Emerging two-dimensional material nanozymes for theranostic nanomedicine

Nanomaterials-based artificial enzymes (nanozymes) with valuable enzyme-like catalytic properties have been booming during the past few years. Promoted by the advances in biological medicine and nanotechnology, nanozymes possess the potential to serve as an emerging agent for biosensing, immunoassays, detection and diagnosis, catalytic therapeutics, and other applications in the biomedicine field. Two-dimensional (2D) nanomaterials are of considerable interest in biomedical applications due to their ultrathin layered structure and unique physiochemical properties. Inspired by the diversified catalytic performance of 2D nanomaterials, scientists extensively have developed 2D materials as bioactive nanozymes for theranostic nanomedicine. Here, recent advances in enzyme-like 2D nanomaterials design and construction are comprehensively presented. Additionally, we exhibit that, with the synergistic effect of catalytic activities and desirable physicochemical performances, 2D nanozymes can serve as versatile platforms with extensive applications from target detection to in vivo theranostic. It is believed that such promising alternatives towards natural enzymes will be of vital significance in the field of nanotechnology and biomedicine.


INTRODUCTION
Enzyme is a natural biocatalyst related to many physiological processes in living organisms known for its high efficiency and selectivity, which is one of most delicate natural products. However, the intrinsic drawbacks of natural enzymes such as fabrication and preservation difficulties, high cost, poor recyclability and laborious preparation, impede their practice applications in biomedical engineering (Lin et al. 2014). Inspired by biomimetic engineering, Ronald Breslow innovatively proposed the notion of artificial enzymes aiming to simulate the catalysis mechanism and process of natural enzyme by using synthetic materials (Breslow and Overman 1970). The rapid growth of researches in nanotechnology brings new opportunities to bionic enzymes owing to the nano-size effect and satisfactory properties of nanomaterials. As early as 2004, Scrimin and co-workers pioneered the term "nanozyme" and opened the door to the favorable integration of nanomaterials and enzymology (Manea et al. 2004).
Nanozyme is used as an umbrella term for nanomaterials or nanostructures with natural enzymelike properties not only representing a certain kind of material, but a variety of materials combinations (Gao et al. 2007;Jiang et al. 2019;Korschelt et al. 2018;Liang and Yan 2019;Manea et al. 2004;Wei and Wang 2013). Up to now, a large number of nanozymes have been constructed to exert on-demand highly and tunable catalytic activity in various fields of chemistry, biology and medicine, by promoting the target chemical reaction (Giljohann et al. 2010;Huang et al. 2011;Kim et al. 2009;Lu et al. 2009;Sun et al. 2012;Wulff and Liu 2012;Xu et al. 2019). The interdisciplinary concept of nanocatalytic medicine (NCM) was introduced by our group to describe the catalytic therapeutic strategies that the chemical reactions can be triggered in situ to generate or eliminate the corresponding species, and further treating various diseases (Lin et al. 2018b;Yang et al. 2019). Notably, nanozymes completely correspond with the emerging perspectives of theranostic practices. Typically, it is well known that the overexpressed reactive oxygen species (ROS) like hydrogen peroxide (H 2 O 2 ), hydroxyl radical (·OH), singlet oxygen ( 1 O 2 ) and superoxide anion (O 2 − ) contribute to oxidative damage-related diseases (Vander Heiden et al. 2009;Zhang et al. 2021;. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in human body are of vital importance in the regulation of ROS contents (Pisoschi and Pop 2015;Sies 2015). By mimicking the ROS scavenging capability of natural enzymes, nanozymes featuring the similar functions can clear the excessive reactive oxygen species, and thus suppressing inflammation Qin et al. 2020;Wu et al. 2021), preventing neurological injury and aging-related diseases Kwon et al. 2018;Singh et al. 2017), etc.
Exploring the structure-activity relationship towards nanomaterials and/or nanostructures has been a longheld goal for materials science. Novoselov and Geim et al. reported the first experimental prototype of twodimensional (2D) single-elemental atom crystalgraphene in 2004, opening a new era of twodimensional materials (Novoselov et al. 2004). Since ultrathin 2D topologic confinement, electrons in nanosheet could only enable to move freely on the nonnano scale inside planar dimension, which endows 2D materials with unique electrical characteristics and excellent physicochemical properties (Gopal et al. 2020;Zhang 2015). In recent, 2D nanomaterials and their potential applications in catalysis and electric industry have been widely discovered, arousing explorations of which to serve as nanozymes ). In addition, owing to desirable biocompatibility and biodegradability, 2D nanomaterials hold great potential in the field of biomedicine Wang and Cheng 2019). Based on exceptional catalytic activity and potentials in future clinical applications, 2D nanomaterials have been reported to exhibit stimuliresponsive and multi-enzyme mimetic activities (Chen et al. 2018;Chong et al. 2016;Gao et al. 2020;Liu et al. 2021;Ren et al. 2019;Shan et al. 2019;Song et al. 2010;Yim et al. 2018). In general, the designable 2D nanomaterials represent a next-generation artificial nanozymes for the unique applications in some specific niches.
Despite the rapid development of 2D nanomaterials, few studies have specifically summarized its applications as nanozymes in the field of biomedicine. To highlight the value of 2D materials in the fields of biology and medicine, this review discusses the endeavors on developing promising enzyme-like twodimensional nanomedicine. We also introduce the latest multifunctional 2D nanozymes and present how they transform their own enzyme-like activities under the activation of the external environment to enhance the therapeutic efficacy for different diseases. Finally, we come up with future perspectives of nanozyme development with a view to improving the discipline system of "nanozymology" and promoting their practical applications (Fig. 1).

TWO-DIMENSIONAL NANOMATERIALS AS NANOZYME FOR BIOMEDICAL APPLICATIONS
2D nanomaterials such as graphene, MXene, transition metal dichalcogenides (TMDs), black phosphorus, layered double hydroxides (LDH), and layered metal oxides, are of increasing attention for their intriguing .. . A n t i-in � la m m a t io n A n t ii n f e c t i o n  Fig. 1 Schematic illustration of emerging two-dimensional material nanozymes for theranostic nanomedicine physiochemical properties (Lin et al. 2018a;Zhang 2015). Surprisingly, the past decades have witnessed growing interest in developing advanced 2D nanomaterials featuring the mimicking potential of natural enzyme functions due to their high catalytic performances. In this section, we will introduce design concepts for multifunctional 2D nanomaterials of enzyme-like activity and their biomedical applications.

Graphene and derivatives-based nanozyme
Graphene is the most representative 2D nanomaterials endowed with unique electronic, optical and other physicochemical properties, which has been widely investigated in nanomedicine and biomedical engineering (Geim and Novoselov 2007). Duo to the lack of catalytic activity, pristine graphene is inadequate to be an artificial candidate material as nanozyme (Guo and Dong 2011). Nevertheless, it provides new opportunities like a few carbon-based nanomaterials including graphene oxide (Song et al. 2010), fullerene (Kroto et al. 1985), carbon nanotubes (Cui et al. 2011) and carbon nanodots (Chong et al. 2016) have been found to possess enzyme-like activity. In specific, upon doping or modification, graphenebased nanomaterials can exhibit biocatalytic functions similar to oxidase, catalase, superoxide dismutase, etc. (Boutorine et al. 1995).
In 2010, Song et al. reported that carboxyl-modified graphene oxides could catalyze the reduction of H 2 O 2 , which demonstrated intrinsic peroxidase-like activity derived from carboxyl-modified graphene oxides noted as GO-COOH (Song et al. 2010). To take advantage of the enzyme-like activity, they designed a colorimetric method for H 2 O 2 and glucose detection. In the presence of H 2 O 2 and peroxidase substrate 3,3',5,5'tetramethylbenzidine (TMB), GO-COOH can change TMB absorbance at 652 nm by a blue chromogenic reaction ( Fig. 2A, 2B). Given that the catalytic activity of GO-COOH is relative with H 2 O 2 concentrations, it is feasible for GO-COOH to detect H 2 O 2 . In addition, by detecting hydrogen peroxide, the product of the glucose oxidase catalyzed reaction, a simple and rapid detection of glucose was successfully developed. The detection activity of GO-COOH was regulated by acidic pH, reaction temperature, and hydrogen peroxide concentration, and the optimal conditions were of pH 4.0, 35.8 °C, and 150 mmol/L after systematic assessment. It could be found that the catalytic activity of GO-COOH towards TMB superior to that of natural enzyme horseradish peroxidase (HRP) under the optimum conditions. As the Fig. 2C shows, the GO-COOH nanozyme presents highly selective and sensitive detection performance of glucose in buffer solution, dilute blood and fruit juice. H 2 O 2 detection is of great significance in the fields of biology, medicine, environmental protection, chemical industry and food engineering. Inspired by the participation of H 2 O 2 in the catalytic reaction of peroxidase, the concentration of H 2 O 2 can be detected selectively by the color reaction of peroxidase-like nanozymes with certain colorimetric substrates. More importantly, the designing concept is not limited to H 2 O 2 peroxide detection. Such study provides a new insight into applying nanozymes in targeting detection and diagnosis.
In order to further promote the applications of graphene, scientists have developed various methods to convert 2D graphene to 0D graphene quantum dots (GQDs). As a typical structural derivative of graphene, quantum dots are of strong photoluminescence property which is essential to several applications like bioimaging or photovoltaics. Additionally, the photoluminescent GQDs could react with either electron acceptor or donor, and subsequently be quenched, which may serve as an emerging free radical scavenging nanoagent. For instance, Chong and coworkers discovered that graphene quantum dots have ROS-regulation function that GQDs could perform superoxide dismutase-like activity to downgrade ROS levels, while generating ROS to produce phototoxicity upon exposure to blue light ( Fig. 2D) (Chong et al. 2016). This exceptional Janus-like ROS-regulated performance simultaneously enables GQDs with oxidative stress relieving capacity and antibacterial or anticancer efficacy. Based on desired antioxidant activity, they explored the effectiveness of GQDs protecting cells from oxidative damage in vitro and in vivo. The cell experiments indicated that the GQDs remarkably improved the survival rate of H 2 O 2 and Xray damage cells (Fig. 2E, 2F). Furthermore, they found that under the exposure of light, GQDs showed completely opposite effect of ROS regulation performance that GQDs elicit phototoxicity under the 405 nm laser irradiation by generating ROS and further inducing cell apoptosis (Fig. 2G). What's more, GQDs could accelerate the inactivation of anti-oxidase in body. This research developed a light-activatable nanozyme and demonstrated the artificial nanomaterials of diverse bio-functions through conditional activation. Since most 2D nanomaterials are known to feature photoelectric characteristics, it is of great significance to develop stimuli-responsive smart nanomedicine based on diverse 2D nanomaterials.

Two-dimensional (2D) transition metal oxides (TMOs) -based nanozymes
During the past few years, transition metal oxides (TMOs) have received great attention and spurred a 2D material-based nanozymes

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range of research efforts. 2D TMOs nanosheets with specific mixed valence, desirable conductivity property, abundant redox combinations, and thermal stability have attracted current interest in various applications such as photoelectric catalysis, sensors, and electrochemical energy storage (Haque et al. 2017;Lee et al. 2018).
Vanadium is a transition metal. It is widely believed that vanadium pentoxide (V 2 O 5 ) is the toxic compound, which is an oxidant and a common component in PM 2.5 (Dill et al. 2004). Interestingly, a great deal of novel properties is emerging as materials are being decreased to nanoscale. V 2 O 5 materials at nanoscale show completely opposite characteristics. For example, a biocompatible nanozyme based on V 2 O 5 nanowires with remarkable glutathione peroxidase (GPX)-like activity was developed for performing ROS scavenging and serving as cytoprotectant against harmful oxidative  (Chong et al. 2016) damage (Vernekar et al. 2014).
Recently, a few investigations have reported that reactive oxygen species (ROS) levels are associated with the replication of human immunodeficiency virus (HIV-1) after infection. Cellular antioxidant machinery has been demonstrated to contribute to the acceleration of HIV-1 reactivation during HIV-1 latency (Diaz et al. 2019;Shytaj et al. 2020). In order to neutralize ROS in HIV-1 infected cells and inhibit viral reactivation, Singh et al. used V 2 O 5 nanosheets (Vs) as glutathione peroxidaselike nanozyme, which is the pioneering therapeutic strategy against HIV infection (Fig. 3A) (Singh et al. 2021). In 2018, Ghosh et al. found that the enzymemimetic activity of V 2 O 5 was dependent on the reactivity of V=O bond and exposed facets on the surface rather than the morphology (Ghosh et al. 2018). A comparison of activities and toxicities of V 2 O 5 in three different morphologies, i.e., nanowires, nanosheets, and ultrathin nanosheets was conducted by Singh and co-workers. As a result, they found that ultrathin V 2 O 5 nanosheets were selective toward H 2 O 2 with high efficiency and the most secure, which also confirmed the unique superiority of 2D nanomaterials in biomedicine applications. As shown in Fig. 3B, the viral RNA levels from Vs treatment groups were obviously lower than the groups of ARVs (antiretrovirals) alone at Day 21. Meanwhile, V 2 O 5 nanosheets could also suppress viral reactivation stimulated by prostratin and bolster the antiviral capacity of immune cells. It is a great discovery that oxidative stress which changes redox signaling promotes the reactivation and multiplication of HIV-1, revealing the relationship between redox metabolism and disease outcomes. These advances together offer a novel therapeutic potential for the HIV-1 adjuvant therapy of diverse antioxidants especially nanozyme. The satisfactory glutathione peroxidase (GPX)-like activity of V 2 O 5 nanosheets may encourage new applications to treat other critical illness with redox imbalance.

Black phosphorus nanosheet-based nanozymes
Black phosphorus nanosheet (BP NS) marks an omnipotent, rising-star nanomaterial with unique properties. Remarkably, BP is also a promising candidate in the multidisciplinary biomedical field due to its good biocompatibility and excellent biodegradation. In the past few years, BP exhibited considerable photothermal therapy (PTT) and photodynamic therapy (PDT) efficacies (Wang et al. 2015). In recent, researchers have been focusing on the enzyme-like activity of BP, owing to its prior chemical reactivity with ROS. This finding introduces BP as a new species of nanozyme for anti-oxidant related diseases therapeutics.
As stated, acute kidney injury (AKI) as a severe illness leading to loss of renal function is largely caused by drugs administration in chemotherapy. In the process of the renal excretion of chemotherapeutic drugs, massive ROS generate and damage renal cells. In order to relieve AKI and protect the renal function, Hou et al. prepared a black phosphorus-based anti-oxidative nanoagent for kidney targeting and ROS neutralizing (Fig. 3C)  . It has been reported that flake-like DNA frameworks have ability to kidney targeting drug delivery (Jiang et al. 2018). The same geometrical framework endows BP NSs with passive targeting to kidney, and the biocompatible nanosheets can degrade into nontoxic phosphorus oxides and/or phosphates. Most importantly, BP NSs display fascinating performance without any sophisticated modifications which facilitates their ultimate clinical translation potential. The blood biochemical indexes of renal function including serum creatinine (CRE) and blood urea nitrogen (BUN) elucidated that the BP NSs are an effective therapeutic antioxidant as a SOD mimic for AKI treatment (Fig. 3D, 3E). Notably, the outstanding biocompatibility and biodegradability of BP NSs promote the further translational research on such a promising 2D nanozyme for AKI treatments. Furthermore, the in vivo imaging diagnosis shows great value in real-time monitoring for AKI . 2D nanomaterials-based contrast agents are also expected to provide multiple applications in diagnosticimaging fields, which will allow innovative theranostics in AKI.
Growing evidence suggests that the elevation of transition-metal ions is the main cause of many neurodegenerative diseases (Barnham and Bush 2014;Jun and Saxena 2007). Except for ROS scavenging, BP NSs may also aid in the regulation of metal ions such as Cu 2+ for neurodegenerative disorder treatment. For example, Chen et al. used BP NSs as a neurodegenerative disorder therapeutic agent which can cross the blood-brain barrier (BBB) and capture excess redoxactive Cu 2+ (Chen et al. 2018). Notably, their work exhibited a synergistic therapeutic strategy that BP nanosheets could effectively enhance the blood brain barrier penetration due to local hyperthermia taking advantage of the high photothermal-conversion efficiency (Fig. 3F). Subsequently, BP nanosheets as neuronal cells protector enables to downgrade the cellular Cu 2+ level and eliminate ROS. Thus, it is believed that black phosphorus nanosheet will be applied as a SOD-like nanozyme which may perform multifunctional nanodrugs rather than just conduct ROS scavenging. 2D material-based nanozymes

2D transition metal chalcogenides (TMCs) nanosheet-based nanozyme
2D transition metal chalcogenides (TMCs) containing transition metal atoms and sulfur atoms are of great interest in different fields especially energy storage, catalysis, electroluminescent devices, bioimaging and nanomedicine (Manzeli et al. 2017). It is novel but highly attractive that 2D TMCs nanomaterials possess  (Singh et al. 2021). C Experimental illustration of black phosphorus nanosheets for acute kidney injury therapy. D, E Blood analysis of mice from different groups. Reproduced with permission . F Photographs and the NIR fluorescence imaging of brains after various treatments (1. Cy5-PEG-BP; 2. Cy5-PEG + NIR; 3. Cy5-PEG-BP + NIR). Reproduced with permission (Chen et al. 2018) the ability to scavenge ROS and/or reactive nitrogen species (RNS) via reducing free radicals similar to superoxide dismutase and nitrate reductase (NR). The intrinsic connection between 2D nanomaterials and natural enzymes has opened the door for providing new strategy of free radical scavenging and ROS or RNS-related disease therapeutic modalities.
As a typical paradigm, a biocompatible enzyme-like nanomedicine based on 2D TMCs nanosheets with effective antioxidative activity was developed for sepsis treatment (Fig. 4A) (Yim et al. 2020). In particular, the functionalized TMCs could reduce cellular ROS and RNS without disrupting ROS-producing enzymes. The results showed that, upon stimulation with lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4), the inflammatory bone marrow-derived macrophages (BMDMs) generated considerable mitochondrial ROS, and WS 2 nanosheet exhibited superior ROS scavenging ability in contrast to that of other types of TMCs (Fig. 4B). As shown in Fig. 4C, the survival fractions of bacteremia mouse model dramatically declined, while that of mice with WS 2 nanosheets treatment is up to 90%. Moreover, ultrathin thickness of the 2D nanosheet endows them easy excretion out of the body. Such excellent therapeutic effect and biocompatibility of 2D TMC nanoagent facilitate their versatile biomedical applications, especially in the treatment of oxidative stress-related inflammatory diseases.

MXene nanosheet-based nanozyme
MXenes are a new class of 2D layered nanomaterials, where M stands for the transition metal, and X is carbon and/or nitrogen. In recent years, MXenes with appealing properties such as photothermal conversion, rich surface functional groups, good hydrophilicity and other biological effects, have made great progresses in the field of biomedical applications (Lin et al. 2018a). A recent report showed that 2D MXenes could react with free radicals, for which it can exert the effects of ROS scavenging like superoxide dismutase and catalase (CAT) .
To explore new biologic roles of MXenes, Ren et al. developed 2D Nb 2 C MXene as a radioprotectant for avoiding ionizing radiation (IR)-induced injury (Fig. 4D) (Ren et al. 2019). It is an unneglectable problem that ionizing radiation (IR) lead to serious health hazards such as acute/chronic radiation syndrome and leukemia. However, suffering from disappointing protective efficiencies and the severe side effects, current radioprotectors do very little to avert IR-induced multiple tissue injuries. MXenes are of great potential in prevention ionizing radiation with higher efficiency and biocompatibility. The CCK-8 assay exhibited that the survival rate of 3T3/A31 cells with the incubation of Nb 2 C-PVP was much higher than that without protection (Fig. 4E). They found that the pretreatment with Nb 2 C-PVP yielded a 100% survival rate of mice after γ-irradiation which evidencing a highly efficient nanozyme for attenuating IR-induced injury in vivo (Fig. 4F). The protective effect is much better than that of other nano-radioprotector like cysteine-protected MoS 2 nanodots of 79% , and Fe@C nanoshields of 90% . Furthermore, they proposed the radicalclearance mechanism of Nb 2 C via DFT calculations. It could be found that [Nb 2 C] site is the favorable adsorption site for ·OH. After continuous attack of ·OH, the hydroxyl groups on the surface can be dehydrated, forming oxygenic nanosheet of NbO x species. This study paves a new avenue for developing MXenes in the field of mimicking natural enzymes for ROS quenching.

2D metal-organic framework (MOF) nanosheetbased nanozyme
As a novel layered nanomaterial, 2D metal-organic framework (MOF) nanosheets come into spotlight owing to the abundant active sites on their surface or pore structure in contrast to traditional 3D MOF materials. Metalloporphyrins have already proven high similarity of their microstructure together with natural enzymes (Feng et al. 2012). Therefore, metalloporphyrin frameworks have been designed and established as biological oxidase-mimicking nanozymes for the catalytic activation of molecular oxygen.
In 2016, Wang et al. synthesized ultrathin bimetallic 2D Co-TCPP(Fe) ((Fe(III) tetra(4-carboxyphenyl) porphins chloride) nanosheets as a promising biosensor for H 2 O 2 detection (Fig. 5A)  . It is common knowledge that the iron-porphyrin derivative exhibits similar effects of hemoglobin. Inspired of the hemeprotein-like activity, researchers designed an electrochemical detection built on Co-TCPP(Fe). As expected, the glassy carbon electrode (GC)/(Co-TCPP(Fe)) n (n = 1-6) electrodes exhibit noticeable reduction peak elucidating the reduction of H 2 O 2 , suggesting the hemeprotein-mimic activity. These results indicated that the catalytic activity of electrode exhibit a strong dependence of H 2 O 2 concentration, showing linear relationships between the H 2 O 2 concentration and current response with H 2 O 2 concentration ranging from 0.4 × 10 −6 mol/L to 50 × 10 −6 mol/L. Furthermore, this Co-TCPP(Fe)-enabled nanozyme biosensor can effectively offer the real-time detection of H 2 O 2 secreted from living cells (Fig. 5B).

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Likewise, their group successfully mimicked enzyme triggering cascade reactions by synergistic cooperation of Au nanoparticles (NPs) and 2D Cu-TCPP(Fe) nanosheets (Huang et al. 2017). In this work, they elaborately prepared Au NPs/Cu-TCPP(Fe) hybrid nanosheets in which AuNPs function as oxidase-like nanozyme, while Cu-TCPP(Fe) is employed as peroxidase-mimic nanozyme (Fig. 5C). They constructed an artificial enzymatic cascade reaction system. Firstly, the hybrid nanozyme can first catalyze glucose to generate hydrogen peroxide. Secondly, the product H 2 O 2 can oxidize TMB to produce a blue-color reaction in the presence of peroxidase-like nanozyme. As depicted in Fig. 5D and 5E, a stronger light absorption at 652 nm demonstrated that the coupled nanozyme system is capable of initiating the TMB oxidation, and the results proved the occurrence of biomimetic cascade catalytic reaction. This study verified that the artificial enzymatic cascade reactions are of great application potential by integrating diverse nanomaterials with enzyme-like properties.

Other 2D families nanosheet-based nanozyme
In addition to the 2D nanomaterials with enzyme-like characteristics mentioned above, there are numerous 2D materials used as nanozymes for catalytic medicinerelated treatments. Niobium dieseline (NbSe 2 ) is a kind of 2D transition metal selenides (TMSs) which holds great promise for electrical devices by virtue of its excellent superconductivity. In 2020, Miao et al. pioneered the application of DNA-capped NbSe 2 as nanozyme in reactive oxygen and nitrogen species (RONS) elimination (Fig. 6A) ). As

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discussed above, RONS signaling molecules are inflammatory signaling molecule which can induce the secretion of inflammatory cytokines and then exacerbate inflammation. It is highly expected that NbSe 2 nanosheets possess the reaction activity with polar molecules, suggesting that the potential of nanosheets as anti-inflammatory nanodrug. As shown in Fig. 6B, NbSe 2 exhibits evident efficacy in antioxidative protection, while H 2 O 2 leads to massive cell apoptosis. Furthermore, these NbSe 2 nanosheets significantly down-regulated the inflammatory cytokines level in rear thigh inflammation mice, suggesting the excellent anti-inflammatory activity in vivo. Moreover, under the 808 nm laser irradiation, the NbSe 2 nanosheets exhibited favorable photothermal tumor eradication and PA imaging contrast enhancement function.
Dehydrogenase (e.g., malate dehydrogenase, NADH dehydrogenase and succinic dehydrogenase) is a class of vital enzymes that mediate oxidation-reduction biochemical reactions and play a key role in tricarboxylic acid (TCA) cycle (Spinelli et al. 2017). With the in-depth studies of disease mechanism, dehydrogenase has been found to associated with immune response and the progression of a great variety of diseases. For example, dehydrogenase may generate excessive ROS and cause oxidative stress in mitochondria, which triggers the inflammatory reaction. A representative study by Gao et al. firstly presented that SnSe nanosheets feature dehydrogenase-like activity, proving a novel type of nanozyme for efficient bio-catalysis (Gao et al. 2020). Colorimetry-based detection was employed to investigate the catalytic activities of SnSe nanosheets. Once receiving transferred hydrogen, the corresponding assay kits will display dramatic color reaction (Fig. 6C). As a result, SnSe exhibited strong ability of dehydrogenation in comparison with other  . C The evaluation of dehydrogenase-like activity of different nanosheets, i.e., CdSe, SnS, SnSe. Reproduced with permission (Gao et al. 2020). D Schematic of NIR-II responsive Cu 2 MoS 4 nanosheets of dual enzyme-like activities. E The photographs of MDR S. aureus-infected mice after different treatments. Reproduced with permission (Shan et al. 2020) similar 2D TMSs, indicating that the Sn elements exist the high catalytic effect in combination with selenium components. Given that SnSe nanosheets displayed enhanced and universal catalytic activity (>75%) in diverse reaction without the assistance of co-enzymes, it is highly expected that SnSe nanozyme will be further exploited for biomedical practices.
In the past decade, various nanomaterials as antibacterial agents have attracted considerable interests, which promote the exploration of potential antibacterial strategies that exploit different properties of materials (Xin et al. 2019). Catalytic medicine based on nanozymes paves a new way for bacterial infections. Aiming at multidrug-resistant (MDR) bacteria, Shan et al. proposed a second near-infrared window (NIR-II) light responsive nanozyme to combat multiple drug resistance (MDR) Escherichia coli (E. coli) and MDR Staphylococcus aureus (S. aureus) using Cu 2 MoS 4 nanoplates (CMS NPs) as an oxidase-and peroxidaselike catalyst whose activity can be modulated by NIR-II laser irradiation (Fig. 6D) (Shan et al. 2020). Cu 2 MoS 4 (CMS) is a neotype ternary layered TMC featuring high photothermal conversion efficiency (37.8%) and the catalytic ROS generation ability. More importantly, they found that the photothermal effect for hyperthermia facilitates ROS production to kill bacteria. Finally, a significant abscess and severe ulceration were identified in control groups showing the infection gradually aggravated without CMS NPs treatment (Fig. 6E).

CONCLUSIONS AND PERSPECTIVES
2D nanomaterials show great promise as substitutes of natural enzymes due to their intrinsic catalytic activity.
Excitedly, the superior catalytic properties of nanozyme fit neatly to the catalytic strategy for many kinds of diseases treatment. This review briefly overviews recent advances in the design and development of nanozyme based on 2D materials focusing particularly on their biological applications including detection, anti-inflammation and anti-bacteria (Table 1).
During the past few years, there have been hundreds of literatures focused on nanozymes and a number of nanomaterials have been developed as nanozyme. Nevertheless, the area of 2D nanomaterials as nanozymes is still in its infancy. Compared to other non-2D nanozymes, 2D nanomaterials have their fascinating advantages thanking to the unique layered structure.
(1) The high specific surface areas enable the 2D nanomaterials with a high density of surface active sites, which is beneficial to enzyme-like catalytic reactions.
(2) Traditional inorganic enzyme-like nanomaterials have the unavoidable drawbacks such as poor chemical/ thermal stabilities, single functionality and poor degradability. Excitedly, 2D nanomaterials possess desirable characteristics like facile modification and functionalization, tunable morphology/structure, plasticity, multivalent interactions, satisfactory biodegradation, and relatively high physiological stability, which endow 2D nanomaterials with great potential for further clinical applications.
(3) The unique inherent optoelectronic properties of 2D nanomaterials facilitate the implementation of stimuli-responsive multifunctional nanodrugs.
As a burgeoning field, the exact concept of nanozyme has not be put forward and it is urgent to establish evaluation standards for enzyme-like catalytic activity of a variety of nanozymes. Meanwhile, the catalytic

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mechanisms have to be comprehensively explored to allow more designs over the enzyme-like catalyst. Although extensive researches have demonstrated that SOD-like nanozymes are effective in treating ROSrelated diseases, the mechanisms of treatment always elude explanation. It is urgent to thoroughly investigate the biological phenomena basing on molecular biology before applying nanozyme-drugs into human body. Moreover, when it comes to nanomedicine in vivo applications, biocompatibility and biosafety are the eternal theme. Comprehensive and long-term bio-safety assessment are indispensable for 2D nanozyme in future clinical applications. Enzyme is a huge family, and thousands of enzymes have been found in the human body. In addition to several enzymes that have been already studied, the development of nanozymes mimicking more types of natural enzymes is needed, which may be a new opportunity to explore and treat intractable diseases. For 2D nanomaterials, we can take advantage of their other unique physicochemical properties to catalyze reactions more than just redox type reactions. It is believed that conditional-activated smart nanozyme that can convert its functions depending upon different diseases will be a hot topic in nanomedicine field. Given that, we believe that 2D nanomaterials will be a huge part of natural enzyme alternatives offering new opportunities for novel nanozymes with a board of applications in detection, diagnostics and catalytic therapy.