single Iron Site Nanozyme for Ultrasensitive Glucose Detection

Nanomaterials with enzyme‐mimicking characteristics have engaged great awareness in various fields owing to their comparative low cost, high stability, and large‐scale preparation. However, the wide application of nanozymes is seriously restricted by the relatively low catalytic activity and poor specificity, primarily because of the inhomogeneous catalytic sites and unclear catalytic mechanisms. Herein, a support‐sacrificed strategy is demonstrated to prepare a single iron site nanozyme (Fe SSN) dispersed on the porous N‐doped carbon. With well‐defined coordination structure and high density of active sites, the Fe SSN performs prominent peroxidase‐like activity by efficiently activating H₂O₂ into hydroxyl radical (?OH) species. Furthermore, the Fe SSN is applied in colorimetric detection of glucose through a multienzyme biocatalytic cascade platform. Moreover, a low‐cost integrated agarose‐based hydrogel colorimetric biosensor is designed and successfully achieves the visualization evaluation and quantitative detection of glucose. This work expands the application of single‐site catalysts in the fields of nanozyme‐based biosensors and personal biomedical diagnosis.     

Recent Advances in Nanozyme Research

As a new generation of artificial enzymes, nanozymes have the advantages of high catalytic activity, good stability, low cost, and other unique properties of nanomaterials. Due to their wide range of potential applications, they have become an emerging field bridging nanotechnology and biology, attracting researchers in various fields to design and synthesize highly catalytically active nanozymes. However, the thorough understanding of experimental phenomena and the mechanisms beneath practical applications of nanozymes limits their rapid development. Herein, the progress of experimental and computational research of nanozymes on two issues over the past decade is briefly reviewed: (1) experimental development of new nanozymes mimicking different types of enzymes. This covers their structures and applications ranging from biosensing and bioimaging to therapeutics and environmental protection. (2) The catalytic mechanism proposed by experimental and theoretical study. The challenges and future directions of computational research in this field are also discussed.     

Defect engineering in nanozymes

Due to the high stability, various synthesis strategies, low cost, and tunable performance, nanozymes have gained much attention as the replacement of natural enzymes. To widen the application, highly active, specific, and robust nanozymes are in need. Recently, defects in nanomaterials have been verified to play a significant role in enhancing catalytic performances. Therefore, the marriage between defect engineering and nanozymes is expected to spark new possibilities. In this review, defect engineering strategies in nanozymes are summarized and the close relationships between defects and nanozyme properties are highlighted. It is anticipated that defect engineering will bring new opportunities to the evolving field of nanozymes.     

Electrochemical biosensor for detection of adenosine based on structure-switching aptamer and amplification with reporter probe DNA modified Au nanoparticles

In the present study, an electrochemical sensing strategy for highly sensitive detection of small molecules was developed based on switching structures of aptamers from DNA/DNA duplex to DNA/target complex. A gold electrode was first modified with gold nanoparticles (AuNPs), and thiolated capture probe was immobilized onto the electrode via sulfur-gold affinity. Then, a "sandwich-type" strategy was employed, which involved a linker DNA containing antiadenosine aptamer sequence and reporter DNA loaded on AuNPs. In the presence of adenosine, the aptamer part bound with adenosine and folded to the complex structure. As a result, the reporter probes together with AuNPs were released into solution and reduced a decrease in peak current. With the enhancement effect of AuNPs, a detection limit as low as 1.8 x 10(-10) M for adenosine was achieved. The sensor exhibited excellent selectivity against other nucleosides and could be used to detect adenosine from real human serum samples.     

Structural Analysis and Intrinsic Enzyme Mimicking Activities of Ligand-Free PtAg Nanoalloys

Nanozymes are nanomaterials with biocatalytic properties under physiological conditions and are one class of artificial enzymes to overcome the high cost and low stability of natural enzymes. However, surface ligands on nanomaterials will decrease the catalytic activity of the nanozymes by blocking the active sites. To address this limitation, ligand-free PtAg nanoclusters (NCs) are synthesized and applied as nanozymes for various enzyme-mimicking reactions. By taking advantage of the mutual interaction of zeolitic imidazolate frameworks (ZIF-8) and Pt precursors, a good dispersion of PtAg bimetal NCs with a diameter of 1.78 ± 0.1 nm is achieved with ZIF-8 as a template. The incorporation of PtAgNCs in the voids of ZIF-8 is confirmed with structural analysis using the atomic pair-distribution function and powder X-ray diffraction. Importantly, the PtAgNCs present good catalytic activity for various enzyme-mimicking reactions, including peroxidase-/catalase- and oxidase-like reactions. Further, this work compares the catalytic activity between PtAg NCs and PtAg nanoparticles with different compositions and finds that these two nanozymes present a converse dependency of Ag-loading on their activity. This study contributes to the field of nanozymes and presents a potential option to prepare ligand-free bimetal biocatalysts with sizes in the nanocluster regime.     

General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.     

Metal-Based Nanozymes with Multienzyme-Like Activities as Therapeutic Candidates: Applications,Mechanisms, and Optimization Strategy

Most nanozymes in development for medical applications only exhibit single-enzyme-like activity, and are thus limited by insufficient catalytic activity and dysfunctionality in complex pathological microenvironments. To overcome the impediments of limited substrate availabilities and concentrations, some metal-based nanozymes may mimic two or more activities of natural enzymes to catalyze cascade reactions or to catalyze multiple substrates simultaneously, thereby amplifying catalysis. Metal-based nanozymes with multienzyme-like activities (MNMs) may adapt to dissimilar catalytic conditions to exert different enzyme-like effects. These multienzyme-like activities can synergize to realize “self-provision of the substrate,” in which upstream catalysts produce substrates for downstream catalytic reactions to overcome the limitation of insufficient substrates in the microenvironment. Consequently, MNMs exert more potent antitumor, antibacterial, and anti-inflammatory effects in preclinical models. This review summarizes the cellular effects and underlying mechanisms of MNMs. Their potential medical utility and optimization strategy from the perspective of clinical requirements are also discussed, with the aim to provide a theoretical reference for the design, development, and therapeutic application of their catalytic effects.     

Immobilization of Au nanoparticles on Au electrode for hydrazine detection: Using thiolated single-stranded DNA as a linker

In this article, we report a method for effective immobilization of Au nanoparticles (AuNPs) on thiolated single-stranded DNA (thiol-ssDNA) modified Au electrode (AuE) surface via coordination interactions between the nitrogen atoms of DNA bases and AuNPs. It suggests that the resultant AuNP-immobilized AuE exhibits notable catalytic performance for hydrazine oxidation and the loading of AuNPs on the AuE surface and hence the effective catalytic area can be tuned by the immobilization time of thiol-ssDNA and adsorption time of AuNPs. This hydrazine sensor has a fast amperometric response time of less than 4 s. The linear range and detection limit are estimated to be from 0.1 mM to 100 mM (r = 0.998) and 0.56 μM at a signal-to-noise ratio of 3, respectively.     

纳米酶:对抗细菌的新策略

由细菌引发的相关疾病和环境污染等问题引起了人们的高度重视,同时随着抗生素的使用,细菌的耐药性逐渐增强,人们急需开发新型抗菌剂。诸如溶菌酶、髓过氧化物酶等天然酶具有显著的抗菌能力,但其作为抗菌剂存在保质期短、生产成本高等缺点,很难大规模生产。因此,人们正探索寻求天然酶的替代品。纳米酶是新一代人工模拟酶,兼具纳米材料独特的理化性质和类酶催化活性,因其结构稳定、生产成本低等优点受到广泛关注。本文综述了纳米酶的抗菌机制和近期抗菌纳米酶的主要研究进展,并对未来该领域的研究进行展望。     

Surface modification of nanozymes

Nanoparticles and proteins are similar in a number of aspects,and using nanoparticles to mimic the catalytic function of enzymes is an interesting yet challenging task.Impressive developments have been made over the past two decades on this front.The term nanozyme was coined to refer to nanoparticlebased enzyme mimics.To date,many different types of nanozymes have been reported to catalyze a broad range of reactions for chemical,analytical,and biomedical applications.Since chemical reactions happen mainly on the surface of nanozymes,an interesting aspect for investigation is surface modification.In this review,we summarize three types of nanozyme materials catalyzing various reactions with a focus on their surface chemistry.For metal oxides,cerium oxide and iron oxide are discussed as they are the most extensively studied.Then,gold nanoparticles and graphene oxide are reviewed to represent metallic and carbon nanomaterials,respectively.Types of modifications include ions,small molecules,and polymers mainly by physisorption,while in a few cases,covalent modifications were also employed.The functional aspect of such modification is to improve catalytic activity,substrate specificity,and stability.Future perspectives of this field are speculated at the end of this review.     

Negatively charged gold nanoparticles as an intrinsic peroxidase mimic and their applications in the oxidation of dopamine

Artificial inorganic peroxidase is of great interest due to its intrinsic advantages over natural counterpart. Negatively charged gold nanoparticles (AuNPs) were discovered to function like a peroxidase in the present study. Two AuNPs in different size were prepared and characterized by TEM, and assayed for peroxidase activity. Its catalytic activity was found to follow Michaelis–Menten kinetics. The negative surface charge notably improves the affinity toward a substrate TMB, proved by the determined kinetic parameters. The particles expressed optimal catalytic activity under mildly acidic environment and resistance to elevated temperature and increased concentration of sodium azide. The origin of the activity was investigated tentatively. Hydrogen peroxide-treated AuNPs exhibited an enhanced activity. EDTA temporarily blocked the activity partially, while thiol groups permanently blocked the activity completely. Tests imply that it is the surface Au⁺ that provides the activity. The successful oxidation of dopamine, as an instance, under the action of AuNPs as a peroxidase was conducted. These studies would lead to a wide range of potential applications.     

Engineering the pH‐Responsive Catalytic Behavior of AuNPs by DNA

Noble metal nanoparticles have attracted much interest in the heterogeneous catalysis. Particularly, efficient manipulation of the responsive catalytic properties of the metal nanoparticles is an interesting topic. In this work, a simple and efficient strategy is developed to regulate the pH‐responsive catalytic activities of glucose oxidase (GOx)‐mimicking gold nanoparticles (AuNPs). Four DNA strands (regulating strands) that differ slightly in sequences are used to interact non‐covalently with citrate‐capped AuNPs, resulting in markedly distinct pH‐dependent catalytic behavior of AuNPs. This is ascribed to the characteristic pH‐induced conformational change of the DNA strands that leads to the different adsorption capability to the NPs surface, as demonstrated by pH‐CD profiles of the respective DNA molecules. The pH‐dependent catalysis of AuNPs is also encoded with structural information of the double‐stranded DNA (including regulating strands and their complementary strands) that has conformation resistant or responsive to pH change. As a result, the catalysis can be programmed into an AND gate, a XNOR gate or a NOT gate, using pH and complementary strand as the inputs, the nanoparticle activity as the output and the regulating strands as the programs. This work can be expanded by engineering the catalytic behavior of noble metal nanoparticles to respond smartly to a variety of environmental stimuli, such as metal ions or light wavelengths. These results may provide insight into understanding ligand‐regulated nanometallic catalysis.     

Direct detection of β-agonists by use of gold nanoparticle-based colorimetric assays

β-Agonists fed to animals for human consumption pose a serious threat to human health. Fast, broad-spectrum detection methods are needed for on-site screening of various types of β-agonists from animal feeds, meats, and animal body fluids. We developed a colorimetric assay that uses gold nanoparticle (AuNP) plasmon absorption to realize quick detection of β-agonists from liquid samples. β-Agonists showed the capability of directly reducing HAuCl(4) into atomic gold, which involved oxidation of the amine or phenol group on the benzene ring of the β-agonists. The resulting atomic gold formed AuNPs spontaneously, which had strong plasmon absorption at 528 nm. The linear relationship between the concentrations of β-agonists and the AuNPs plasmon absorbance granted quantitative determination of β-agonists in solution. The AuNPs colorimetric assay showed different sensitivities toward β-agonists with different substituent groups on the aromatic ring. β-Agonists with phenol groups had a lower limit of quantitation (LOQ) than those with amine groups. High-resolution transmission electron microscopy (TEM) images revealed the sizes of the AuNPs were in the range 15-25 nm, while X-ray energy-dispersive spectroscopic data suggested the smaller particles observed in TEM with lower contrast may be salt particles from the buffer solution. The developed colorimetric assay can potentially be used for the detection of β-agonists and their analogues from serum, urine, and other liquid samples in the presence of interference from common antibiotics and glucose.     

Nano‐Gold as Artificial Enzymes: Hidden Talents

Creating artificial enzymes that mimic the complexity and function of natural systems has been a great challenge for the past two decades. In this Progress Report, the focus is on recently discovered “hidden talents” of gold nanomaterials in artificial enzymes, including mimicking of nuclease, esterase, silicatein, glucose oxidase, peroxidase, catalase, and superoxide dismutase. These unexpected enzyme‐like activities can be ascribed to nano‐gold itself or the functional groups present on surrounding monolayer. Along with introducing the mechanisms of the various enzyme‐like activities, the design and development of gold‐based biomimetic catalysts, the search for efficient modulators, and their potential applications in bionics, biosensing, and biomedical sciences are highlighted. Eventually, it is expected that the rapidly growing interest in gold‐based nanozymes will certainly fuel the excitement and stimulate research in this highly active field.     

High‐Performance Integrated Enzyme Cascade Bioplatform Based on Protein–BiPt Nanochain@Graphene Oxide Hybrid Guided One‐Pot Self‐Assembly Strategy

Nanozymes provide new opportunities for facilitating next generation artificial enzyme cascade platforms. However, the fabrication of high‐performance integrated artificial enzyme cascade (IAEC) bioplatforms based on nanozymes remains a great challenge. A facile and effective self‐assembly strategy for constructing an IAEC system based on an inorganic/protein hybrid nanozyme, β‐casein‐BiPt nanochain@GO (CA‐BiPtNC@GO) nanohybrid with unique physicochemical surface properties and hierarchical structures, is introduced here. Due to the synergetic effect of the protein, GO, and Bi³⁺, the hybrid acts as highly adaptable building blocks to immobilize natural enzymes directly and noncovalently without the loss of enzyme activity. Simultaneously, the CA‐BiPtNC@GO nanohybrid exhibits outstanding peroxidase‐mimicking activity and works well with natural oxidases, resulting in prominent activity in catalyzing cascade reactions. As a result, the proposed IAEC bioplatform exhibits excellent sensitivity with a wide linear range of 0.5 × 10^‐6 to 100 × 10^‐6 m and a detection limit of 0.05 × 10^‐6 m for glucose. Meticulous design of ingenious hierarchically nanostructured nanozymes with unique physicochemical surface properties can provide a facile and efficient way to immobilize and stabilize nature enzymes using self‐assembly instead of chemical processes, and fill the gap in developing robust nanozyme–triggered IAEC systems with applications in the environment, sensing, and synthetic biology.     

Dynamically Programmed Switchable DNA Hydrogels Based on a DNA Circuit Mechanism

Biological stimuli‐responsive DNA hydrogels have attracted much attention in the field of medical engineering owing to their unique phase transitions from gel to sol through cleavage of DNA cross‐linking points in response to specific biomolecular inputs. In this paper, a new class of biological stimuli‐responsive DNA hydrogels with a dynamically programmed DNA system that relies on a DNA circuit system through cascading toehold‐mediated DNA displacement reactions is constructed, allowing the catalytic cleavage of cross‐linking points and main chains in response to an appropriate DNA input. The dynamically programmed DNA hydrogels exhibit a significant sharp phase transition from gel to sol in comparison to another DNA hydrogel showing noncatalytic cleavage of cross‐linking points due to synchronization of the catalytic cleavage of cross‐linking points and the main chains. Further, the sol–gel phase transitions of the DNA hydrogels in response to the DNA input are easily tunable by changing the cross‐linking density. Additionally, with a structure‐switching aptamer, DNA hydrogels encapsulating PEGylated gold nanoparticles can be used as enzyme‐free signal amplifiers for the colorimetric detection of adenosine 5′‐triphosphate (ATP); this detection system provides simplicity and higher sensitivity (limit of detection: 5.6 × 10^?6 m at 30 min) compared to other DNA hydrogel‐based ATP detection systems.     

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.