盧劍天 鄒金輝 趙博霖 張玉微

（廣州大學(xué)化學(xué)化工學(xué)院， 廣州 510006）

Natural enzymes， proteins or RNAs with high selectivity and high catalytic activity， are of significance in some fields including disease therapy， clinical diagnosis， food industry， water treatment and textile productionetc［1-2］. Natural enzymes have attracted a great number of researchers to put their attention no whetherin vivoorin vitro. Researchers have made a great progress in the application of natural enzymes owing to their merits.However， they suffer from some problems such as high cost， low stability and hard to mass produce， which will restrict the development of natural enzymes. To solve these problems， a lot of efforts have been made to improve their properties， but the progress is not satisfactory［3］. Interestingly， a concept “artificial enzyme”has been proposed by Ronald Breslow［4］. In the past few decades， a great amount of artificial enzymes with great stability， reusability， ease of preparation and have been synthesized， showing similar rate constants and specificities to natural enzymes［5］.

Nanozymes， a kind of artificial enzymes， was first suggested to describe the monolayer of thiol-protected gold nanoclusters with significant ribonuclease activity by Scrimin and coworkers［6］. Subsequently，nanomaterials with enzyme-like activity were described by nanozyme as a consensus and attracted a great many researchers to work on the application of nanozymes. Due to the merits of easy preparation， low cost， good stability and ease to tuning the activityetc， nanomaterials with enzyme-like activity have attracted much attention， including metal， metal oxide， carbon-based material and so forth.

Nanozymes always exhibiting catalytic activity have been reported to resemble the functions of various enzymes-like oxidase， peroxidase， catalase， laccase and superoxide dismutase［1］. The enzyme-like activity of nanozymes can be affected by particle size， shape， and surface facets that can be utilized as feasible strategies to adjust the activity of nanozymes［7］. Compared with natural enzyme， nanozyme exhibits some advantages in terms of high stability， large specific surface area， low cost and tunable catalytic activity and types but the activity of nanozymes still a little lower than natural enzyme［8-9］. Furthermore， great progress has been made in disease therapy， heavy metals determination， organic compound degradation as well as agriculture with using nanozyme. Especially， sensing application with the use of nanozymes that have great promise for commercialization can give the comparable responses as commercial measurement method. In this paper， we introduce the classification of nanozymes as well as several strategies for improving the catalytic activity of nanozymes. What′s more， we also summarize recent progress of in sensing， including the detection of small biomolecules， biomacromolecules， phenolic pollutants， organophosphorus pesticdes and ions and put forward the challenge nanozymes are facing.

1 Classification of Nanozymes

Since Fe3O4nanoparticles were found to have peroxidase-like activity， a great number of nanomaterials with enzyme-like activity were investigated such as novel metals， metal oxides， metal sulfides， carbon-based materials and other complexes. In order to gain a deeper understanding of nanozymes， it′s necessary to classify the nanozymes into certain types according to their enzyme-like activity. Till now， there are two major categories of nanozyme， namely oxidoreductase and hydrolase［10］.Nevertheless， most of researches focus on oxidoreductase while the researches on hydrolase are only a few. Moreover， both oxidoreductase and hydrolase can be subdivided into several types， for example，oxidoreductase can be subdivided into oxidase， peroxidase， catalase， superoxide dismutase， glucose oxidase and laccaseetc， and hydrolase can be subdivided into nuclease， esterase， phosphatase， protease and ureaseetc［11］. Despite a great progress has been made in the exploration of various active nanozymes，it is still far from sufficient because the more than 7000 kinds of enzymes have been discovered while the classification of nanozyme are less than 100. The two can′t be taken into comparison simultaneously.Therefore， we need to pay attention to explore more nanozymes with various activities and further find out nanozymes with multi-activity.

2 Adjustment on the Catalytic Activity of Nanozymes

2.1 Size Effect on Reactivity of Nanozymes

Size dependence is a key factor for the property of nanomaterials as well as the enzyme-like activity of nanozymes［12-16］. For single nanozyme， the smaller nanozymes possess weaker catalytic activity， while the smaller nanozymes show higher catalytic activity the same mass amount as others because of the larger total specific surface areas. Chen et al. have prepared Fe-N/C single-atom nanozymes with four different sizes by carbonization of the bimetallic Fe-Zn ZIFs precursors［17］. By modulating the molar ratios of methanol and metal salts， different sizes of samples could be obtained and the sample with size of 120 nm exhibited the highest oxidase-like activity. However， the smallest nanozymes obtained here were 35 nm and show weaker activity than the nanozymes of 120 nm owing to significant agglomeration of nanoparticles （NPs） with less active sites exposed （Fig. 1）. In addition， Xi et al. reported the size effect on Pd-Ir core-shell nanoparticles with four different sizes but identical shapes and surface structures［18］.Enzyme-linked immunosorbent assay （ELISA） was carried out to estimate the size effect of nanozyme with using Pd-Ir NPs as model. A result obtained from ELISA was that smaller Pd-Ir NPs offer higher detection sensitivities and revealed the size effect of Pd-Ir NPs. These works demonstrate that the catalytic performance of nanozymes is size-dependent and in general higher as nanozymes smaller. Hence， the activity of nanozymes can be tuned by modulating the size of nanozymes and to prepare smaller particles may be a good choice but this is not always the case because agglomeration may be a factor that will decrease the activity of nanozyme. The size effect of nanozymes has been reviewed in many articles， it will not be reviewed in detail in this work.

2.2 Morphology

A series of studies have confirmed that the catalytic performance of nanozymes can be optimized by the rational design of their morphology［19-23］. The reason for optimizing the catalytic performance by tuning the morphology may be that the morphology of the nanozymes is determined by the exposed crystal planes with specific atomic arrangements， and different crystal planes will exhibit different electronic structures，which significantly endows the nanozymes with various catalytic performance. As previous reported， Wei et al. prepared CeO2nanocubes and nanorods by a hydrothermal method and the peroxidase-like activities of two samples were investigated［19］. Under the same condition， CeO2nanorods exhibited higher catalytic activity than CeO2nanocubes at different pH. As proved by XPS and Raman analysis， there were more Ce3+on the surface of the nanorods and the high concentration Ce3+enhanced the reducibility of ceria.Furthermore， the specific surface areas of nanorobs calculated by the BET method were much larger than that of nanocubes， so there were more active sites on the surface of ceria nanorobs. It demonstrated that the peroxidase-like activity of nanoceria was morphology-dependent. In addition， Huang et al. have also studied the effect of different morphology on the oxidase-like catalysis of nanoceria by synthesizing three different samples， namely CeO2nanorods， CeO2nanocubes and CeO2nanopolyhedras［24］. As is revealed by their study， CeO2nanocrystals show morphology-dependent oxidase-like catalytic activity， following the order： rods＞polyhedra＞cubes. The study has proved that the oxidase-like catalysis of nanoceria relied on morphology and the enhanced activity of nanoceria could be attributed to the unique ［110］ facets on CeO2nanorobs （Fig. 2）. In addition to nanoceria， various nanozymes have also been investigated. For instance， Vernekar et al. have proved that the oxidase-like activity of MnFe2O4was morphologydependent. Among three morphologys of nanozymes， nanowires， nanosheets and nanooctahedra，MnFe2O4nanooctahedra with the ［111］ facets presented the highest activity owing to the fact that the overall arrangement of atoms in the plane and sub-layer which might result in favorable electronic interactions， creating a site for substrate binding［25］. The results of these papers further confirmed that morphology plays an important role in influencing the activity of nanoenzymes， and the specific rules and mechanisms should be studied in the future.

2.3 Composition

There are some papers having proved that bimetallic and mutimetallic nanostructures possess better enzyme-like activity owing to synergistic effect among the compositions or the emergence of new properties.Singh et al. developed a bimetallic nanozyme with Pd nanoparticles grown on Au nanorob （AuNRs） surface preferentially on end facets［26］. With certain Pd∶Au ratio， Pd nanoparticles preferred to disperse at end of AuNRs that allowed the detection of malathion due to the interaction between the uncovered surface of AuNRs and sulfanyl group （R-S-R′） of malathion. In Comparison to pure AuNRs， Pd@AuNRs exhibited higher catalytic activity on account of the combination of peroxidase potential originated from both compositions and larger catalytic surface. Furthermore， the Au@Pt nanozymes with core-shell structure were formedviaa seedmediated method by Wu et al.［27］. With different amount of Pt adding， Au@Pt presented different catalytic activity and the good peroxidase-activity could be found on Au@Pt2.5% （Fig.3）. Compared with AuNRs，both Au@Pt2.5% and Au@Pt25% showed higher Raman signals due to the leading role of the high catalytic of Pt for producing high Raman signals. Taking advantage of the finite difference time domain （FDTD）simulations， a fact could be found that the growth of Pt would damp the electric field of AuNPs and further affect SERS signals but the signals did not show a monotonic decrease tendency. The work provided an effective strategy to develop rational design of nanozymes with sensitive SERS properties. Recently， Lv et al.provided a strategy for alcohol detection by coupling Au@PtRu and alcohol oxidase for the first time［28］. The Au@PtRu nanozymes with silkworm cocoon-like structures exhibited superior catalytic activity compared with the Au and Au@Pt nanorobs due to more hydroxyl radicals generated from the hydrolysis of hydrogen peroxide catalyzed by the trimetallic nanozymes.

Except for bimetallic or mutimetallic nanomaterials， the metal/nonmetal composites have also attracted much interest owing to their enhanced catalytic activity. Taking enhanced activity of metal/nonmetal composites into consideration， Jin et al. provided a fast and sensitive colorimetric method for detecting glutathione （GSH） with TiO2/C-QDs as oxidase mimics［29］. With few-layered Ti3C2TxMXene nanosheets as raw materials， a one-step hydrothermal method was used to prepare TiO2/C-QDs in which QDs of TiO2were grownin situon a carbon matrix. TiO2/C-QDs nanozymes shower higher catalytic activity than each component because of the synergistic effect. It′s interesting that TiO2can enrich the dissolved oxygen and catalyze oxygen to generate reactive oxygen species （ROS） but with low electron mobility. Such limitation can be solved by TiO2loaded on C-QDs because C-QDs can boost the electron mobility of TiO2. Furthermore， Sun et al.constructed a colorimetric assay for ascorbic acid by using IrO2/GO nanozymes as a peroxidase mimic［30］. The monodisperse IrO2nanoparticles were prepared by pulsed laser ablation in ethanol but suffered from aggregation easily that made the catalytic activity of IrO2disappointing （Fig.4）. In order to avoid aggregation，polyallylamine-modified GO nanosheets with remarkable hydrophilicity and high surface area were chosen as support of IrO2nanoparticles to form IrO2/GO with good dispersibility. As a result， IrO2/GO nanozymes exhibited higher activity than only IrO2nanoparticles and even horseradish peroxidase. These works have confirmed that taking the shortcomings of nanozymes into consideration， we can find suitable materials to couple with nanozymes to overcome the problem.

2.4 Surface Coating and Modification

Surface coating and modification is an effective way to tune the catalytic activity of nanozymes due to the change of the surface property［31-34］. There are some general strategies that have significant effect to modulate the surface property of nanozymes such as surface charge modulation， redox potential modulation， surface acidity modulation， surface blocking by polymers， adsorption of specific molecules and covalent modification［35］. Naha et al.［36］have made a work that using the dextran-coated iron oxide nanozymes （Dex-NZM）as antibiofilm agent to treat an oral disease. In comparison to uncoated iron oxide nanozymes， Dex-NZM with similar core size showed lower activity but higher dispersibility and more stable in phosphate buffered solution（PBS） and saliva. As for selectivity， the work gave a result that Dex-NZM with no adverse effect couldn′t bind to both oral gingival and fibroblast cells， while uncoated NZM bound very strongly and in high amounts that resulted in significant reductions in cell viability at 24 h. Bacterial killing and exopolysaccharides （EPS）degradation within biofilms could be evaluated by high-resolution confocal fluorescence imaging combined with quantitative computational analysis. And the result could be that there was a great number of bacterial deaths and significant degradation of the EPS， revealing the excellent antibiofilm effects of the Dex-NZM.Although surface coating can reduce the catalytic activity of nanozymesviareducing surface contract between nanozymes and substrates， it′s necessary to get surface coating to enhance some properties of nanozymes including selectivity， dispersibility and biocompatibility. In contrast， Lu et al. have manifested that the peroxidase-like activity of Co3O4could be enhanced by surface coating withβ-cyclodextrin （β-CD）nanoparticles. Co3O4@β-CD NPs could be formed withβ-CD effectively modified Co3O4NPs by a one-pot synthesis method［37］. With certain amount ofβ-CD （6.45%）， Co3O4@β-CD showed the highest catalytic activity on account of more tetramethylbenzidine （TMB） absorbed on the cavity ofβ-CD and enhanced the electron transfer.However， with more than 6.45%β-CD， the catalytic activity of Co3O4@β-CD began to decrease because the active sites were covered byβ-CD. As a result， the peroxidase-like activity of Co3O4@β-CD nanozymes exceededβ-CD and Co3O4owing to the synergistic effect betweenβ-CD and Co3O4， revealing that selecting appropriate materials to modify nanozymes can achieve enhanced catalytic performance of nanozymes

Modifying with ions can be another effective way to modulate the activity of nanozymes， changing the surface charge of nanozymes. Liu et al. studied fluoride capping nanoceria with boosting oxidase-like activity，using to probe F-（Fig.5）［38］. By measuring theζ-potential of nanoceria， a result could be obtained that the surface positive charge decreased gradually and even converted to negative charge with more and more adding，indicating the successful adsorption of F-on nanoceria surface. As the work presented， the promote effect of F-have better performance on TMB instead of 2，2′-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）diammonium salt （ABTS） owing to the electrostatic interaction between cationic TMB and F-capping nanoceria. However， this simple surface charge model was just the one reason of the promote effect. Except the surface charge model，the other two reasons could be the low surface energy of fluorinated surfaces that could inhibit the adsorption of product and electron sponge mechanism for nanoceria nanozymes because F-is a strongly electron withdrawing ligand. Inspired by the previous work， Song and co-workers designed fluoride capped V6O13-reduced graphene oxide nanocomposites （NCs） with high oxidase-like activity［39］. Using TMB as a chromogenic substrate， the V6O13-rGO NCs proposed here presented higher oxidase-like activity than V6O13，rGO， and the mixture of V6O13and rGO due to the synergy reaction of V6O13and rGO. Moreover， the oxidase-like activity of V6O13-rGO NCs in the presence of F-could be enhanced 2.8-times higher than that of V6O13-rGO NCs without F-and the reasons could be the same as mentioned above that augmented affinity between the surface of the nanozymes and the substrate of TMB by changing the zeta potential of nanozymes with F-capping and facilitating the electron transfer between substrates， dissolved oxygen， and the nanozyme.

More surprisingly， as one strategy of surface modification， molecular imprinting polymers （MIPs） are able to perform as substrate binding pockets on nanozymes and further to improve the activity and selectivity of nanozyme. Recently， an emerging Fe3O4nanozymes with enhanced catalytic activity and selectivity by creating molecular imprinting polymers have constructed by Zhang et al.［40］. At the beginning， using acrylamide and NIPAAm as monomers and MBAAm as a cross-linker to the Fe3O4NPs and TMB（or ABTS）mixture formed the the nanogel products and the TMB and ABTS imprinted gels are named T-MIP and A-MIP，respectively. With TMB as substrate， the T-MIP nanogels showed higher activity and selectivity than naked Fe3O4NPs which were superior to A-MIP， indicating high activity and selectivity achieved. With ABTS as substrate， an opposite result could be obtained here. Taking the electrostatic interaction into consideration， a new strategy that using cationicN-［3-（dimethylamino）propyl］methacrylamid （DMPA） or anionic 2-acrylamido-2-methyl-1-propanesulfonic acid（AMPS） as part of the monomer mixtures was proposed and the nanogels could be named MIPneg and MIPpos corresponding to the component of AMPS and DMPA （Fig. 6）. Take advantage of TMB as substrate， the order of the activity and selectivity of nanozymes could be concluded as following：T-MIPneg＞T-MIP＞Fe3O4＞A-MIP＞A-MIPpos. The contrast result could be observed with ABTS as substrate that T-MIPneg＜T-MIP＜Fe3O4＜A-MIP＜A-MIPpos. Not only the activity and selectivity of Fe3O4nanozymes， but that of Au and CeO2also could be furthered by engineering molecular imprinting polymers on nanozymes. Thus， engineering molecular imprinting polymers on nanozymes can be an effective way to improve activity and selectivity of nanozymes and choosing the substance with opposite charge to substrate as monomer shows further enhancements.

3 Recent Research Progress of Nanozymes in Sensing

Although traditional analytic methods have been successfully used in sensing such as inductively coupled plasma mass spectrometry （ICP-MS）， atomic emission spectroscopy （AES）， high-performance liquid chromatography （HPLC）， electrochemical sensors. But they are suffering from a number of problems including time-consuming sample preparation， required sophisticated instrumentation， high cost and highly trained experts. Thus， great efforts have been made to fabricate a simpler method to achieve on-site analysis.Colorimetric or fluorescent strategies are regarded as ideal methods that have significantly potential application in sensing， possessing the virtues of rapid response， low cost， portability and time saving. Colorimetric assay and fluorescent assay can be achievedviacomparing the change of color or the change of fluorescence. To date， a great number of colorimetric assays and fluorescent assays were constructed with using nanozymes，which can catalyze the conversion of colorimetric substrate and fluorescent substrate. As reported， there are some substances like antioxidants， ions， pesticides， phenolic pollutants and so on that can be detected by colorimetric assay and fluorescent assay with the use of nanozymes［41-42］.

3.1 Detection of Small Biomolecules

Small biomolecules play a vital role in maintaining various metabolic reactions and biological functions in human life. Various of small biomolecules play different roles in human body， such as ascorbic acid，cysteine， glutathione and glucose. The detection of small biomolecules has aroused significant interest in various fields， including disease diagnosis and medicine.

3.1.1 Detection of Ascorbic Acid

Ascorbic acid （AA） plays a key role in many physiological and biochemical processes including prevention of scurvy， improving immunity， promoting collagen synthesis， antioxidant，etc［43］. Therefore，constructing a colorimetric assay with a rapid response for ascorbic acid is essential for human health. As mentioned above， Sun et al. reported a colorimetric assay for AA in the presence of IrO2/GO which can be served as peroxidase mimic［30］. In this colorimetric assay system， AA can give sensitive response within the concentration range of 5～70 μmol/L with the detection limit of 324 nmol/L. The peroxidase-like activity should derive from electron transfer mechanism instead of generation of hydroxy radicals as they investigated. Wang et al. fabricated a ratiometric fluorescence system to detect ascorbic acid by combining inherent oxidase and ascorbate oxidase mimetic activity of CuO NPs. The limit of detection of this ratiometric assay is 0.16 μmol/L［44］.Liu et al. developed a Co-N-C simple-atom nanozymes for colorimetric discrimination of antioxidants，including glutathione （GSH）， AA， cysteine （Cys）， tannin （TA）， catechin （C）， dopamine （DA） and uric acid （UA）， which exhibits a broad application prospect in medical dianosis［45］.

3.1.2 Detection of Cysteine

As one of the biothiol exiting in mammal， cysteine plays an essential role in many physiological and pathological processes. An abnormal concentration level of cysteine will trigger relevant diseases including liver injury，osteoporosis， skin lesions， cardiovascular problems and so on［46］. Owing to the high reducibility of cysteine， a series of colorimetric and fluorescent assays have been developed for the detection of cysteine induced by nanozymes. For example， a colorimetric determination ofL-cysteine （L-Cys） with the assistance of VS4-H2O2-TMB system was reported by Chen group （Fig.7）［47］. The mechanism can be concluded as following： 1） VS4 submicrospheres catalyze the H2O2to produce OH－， which further can oxide TMB to generate a blue products oxTMB； 2） oxTMB can be reduced to form the colorless products TMB byL-Cys. The liner range of colorimetric determination ofL-Cys is from 5 to 100 μmol/L and the limit of detection is 2.5 μmol/L. Although the selectivity of this reaction system towardL-Cys is good enough， the method proposed here has not been taken detection on real sample and thus can′t evaluate its practicality. Li et al. prepared cerium nanotube combined with aspartic acid （Asp/Ce-NT） used for colorimetric assay of cysteine［48］. In this work， Asp/Ce-NT showed oxidase-like activity that could be used to oxide TMB without H2O2and enzymes. The catalytic activity of Asp/Ce-NT could be influenced by several factors including the radio of Asp to Ce（NO3）3during synthesis procedure and the chirality of Asp. The result indicated thatL-Asp/Ce-NT exhibits more excellent activity than D-Asp/Ce-NT. The colorimetric assay system in the paper showed high sensitivity and selectivity that the linearity response to the concentrate of cysteine range from 0.08 to 10 μmol/L and the detection limit is as low as 33.2 nmol/L. A penguin shape Cu/Mo metal organic framework was found possesses intrinsic peroxidase-like activity and employed to detectL-Cys， the limit of detection （LOD） of this assay is 0.158 μmol/L［49］.

Florescent assays have been also constructed to detect cysteine. Jin and coworkers developed a Cobalt-based metal organic frameworks （ZIF-67）as oxidase mimic used for fluorescence“turn-on”assays ofL-Cys［50］. ZIF-67 could catalyze the oxidation of amplex red to create a fluorescent resorufin and further a non-fluorescent resazurin.L-Cys，however， could reduce resazurin to generate resorufin that provide a great possibility to construct a fluorescence “turn-on” assay. As the paper confirmed， the fluorescence was time-varying that the emission intensity reached a maximum at 80 min and then fell off after 80 min. It made a little difficult to determineLCys， thus should be optimized in the future study. In addition， a novel three-in-one CuBDC nanozyme，possessing both cysteine oxidase- and peroxidase-mimicking activities， was constructed for the fluorescent assay of cysteine by Li et al （Fig. 8）［51］. Surprisingly， the non-fluorescent CuBDC with TA as ligand could be “turn on” by H2O2originated from the hydrolysis of cysteine. The active Cu2+made two contributions in this cascade reaction： 1） to perform like cysteine oxidase to catalyze the conversion of cysteine to H2O2； 2） to mimic peroxidase to catalyze H2O2to produce OH-. As a platform for cysteine sensing， the CuBDC could be used to detect cysteine without any additional fluorescent labels and a wide linear range was obtained.

3.1.3 Detection of Glutathione

Glutathione is a thiolated tripeptide and its abnormal level will cause some diseases such as such as liver damage， cancer and senile dementia［52］. Thus， it′s of significance to detect glutathione in clinical diagnosis.Li and co-workers have fabricated a simple colorimetric assay to be applied to determine glutathione （GSH）based on the ultrasmall Co3O4nanocrystals［53］. As the work demonstrated， the oxidation of TMB （oxTMB）could be attributed to the catalase-like activity of Co3O4nanocrystals which could catalyze the decomposition of H2O2. With GSH adding increasingly， the absorbance decreased gradually， implying the reduction of oxTMB.Although amino acids， Cys and Hcy， would interfere with the detection of GSH， formaldehyde solution could be used to eliminate interference and a satisfying result was gotten. The colorimetric assay reported here was sensitive and selective to GSH and had potential application in real examples. Recently， Ganganboina et al.have developed a colorimetric biosensor for sensitive and selective detection of nanomolar GSH based on the biomimic oxidase activity of V2O5nanosheet （V2O5NS） （Fig. 9）［54］. It′s the first time to prove the application of V2O5NS as nanozyme for the detection of glutathione. As showed in Fig. 9， TMB could be oxidized to form ox-TMB catalyzed by V2O5NS and then ox-TMB could be reduced to TMB after adding GSH. Based on this，V2O5NS was able to detect GSH in a linear range from 10 to 500 nmol/L， and the limit of detection （LOD） was 2.4 nmol/L. Though the LOD was lower than most of previous reports， the linear range is several times lower than the normal range in human serum， having to dilute the human serum to meet the test range. Therefore，further study should be carried out to expand the detection range to avoid unnecessary step.

The ratiometric fluorescent assay is more accurate and effective because it can minimize the interference from background factors and provide a built-in correction［55］. It′s of great desire to develop a ratiometric fluorescent sensor. Lately， Hu et al. proposed a ratiometric fluorescent strategy for the detection of glutathione using MnO2nanowires as nanozyme［56］. In this sensor， MnO2nanowires could“turn on” the fluorescence of non-fluorescent VB1and quench the fluorescence of Cu/Ag nanoclusters （NCs） concurrently by oxidation reaction， thus realizing ratiometric fluorescent sensing. Nevertheless， GSH can reduce the MnO2nanowires to Mn2+and decreasing their quenching capacity and oxidase-like activity， bringing about the recovery of the fluorescence of Cu/Ag NCs while the fluorescence of oxVB1was quenched （Fig.10）. Inspired by the change of the fluorescence， the ratio F560/F410versus GSH concentration was used for calibration and a linear relationship in the range of 10 to 70 μmol/L was obtained with the detection limit for glutathione of 6.5 nmol/L.

圖1 Fe-N/C 催化劑（a） 2200、 （b） 400、 （c） 120和（d） 35 nm 下的SEM 圖； （e） Fe-N/C 催化劑的尺寸與氧化活性的關(guān)系圖［17］Fig.1 SEM images of Fe-N/C catalysts with size of 2200（a）， 400（b）， 120（c） and 35 nm（d）， respectively；（e） The size-dependent oxidase-like activities of Fe-N/C catalysts with different sizes［17］

圖2 揭示觸發(fā)分子對CeO2的面選擇性響應(yīng)以提高納米酶的活性［24］Fig. 2 Facet-selective response of trigger molecule to CeO2 ［110］ is revealed for up-regulating oxidase-like activity of nanoceria［24］

圖3 通過控制Pt的含量來理性設(shè)計(jì)高性能的Au@Pt納米粒子雙功能納米酶［27］Fig.3 Rational design of high-performance Au@Pt NP bifunctional nanozymes by controlling the Pt amount［27］

圖4 多環(huán)芳烴穩(wěn)定的IrO2/GO納米復(fù)合材料的制備及其對過氧化物酶AA檢測示意圖［30］Fig. 4 Schematic illustration of the preparation of PAH stabilized IrO2/GO nanocomposites and the colorimetric detection of AA based on the peroxidase-like activity of IrO2/GO nanocomposites［30］

圖5 （A） F-捕獲的被氧化酶倒轉(zhuǎn)的納米氧化鈰示意圖； （B）納米氧化鈰粒徑分布和透射電子顯微鏡圖；（C） ABTS （0.5 mmol/L）和（D） TMB （1 mmol/L）的紫外吸收光譜圖［38］Fig.5 （A） A scheme showing F--capped nanoceria with improved oxidase turnovers； （B） DLS size distribution and a TEM image （inset） of nanoceria； UV-Vis spectra of （C） ABTS （0.5 mmol/L） and （D） TMB （1 mmol/L） oxidation by nanoceria［38］

圖6 （A） Fe3O4 納米粒子、（B） T-MIPneg 和（C） A-MIPpos 納米凝膠對TMB 和ABTS 的氧化的光學(xué)照片圖；（D） Fe3O4納米粒子印記TMB的示意圖［40］Fig. 6 Photographs showing the activity and specificity of （A） Fe3O4 NPs， （B） T-MIPneg and （C） A-MIPpos nanogels for oxidation of TMB and ABTS with or without H2O2； （D） A scheme of imprinting TMB on Fe3O4 NPs［40］

圖7 提出的L-半胱氨酸的比色檢測機(jī)制示意圖［47］Fig.7 Proposed mechanism for the colorimetric detection of L-Cys［47］

圖8 CuBDC級聯(lián)半胱氨酸氧化酶和過氧化物酶模擬活性和刺激響應(yīng)的熒光示意圖［51］Fig. 8 Schematic illustration of the cascade cysteine oxidase- and peroxidase-mimicking activities and stimulusresponsive fluorescence of CuBDC［51］

圖9 以TMB作為指示劑的V2O5納米片比色法檢測谷胱甘肽的示意圖［54］Fig. 9 Schematic illustration of V2O5 nanosheet based colorimetric assay for glutathione detection using TMB as the indicator［54］

圖10 基于MnO2-Cu/Ag NCs-VB1體系的谷胱甘肽比率熒光檢測的傳感器示意圖［56］Fig.10 Schematic illustration of the ratiometric fluorescent sensor for GSH based on MnO2-Cu/Ag NCs-VB1 system［56］

圖11 （A） MA-Hem/AueAg納米復(fù)合材料納米酶的制備流程示意圖和（B）葡萄糖催化比色測試圖［58］Fig. 11 Schematic illustration of（A） the fabrication procedure of MA-Hem/AueAg nanocomposite nanozymes and（B） the catalysis-based colorimetric test for glucose［58］

3.1.4 Detection of Glucose

A series of diseases in human body including diabetes， cardiovascular disease and dysfunction of various tissues are relevant to glucose level［57］. Fabricating a simple， sensitive and selective colorimetric assay for glucose is of importance for clinic diagnosis. It has reported that a colorimetric assay can be constructed by a cascade reaction with nanozyme and glucose oxidase together. Liu and colleagues prepared a novel nanozymes， melamine（MA）-Hemin （Hem）/Au-Ag nanocomposites，viathe in-site minerization of Au-Ag into Hem-coupled MA polymer matrix which can be attributable to the covalent attachment of Hem to MA by the cross-linking chemistry and a colorimetric sensor was constructed by combining MA-Hem/Au-Ag nanocomposites with glucose oxidase （Fig.11）［58］. In comparison to pure Hem， the MA-Hem/Au-Ag nanozymes presented four times higher peroxidase-like activity owing to the enhanced function of the electron transferring caused by Au-Ag， thus exhibiting higher sensitivity toward glucose by constructing a colorimetric probe consisting of glucose oxidase， 3，3′，5，5′-tetramethyl benzidine（TMB）， H2O2and MA-Hem/Au-Ag. Under the optimal conditions， glucose could be detected with a liner range from 0.0050 to 2.0 mmol/L and the limit of detection is about 1.8 μmol/L in blood sample. Adeniyi et al. have devoted their effort to design a novel nanozyme，nanomagnet-silica nanoparticles decorated with Au@Pd， for enhanced peroxidase-like activity and colorimetric glucose sensing with a cascade reaction［59］. The peroxidase-like activity of Fe3O4@SiO2-NH2-Au@PdNPs could be attributed to an electron transfer process instead of the Fenton-like reaction. The multiple active catalytic sites and the synergistic effect of the Au@PdNPs played crucial roles for the high sensitivity of glucose sensor with the LOD of 60 nmol/L and the LOQ of 200 nmol/L. A linear range from 0.010 to 60 μmol/L obtained here was wide but lower than the concentration of glucose in the serum sample of a healthy individual （3.0～8.0 mmol/L） and diabetes patients （9.0～40.0 mmol/L） and then an additional step of dilution might be carried out.

Except for coupling glucose oxidase with nanozyme for determining glucose， a new way to detect glucose with the dual activity of nanozyme is put forward. For instance， chemically modified graphitic carbon nitride-chitinacetic acid hybrid （MGCN） based nanozymes for glucose sensing have been reported for the first time［60］. The metal-free MGCN processed dual activity， glucose oxidase-like- and peroxidase-like activity， and could be used to trigger the cascade reaction（Fig. 12）. The author has fabricated a glucose-paper strip sensor for glucose sensing based on the dual roles MGCN played and the results could be identified with naked eyes. The MGCN have also been investigated in real sample with results as similar as that obtained from a standard market glucometer.

圖12 （a） GOx葡萄糖氧化和H2O2介導(dǎo)的HRP催化TMB氧化的反應(yīng)機(jī)制； （b）非酶葡萄糖識別采用仿生納米酶級聯(lián)法MGCN進(jìn)行葡萄糖氧化，隨后原位生成H2O2和幾丁質(zhì)AcOH分解H2O2和TMB氧化［60］Fig. 12 （a） Enzymatic scheme of GOx and HRP with H2O2-mediated TMB oxidation；（b） Non-enzymatic glucose recognition using a biomimetic nanozyme cascade method of MGCN for glucose oxidation with subsequent in-situ generation of H2O2 and chitin-AcOH for decomposition of H2O2 and TMB oxidation［60］

圖13 利用CoOOH納米片和OPD進(jìn)行堿性磷酸酶活性熒光和比色雙模式測定的示意圖［61］Fig. 13 Scheme depiction of the fluorescence and colorimetric dual-mode assay of alkaline phosphatase activity by using CoOOH nanoflakes and OPD［61］

圖14 基于Pd/g-C3N4的比例熒光測定乙酰膽堿酯酶活性的示意圖［62］Fig.14 Illustration of the Pd/g-C3N4 based ratiometric fluorescence strategy for determination of AChE activity［62］

In conclusion， various of nanozymes exhibit excellent performance in the field of small molecule detection， like ultra-low detections limits and wide linear range， which made the nanozymes even better than natural enzymes. However， nanoenzymes still face some problems need to be solved， such as， the detection mechanism needs further study， the detection analytes need to be more diverse， the anti-interferences should be further improved. Therefore， how to further customize the activity of the nanozymes and give it higher specificity and sensitivity for the detection of small molecule compounds with biological activity are two important questions for future research.

3.2 Detection of Biomacromolecules

Detection of biomacromolecules from various complex matrices is significant in the field of biology due to their important functions in numerous biological processes. The nanozymes have been widely used to analyze and detect various biomacromolecules， such as acid/alkaline phosphatase （ACP/ALP）， AChE， glucosidase and other biomarkers.

3.2.1 Detection of Alkaline Phosphatase

Alkaline phosphatase （ALP） is an important enzyme that can catalyze hydrolysis and transphosphorylation，which is widely found in the liver， bone， intestine， kidney. It is commonly associated with a variety of human diseases， such as diabetes， bone disease， and liver disease. Therefore， the construction of rapid and accurate ALP detection method is particularly important for disease diagnosis. Liu et al. developed a novel fluorescence and colorimetric dual-mode for ALP activity using the CoOOH nanoflake［61］. Based on the principle ofo-phenylenediamine （OPD） can be oxidized to OxOPD and ascorbic acid （AA） can reduce the CoOOH to Co2+， the fluorescence and colorimetric dual-mode detection of AA was achieved （Fig.13）. Besides，ALP can makeL-ascorbic acid-2-phosphate （AAP） hydrolyze to AA. With the help of AAP， the selective and sensitive dual-mode assay of ALP activity can be realized using fluorescence and UV-Vis absorption spectroscopies. The dual-mode quantitative assay of ALP can achieve a low detection limit of 0.026 U/L using the fluorescence method and 0.032 U/L using the colorimetric method.

3.2.2 Detection of Acetylcholinesterase

Acetylcholinesterase （AChE） is an important hydrolase that controls the content of acetylcholine， and the content of the AChE is directly related to the Alzheimer′s disease. Therefore， it is important to develop a reliable and simple method for AChE activity detection and inhibitor screening. Zhang et al. developed a method by combining Pd/g-C3N4witho-phenylenediamine （OPD） to realize the detection of AChE using the ratiometric fluorescence assay［62］. This strategy is suitable for serology test benefitting from the reliable signal output.

The high porosity， tunable topology and easy functionalization of MOFs make them suitable for various biomacromolecular sensing applications. The combination of MOFs with other nanomaterials（such as，g-C3N4， AuNPs， PtNPs and others） produces a synergistic effect in biomacromolecular sensing.The structure and properties of MOFs can be engineered by changing the organic ligands or metal clusters.MOFs often play multiple roles in biosensors such as nanoprobes， nanozymes and nanocarriers.

Although nanozymes has made significant progress in biomacromolecular sensing， the challenges of nanozymes biosensors are gradually emerging. Due the applications in the biological field， the development of stable， low-toxicoty， biocompatible and flexible nanozymes is key to improving the reproducibility of nanozymes biosensors. Therefore， the advanced synthetic strategies to increase the accessible active sites of nanozymes is crucial for enhancing the performance of biosensors.

3.3 Detection of Phenolic Pollutants

Phenolics， highly toxic compounds， seriously endanger ecological environment， human health， animal and plant life and will interfere with the endocrine system of organisms and bring about problems such as reproductive disorders， abnormal development and weakened immune function［63-64］. Wang et al. proposed CH-Cu as a bioinspired laccase-mimicking nanozyme via the coordination of Cu+/Cu2+with a cysteine（Cys）-histidine （His） dipeptide （Fig. 15）［65］. The laccase-like activity of CH-Cu was demonstrated with 2，4-DP and 4-AP and could be attributed to the CH-Cu nanoparticles instead of Cu2+decomposed from CH-Cu. As confirmed， CH-Cu could play a degradation effect on various phenolic pollutants and showed higher activity than natural laccase because there were more active sites in a single particle. Xu et al. synthesized a Cu-based laccase mimic to oxidize phenolic pollutants， including 2，4-dichlorophenol， phenol，p-chlorophenol，2，6-dimethoxyphenol， hydroquinone，o-nitrophenol ando-aminophenol hydroquinone［66］. The Cu nanozyme is relatively stable and applicable for the treatment of phenolic pollutants.

圖15 模擬CH-Cu 納米酶的漆酶制備示意圖，該納米酶與天然漆酶的催化中心相似。 漆酶的PDB 代碼為1V10［65］Fig. 15 Schematic illustration of the preparation of laccase mimicking CH-Cu nanozymes， which resembles the catalytic center of natural laccase. PDB code of the laccase is 1V10［65］

圖16 毒死蜱農(nóng)藥的傳感檢測步驟示意圖［69］Fig.16 Schematic represents the steps involved in chlorpyrifos sensing［69］

Beside complex copper containing materials， coral-like silver citrate （AgCit） was reported possessing excellent laccase-like activity of oxidizing phenolic substrates and the endocrine hormone adrenaline.Comparing with natural enzyme， this AgCit has a higherυmaxand lower Km value using adrenaline as a substrate. A sensor for phenolic substrates was designed based on the colored oxidation products of phenolic substrates produced by the AgCit laccase mimic at the expense of oxygen［67］.

3.4 Detection of Organophosphorus Pesticides

Organophosphates as pesticides play an essential role in controlling diseases and improving the productivity of agricultural crops but they will do harm to environment and human health， causing neurotoxicityviainhibiting the activity of acetylcholinesterase［68］. Based on their previous work， Singh et al. prepared bimetallic nanozyme， Pd@AuNRs to determine malathion in the presence of hydrogen peroxide with OPD as substrate.Malathion could quench the catalytic activity of Pd@AuNRs owing to the interaction between sulfanyl group（R-S-R′） of malathion and AuNRs surface and further inhibited the oxidation of malathion［26］. Compared with other organophosphorus compounds and metal salts， the detection of malathion shows much more sensitive due to the unique sulfanyl group on malathion， indicating the Pd@AuNRs were selective toward malathion. The limit of detection was 60 ng/mL and the potential application in real sample was validated in tap water with the recovery between 80% and 106%.

By introducing a chlorpyrifos specific aptamer， Weerathunge and colleagues designed a chlorpyrifos aptasensor for the detecti on of chlorpyrifos by using Tyrosine-capped silver nanoparticles as nanozymes［69］. As seen in Fig. 16， the weak non-covalent interaction of a pesticide-specific Chl aptamer with the Ag nanozyme could decrease the peroxidase-like activity of Ag nanozyme and the oxidation of TMB couldn′t be triggered，while the catalytic activity of Ag nanozyme could be recovered in the presence of chlorpyrifos which can bind to the chlorpyrifos specific aptamer more strongly than Ag nanozyme. The selectivity of chlorpyrifos aptasensor toward chlorpyrifos was tested among various pesticides including the organophosphates class and the other class and a gratifying outcome was acquired for the detection of chlorpyrifos. Along with the increasing concentration of chlorpyrifos， the absorption values increased gradually and exhibited a linear relationship ranging from 35 to 210 mg/L. The limit of detection was 11.3 mg/L while the limit of quantification was 34.1 mg/L， indicating the chlorpyrifos aptasensor sensitive enough to chlorpyrifos. Finally， the current sensing platform has been proved robust in the river water sample.

More recently， Huang et al. have fabricated a colorimetric paper sensor for sensing organophosphorus pesticides （OPs） based on an AChE-MnOOH nanozyme cascade catalysis （Fig.17）［70］. In fact， the oxidaselike activity ofγ-MnOOH nanowires （NWs） could be passivated by thiocholine （TCh） produced by acetylcholinesterase （AChE） acetylthiocholine iodide （ATCh）. Taking advantage of ICP-MS and two probe reactions，γ-MnOOH nanowires have been demonstrated to degrade to produce Mn2+ions induced by AChEATCh reaction. Nevertheless， AChE enzyme could be passivated by organophosphorus pesticides （Ops），and AChE-ATCh reaction was inhibited to produce TCh so that the oxidation of TMB could be achieved owing to the oxidase-like activity ofγ-MnOOH nanozymes without disintegration. Hence， a facile paper sensor was proposed for sensing OPs based on the cascade reaction of ATCh and degradableγ-MnOOH nanozyme. Both omethoate and dichlorvos could quantitatively visualize in the certain concentration with a LOD value of 10 ng/mL and 3 ng/mL， respectively. Although the selectivity of MnOOH-AChE platform toward OPs was not satisfactory enough， it might be used to detect both OPs and carbamates as the author considering.

圖17 基于可降解MnOOH納米酶的乙酰膽堿酯酶活性與農(nóng)藥的傳感原理圖［70］Fig.17 The sensing principle of AChE activity and pesticides based on degradable MnOOH nanozyme［70］

圖18 基于汞促進(jìn)AuNPs納米酶活性的AuNZ-PAD對Hg2+離子的比色傳感機(jī)制示意圖［72］Fig.18 Schematic illustration of the AuNZ-PAD colorimetric sensing mechanism for Hg2+ ions based on the mercurypromoted nanozyme activity of AuNPs［72］

圖19 濃縮-檢測一體化戰(zhàn)略示意圖［74］Fig.19 Illustration of enrichment-detection integration strategy［74］

3.5 Detection of Ions

3.5.1 Detection of Mercury（） Ions

As is known to all， Hg2+will do harm to human health，causing a variety of diseases such as acrodynia， kidney failure， and cognitive and motor disorders as well as serious impacts on the nervous and metabolic systems［71］. Hg2+can accumulate in living organisms and denature enzymes and proteins by binding to their S-containing ligands 55； thus， it is urgent to develop a rapid and on-site method to trace Hg2+level in environment. Researchers have made progress on constructing the inexpensive and simple colorimetric assays for Hg2+sensing with nanozyme as catalyst. There are several Hg2+nanosensor developed with using Au nanozymes owing to the formation of Au-Hg amalgam. For example， a facile gold nanozyme based paper chip was fabricated by Han′ group （Fig. 18）［72］. Based on the formation of Au-Hg amalgam， a sensitive colorimetric assay for Hg2+could be achieved with the detection limit of 30 μg/L.Interestingly， with increasing the number of drops of test sample， a lower detection limit of 1.2 μg/L could be obtained.

Instead of Au nanomaterials， Niu et al. designed a colorimetric method for Hg2+detection by using the cysteine-decorated ferromagnetic particles （Cys-Fe3O4）， with a detection limit of 5.9 pmol/L and the quantification limit of 19.7 pmol/L［73］. The composite nanozyme was prepared with cysteine modified on the surface of Fe3O4. Although Cys-Fe3O4had almost no peroxidase-like activity because the active sites were sheltered by cysteine， Hg2+could combine two cysteine molecules despoiled from the surface of Fe3O4to recover the peroxidase-like activity of Fe3O4. Taking advantage of this property， a sensitive， specific and stable nanosensor for Hg2+was constructed.

Fang group fabricated a portable Hg2+nanosensor comprising of CuS and an RGB sensor to achieve on-site and real-time analysis of mercury contamination with high sensitivity （Fig. 19）［74］. Unlike traditional Hg2+sensors suffering from high cost and dependence on sophisticated instruments， the novel Hg2+nanosensors could be used to detect Hg2+in the actual sample with the merits of high sensitivity， low cost，Portability and real-time performance. It′s the first time to find that the CuS nanozyme acts as the recognition unit， enrichment carrier， and signal amplifier in portable Hg2+nanosensor with no need of cumbersome desorption processes and sophisticated instruments. Surprisingly， the detection of Hg2+showed a wide liner range from 50 ng/L to 400 μg/L and the limit of detection is almost minimum about 50 ng/L compared with other Hg2+sensors as previous reported.

3.5.2 Detection of Other Metal Ions

As a high poisonous compound， Cr（Ⅵ） poses a threat to the environment and human health， causing the wide distribution in water， soil， and the biosphere and bringing about important chromosomic aberration by modifing the deoxyribonucleic acid （DNA） transcription process［75-76］. The Boukherroub group designed the fluorescent sensing systems by CuS-frGO nanozymes where CuS was deposited on functionalized reduced graphene oxide， showed excellent peroxidase-mimicing activity［77］. In the presence of Cr6+， the peroxidase-like activity of CuS-frGO nanozymes could be enhanced due to more hydroxyl radicals produced by the breakdown of H2O molecules which were formed by the decomposition of H2O2catalyzed by Cr6+as seen in Fig. 20. The fluorescent intensity of TA-OH， the product of the reaction between terephthalic acid （TA） and hydroxyl radicals， increased with a linear range of 0～200 nmol/L and the limit of detection for Cr6+was 26.60 nmol/L by using CuS-frGO nanozymes as catalysts.

圖20 CuS 納米粒子在離子存在時(shí)在酸性介質(zhì)中催化H2O2分解生成?OH-自由基并與對苯二甲酸反應(yīng)形成熒光活性TA-OH配合物的示意圖［77］Fig. 20 Representation of the decomposition of H2O2 catalyzed by CuS nanoparticles in the presence of ions in acidic medium to generate ·OH- radicals and formation of fluorescent active TA-OH complex upon reaction with terephthalic acid［77］

圖21 基于CuO納米粒子自級聯(lián)催化反應(yīng)的Ag+離子檢測機(jī)理示意圖［81］Fig.21 Schematic illustration of detection mechanism of Ag+ ions based on CuO NPs as self-cascade catalytical reaction［81］

圖22 （A）用于催化H2O2輔助的AR 氧化為試鹵靈的CoOxH-GO 納米雜化物的制備， （B）葡萄糖氧化偶聯(lián)CoOxH-GO納米雜化物過氧化物酶用于葡萄糖檢測， （C）基于抑制CoOxH-GO納米雜化物酶活性的CN-離子檢測， （D）用于氰化物離子傳感的CoOxH-GO/N+M的制備［87］Fig. 22 Schematic representation of （A） the preparation of CoOxH-GO nanohybrid for the catalysis of H2O2 mediated oxidation of AR to resorufin， （B） glucose oxidase coupled with peroxidase-like CoOxH-GO nanohybrid for the detection of glucose， （C） detection of CN- ions based on the inhibition of the enzymatic activity of CoOxH-GO nanohybrid and （D） the fabrication of CoOxH-GO/N+M for sensing of cyanide ions［87］

Although silver is extensively being used in electrical， photographic industries， pharmaceutical， and cosmetic applications， it′s no doubt that Ag+is a kind of high toxic pollutions that can be discharged into surface water， leading to bioaccumulation and toxicity［78-79］. It has been reported that excessive exposure to Ag+can cause a series of health problems comprising brain and liver damage， skin oxidation， heart enlargement，growth retardation which can be attributed to the combination between Ag+and amine， imidazole as well as carboxyl groups of various metabolites， inactivating sulfhydryl enzymes［80］. Therefore， a colorimetric assay or a fluorescent assay is of importance to be constructed to detect Ag+. He et al. has reported a fluorescent assay for Ag+detection with using CuO as glutathione-oxidase- and peroxidase-mimicing enzyme［81］. As nonfluorescent terephthalic acids can react with hydroxyl radicals to form TA-OH complexes with strong fluorescence， a fluorescent detection for Ag+was constructed owing to the inhibitory effect on the formation of TA-OH. It′s surprising that the limit of detection for Ag+was as low as 37 pmol/L which was almost the lowest among the fluorescent or colorimetric methods. The lowest LOD could be attributed to the roles Ag+played in the reaction and the roles could be summarized as following： 1） Ag+induced the aggregation of CuO； 2） the coordination of GSH reduce and Ag+induced the aggregation of CuO； 3） Ag+could enhance the decomposition of H2O2to form H2O and O2instead of OH-； 4） the fluorescence of TA-OH could be quenched by Ag+（Fig. 21）. Taking together， a strong inhibitory effect of Ag+for the “turn on” fluorescent reaction could be developed. The work has provided a new aspect to construct a colorimetric or fluorescent sensor by design a multifunctional nanozyme and a high sensitivity could be achieved by taking advantage of multiroles of target analyte.

3.5.3 Detection of Nonmetallic Ions

Sulfide is extensively used in many applications such as conversion into sulfur and sulfuric acid， dyes and cosmetic manufacturing， production of wood pulp but showing high toxicity in human body that will cause some problems including Alzheimer′s disease， Down′s syndrome， respiratory paralysis， and hyperglycemia［82-83］.Therefore， fabricating a colorimetric sensor for sulfide anion can be of interest because it can be allowed to evaluate the environmental quality on site. By introducing MoS2to form the heterojunction withg-C3N4viaonepot bottom-up co-calcination， Liu and coworkers designed a novel 2D/2D heterojuncted MoS2/g-C3N4nanocomposite to detect sulfide ions efficiently in the presence of 3，3′，5，5′-tetra-methylbenzidine （TMB）［84］.With the synergetic effect between MoS2andg-C3N4， MoS2/g-C3N4exhibited higher peroxidase-like activity than each of MoS2and g-C3N4. The roles sulfide ions played in the MoS2/g-C3N4-H2O2-TMB system can be Attributable to competitive effect between S2-and the reduction of oxTMB in the precence of S2-. An excellent wide linear range could be obtained under pH=4 at room temperature and the limit of detection of S2-as low as 37 nmol/L showed more sensitive and selective than previous reports for S2-detection.

As a well-known toxic ions， cyanide show great threat to human， giving rise to hypoxia， respiratory failure， endocrine disorders， nervous system lesion， vascular necrosis， and even death［85］. The reason for the toxicity of cyanide can be chalked up to its ability to bind to the active site of cytochrome oxidase and inhibit cellular respiration［86］. Owing to its wide use in industrial fields， it′s urgent to develop a convenient， low-cost and rapid-response method for cyanide detection. Lien et al. revealed that the synergistic effect of cobalt hydroxide/oxide-modified graphene oxide （CoOxH-GO） hybrid on peroxidase-like activity due to the enhanced electron transfer between substrates and the nanozymes with GO acting as a mediator（Fig.22）［87］. Based on the inhibitory effect of CN-， a highly sensitive and selective colorimetric sensor was constructed for CNdetection. As a result， the leaching of Co from CoOxH-GO nanozymes induced by CN-could be the reason for the decline of peroxidase-like activity. Making use of porous nylon membrane， a membrane-based probe was fabricated for probing CN-and the LOD of 100 nm was obtained for CN-which was lower than the maximum level of CN-developed by WHO. In contrast with other laboratorial wastes， the membrane-based probe for CNshowed the most colorless， proving the potential application on real sample.

Arsenic（Ⅴ） is one of the most toxic elements present in water， mainly exhibiting as H2AsO-4and divalent HAsO2-4species in upper oxygenated groundwater［88］. It can interact with enzymes or proteins containing nitrogen， oxygen and sulfur， inhibiting the catalytic activity of enzymes or inactivating proteins［89］. In order to prevent As（Ⅴ） to do harm to human health， a sensitive and selective method should be constructed for As（Ⅴ） detection. Wen with his coworkers reported a colorimetric sensor for the detection of As（Ⅴ） based on CoOOH nanozyme with ABTS as chromogenic substrate （Fig.23）［90］. Actually， CoOOH possessed peroxidase-like activity that could be used to catalyze ABTS convert into green-colored oxidation products. However， an inhibitory effect on the peroxidase-like activity of CoOOH nanozyme could be observed while adding As（Ⅴ） into the solution on account of electrostatic attraction and surface complexation between CoOOH nanozymes and As（Ⅴ）. Taking the property of As（Ⅴ） into consideration， a sensitive colorimetric sensor was proposed and it wouldn′t be interfered by other ions except for PO3-4. Although PO3-4had interference effect toward As（Ⅴ） detection， the problem could be overcameviaadding Ca2+to form precipitation with PO3-4. Therefore， a sensitive and specific colorimetric assay for As（Ⅴ） could be obtained and might be used to sense As（Ⅴ） in real sample.

圖23 CoOOH納米片作為探針用比色法檢測砷酸鹽的示意圖［90］Fig.23 Illustration of the colorimetric method of CoOOH nanoflakes as probe for arsenate detection［90］

In a word， the nanozymes are very promising in environmental pollutant monitoring and analysis. In the future， researchers should develop more environmentally friendly and economical non-metal-based nanozymes to solve the problem of environmental pollution. In particular， nanoenzymes， which have multiple functions such as sensing and adsorption， are highly desirable in addressing environmental pollution.

4 Conclusion and Outlook

As substitute of natural enzymes， nanozymes have received great interest from researchers since 2007. In comparison to enzymes， nanozymes have the advantages of easy mass production， high stability， tunable catalytic activity and types， low cost and so forth. As investigated， they show great potential in many fields including biological imaging， cancer diagnosis and treatment， food testing， environmental protection andetc.It has been demonstrated that nanozymes could be used in sensing application with high sensitivity， stability and selectivity and even better than some natural enzymes. But if we want to make nanozymes have practical application in sensing， some challenges nanozymes are facing should be realized. First， the activity and selectivity of nanozymes should be further improved. Some nanozymes show the activity comparable to enzymes or even better but the selectivity of nanozymes is less than that of enzymes. In other hand， if the selectivity of nanozymes is enhanced by surface modification， the activity of nanozymes may not seem to be much of an improvement or even worse. Thus， it′s urgent to find a new strategy to get balance between the activity and selectivity. Second， only a few papers have investigated environmental toxicity and biotoxicity of nanozymes which may be derived from the leaching ions of nanozymes. It′s of great significance to ensure nanozymes with no toxicity that can′t contaminate environment and do harm to living organisms. Third， the types of nanozymes need to be explored more and nanozymes with multifunctional enzyme-like activity are also attractive in the further study. As we known， there are only two major categories of nanozymes， oxidoreductases and hydrolases， and large quantities of enzymatic reactions are unable to occur with nanozymes as catalyst owing to lack of corresponding activity. More importantly， sometimes if we want to detect some substances like glucose， organophosphorus pesticides and so on， a cascade reaction should be constructed by combining natural enzymes with nanozymes which will bring some problems such as unable to work in the same conditions and high cost. Finally， the cyclic utilization of nanozymes is one of the major problems in particle application.Although some strategies have been proposed to improve the cyclic utilization of nanozymes， it′s not satisfactory enough due to the loss of activity in recycling progress. Nanozymes have showed great promise in sensing application and they will be used in real sample soon if the problems are solved.

NotesThe authors declare no competing financial interest.