State of the art and applications in nanostructured biocatalysis

Abstract

Enzymes have been widely studied for their excellent efficiency, selectivity and environmentally benign nature. However, the development of their biocatalytic applications is severely limited by the lack of stability and reusability. Nanostructured materials have been demonstrated as efficient hosts for the immobilization of enzyme due to their large surface areas as well as higher activities and long-term stability. Furthermore, nanomaterials with enzyme-like characteristics are explored as highly stable and low-cost alternatives to mimic the structures and functions of naturally occurring enzymes. This review offers an overview on the research status of enzymatic immobilization adapted from nanostructured materials and nanozymes in numerous fields, from biosensing and biocatalysis to environmental remediation, with emphasis focused on current challenges and future directions in highly selective and efficient biocatalysis.

Keywords:

• Nanostructured materials
• biocatalysis
• enzyme immobilization
• nanocarrier
• nanozyme

Introduction

Biological catalysis is the application of enzymes, microbial cells or plant and animal cells as biocatalysts for the catalytic reaction [1]. The spectacular advances of biocatalysis in the faster reaction rates, higher substrate specificity and greener reaction process (mild pH, temperature and pressure) have led to an exponential growth in the number of enzyme catalysts. However, they also have disadvantages such as unstable structure, aggregation and high cost of enzyme separation and purification [2–4]. Therefore, the immobilization of enzymes is a promising technique to allow the catalysts to be retained within the reactor while reagents flow through the system. Enzyme immobilization decreases the required quantity of enzyme, simplifies product work-up and potentially increases enzyme stability [5, 6]. At present, the preparation of immobilized enzymes has been generally classified into two major categories including physical and chemical immobilization methods such as adsorption, entrapment [7, 8], covalent binding and cross-linking [9, 10] (Figure 1).

Figure 1. Representive methods applied during the enzymatic immobilization. Absorption (a). Entrapment (b). Covalent attachment (c). Cross-linking (d).

Nanostructured material is a key issue for enzymes immobilization. The immobilized materials are generally organic macromolecular polymers (Chitosan, cellulose) or inorganic materials (SiO₂) in the past [11]. However, the stability of enzymes immobilized in these materials is relatively low, and due to the small surface area, their enzyme load has been limited. Nanomaterials have desirable characteristics such as their good biocompatibility, large surface area, high mass transfer efficiency, high enzyme load, hardness and defined geometry [3, 5, 12–15]. At present, nanomaterials are used for the immobilization of enzymes including magnetic nanomaterials [16] and non-magnetic nanomaterials, in which non-magnetic nanomaterials can be divided into inorganic nanoparticles (Au nanoparticles, mesoporous silica nanoparticle, carbon nanotubes) [17–19], metal-organic frameworks (MOFs) [7, 20].

Some nanomaterials with enzyme-like kinetics characteristics can efficiently catalyze specific reactions under mild conditions, and are termed nanozymes [21]. Studies have attempted to apply them to manipulate enzymatic reactions in a wide range of applications [22–24]. In 2007, Yan’s group first reported that the Fe₃O₄ magnetic nanoparticles (MNPs) had peroxidase-like activity [25]. The MNPs had Michaelis–Menten kinetics characteristics similar to the natural enzyme, and the affinity (K_M) with TMB (3,3′,5,5′-tetramethylbenzidine) was even 40 times higher than that of horseradish peroxidase (HRP) [26]. At present, the nanomaterials with enzyme-like activity include metal elements, metal oxides, carbon-based frameworks and MOFs [27–30]. Different types of enzyme activities have been mimicked by nanomaterials including peroxidase, catalase, oxidase and superoxide dismutase (SOD) (Figure 2). The characteristics of nanomaterials as enzymes are extraordinarily suitable for large-scale applications which have high stability, excellent reusability, low cost and green nature.

Figure 2. Recent applications of nanozymes and proposed mechanisms (A: substrate; POD: peroxidase; CAT: catalase; OXD: oxidase; SOD: superoxide dismutase.). Reprinted with permission from Ref. [21], Huang, Y., Ren, J. and Qu, X., 2019. Nanozymes: Classification, catalytic mechanisms, activity regulation, and applications. Chemical Review, 119(6), pp.4357–4412.

Table 1 lists both the advantages and disadvantages of nano-carrier immobilized enzymes and nanozymes. This review begins with a survey of the recent developments and application of the two types of catalysts. The challenges and prospects are also discussed.

Table 1. Advantages and disadvantages of enzyme immobilization and nanozyme.

	Enzyme immobilization	Nanozyme
Advantages	Excellent substrate specificity	High stability
	High reaction selectivity High catalytic activity	Good reproducibility of results
	Wide range of catalysis	Easy separation
	Design catalysis activity via protein engineering	Good reusability
	Good biocompatibility	Low cost
		Multi-enzyme mimetic activities in the same nanomaterial
Disadvantages	Loss of enzyme activity	Limited range of catalysis
	Limited transfer of substrates and products	Low substrate specificity
	High cost	Low reaction selectivity
	High energy and time consumption of immobilization processes	Potential biotoxicity
	Low durability

Nano-carrier immobilized enzymes

Immobilization of enzymes is a promising solution to overcome the disadvantages of the natural enzymes. The stability of enzymes can be greatly improved by physical adsorption or covalent crosslinking, and the enzymes can also be recycled through simple methods. Moreover, a relative high enzyme-load can be achieved by benefiting from their high surface area. As shown in Table 2, nanomaterials have been used to immobilize enzymes such as horseradish peroxidase (HRP), laccase, versatile peroxidase, polyphenol oxidase and β-galactosidase. These immobilized enzymes have been used in the fields of hydrogen peroxide (H₂O₂) detection, depolymerization of lignocellulosic biomass into vanillin, oxidation of phenolic compounds and transglycosylation of lactose.

Table 2. The applications and performances of nanomaterial immobilized enzymes.

Nanomaterial^a	Enzyme	Immobilization method^b	Application	Performance study	Ref.
Fe3O4 - chitosan	Laccase	GA covalent coupling	Degradation of chlorophenol	Retained more than 76% of its initial activity after 10 cycles	[16]
Fe3O4- polydopamine	Horseradish peroxidase	Covalent attachment	Detection of H2O2	Linear range: 0.5-30 μmol/L; Detection limit: 0.23 μmol/L	[31]
Fe3O4/SiO2- tannic acid	Catalase	APTES/GA covalent coupling	Stability enhancement	Maintained about 55% of its initial activity after 9 cycles	[32]
Fe3O4 -CMC-IL	Penicillin G acylase	EDC/NHS covalent coupling	Stability enhancement	Maintained 92.6 % of its initial activity after 10 cycles	[33]
Fe3O4 -M-MWNTs	Porcine pancreas lipase	Physical adsorption	Synthesis of substituted 2H-chromenes	The yield remained above 83% after 5 cycles	[34]
Fe3O4/h-CNT /ZrO2	Glucose oxidase	GA covalent coupling	Detection of glucose	Linear range: 0-1.6 mmol/L; Detection limit: 6.9 μmol/L	[35]
MSMS	Laccase and versatile peroxidase	–	Conversion of lignocellulosic biomass to vanillin	Yield of vanillin reached 27% within 6 h	[36]
AuNPs-MoS2	Laccase	Nafion coating	Detect catechol	Linear range: 2-2000 μmol/L; Detection limit: 2 μmol/L; Remained about 90% of its initial response after 3 months	[37]
AuNPs	Horseradish peroxidase	Physical adsorption	Immunoassay for chloramphenicol	Linear range: 0.001–10 ng/mL; Detection limit: 0.33 pg/mL; Stable up to 1 month	[38]
AuNPs-Pt-DEBP. AgNPs-Pt-DEBP. AuNPs-BI. AgNPs-BI	Pseudomonas fluorescens lipase	Physical adsorption	Stability enhancement	–	[39]
Chitosan -AuNPs/ montmorillonite.	Polyphenol oxidase	Physical adsorption	Oxidation of phenolic compounds	At pH 3 and 11, retained 53.1% and 75.4% of its activity within 8 h. Maintained about 59.1% of its initial activity after 10 cycles	[40]
Carbon nanofibers–AuNPs	Tyrosinase	GA crosslinking	Detection of ferulic acid in cosmetics	Linear range: 0.1-1.6 μmol/L; Detection limit: 2.89 nmol/L; Quantification limit: 9.64 nmol/L	[41]
SiO2 NPs	β-galactosidase	Encapsulation	Transglycosylation of lactose into N- acetyllactosamine	No significant reduction in the molar yield was observed after 8 cycles	[42]
SiO2- nanocrystalline cellulose- polyethersulfone	Candida rugosa lipase	GA crosslinking	Synthesis of pentyl valerate	Yield reached 91.3% within 3 h	[43]
Mesoporous SiO2-NH2	C. antarctica lipase	GA crosslinking	Synthesis of ethyl levulinate	Yield maintained above at 68 % after 7 cycles	[44]
Macroporous SiO2	Phospholipase D	Physical adsorption	Synthesis of phosphatidylserie	Remained about 90% of its initial activity after 28 days. Yield maintained at 79.3% after 12 cycles	[45]
Mesoporous SiO2-NH2	Xylanase	Physical adsorption	Lignocellulosic hydrolysis	The yield remained about 75% after 9 cycles	[46]
UiO-66-NH2	Lipase AYS	Precipitation-crosslinking	Kinetic resolution of 2-(4-Methylphenyl) propanoic acid enantiomers	Yield reached 72.57% with 93.20% of enantiomeric excess	[47]
UiO-66-porous polyvinylidene fluoride	Lipase	Physical adsorption	Separation from reaction system	Retained 70 % of its initial activity after 100 min at 70 °C. Remained above 50% of its initial activity after 5 cycles	[48]
UiO-66	Pseudomonas fluorescence lipase	Covalent cross-linking	Catalytic enantioselective hydrolysis and transesterification reaction	No significant reduction in the activity was observed after 3 cycles	[49]
ZIF-8	β- galactosidase and glucose oxidase	Encapsulation	Fluorescent detection of lactose	Linear range: 125-1000 μmol/L; Detection limit: 41.67 μmol/L; Remained about 76.83 % of its initial activity after 3 days	[50]
ZIF-8- cellulose acetate	Laccase and glucose oxidase	Encapsulation	Detection of glucose	Linear range: 1-10 mmol/L; Detection limit: 5.35 μmol/L; The time of stable working up to 15 h	[51]
SOM-ZIF-8	Horseradish peroxidase	Encapsulation	Biodegradation of hazardous dyes	Remained about 85% of its initial activity after 5 cycles	[52]
ZIF-8-GO and CABDC-GO	Lysozyme	Encapsulation	Development of a large substrate biocatalysis system	–	[53]
ZIF-8-Fe	Glucose oxidase	Biomimetic mineralization	Development of a cascade catalytic reactor	Retained 92 % of its initial activity after 30 min at pH = 4. Reused at least 5 times	[54]
ZIF-8	Trehalose synthase and glucose isomerase	Encapsulation	Coproduction of trehalose and fructose	Yield of trehalose reached 67.82% within 16.8 h. Yield remained above 50% after 20 cycles	[55]

a IL: ionic liquid. M-MWNTs: magnetic multiwalled carbon nanotubes. h-CNT: hybrid carbon nanotubes. MSMS: magnetic silica microspheres. Pt-DEBP: trans, trans-[dithiodibis(tributylphosphine) diplatinum (II)-4,4′-diethynylbiphenyl]. BI: 4,4′-dithiol-biphenyl. SOM: single-crystal ordered microporous. GO: graphite oxide.

b GA: glutaraldehyde. APTES: 3-aminopropyltriethoxysilane. EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. NHS: N-hydroxysuccinimide.

Magnetic nanomaterials

Magnetic nanomaterials are composed of iron oxide nanoparticles (NPs) with superparamagnetic properties, which have been extensively studied due to their stability and easy separation [56]. The enzymes are immobilized on magnetic nanomaterial by covalent bonds and their stability can be improved in complex environments, which could reduce the leakage of enzymes and their cost of industrial application, and improve their catalytic activity [57]. With an external magnetic field, they can be separated easily and rapidly [58].

Sardaremelli et al. [31] reported the use of Fe₃O₄ magnetic nanoparticles (MNPs) which were coated with polydopamine (PDA-MNPs) to support HRP for the detection of H₂O₂. The PDA-MNPs were cast on the surface of the glassy carbon electrode (GCE) that was modified by poly (L-arginine/toluidine blue), so that they could be separated and recycled easily. With the excellent specificity of HRP, the bio-electrochemical sensor has the detection range of 0.5–30 μmol/L with a low limit of 0.23 μmol/L. And there is no significant change in the detection reproducibility after 30 cycles.

Chlorophenol represents an important intermediate of pesticides that is difficult to degrade in the environment and would get enriched in organisms through natural circulation. Therefore, it would irritate the skin and mucous membrane, and even seriously damage the nervous and respiratory system [16]. Zhang et al. [16] completed the immobilization of laccase with chitosan-coated Fe₃O₄ MNPs (Laccase-Fe₃O₄@CTS), which then oxidize chlorophenols to produce quinones to achieve the rapid biodegradation of chlorophenol (Figure 3). Chitosan could introduce a large number of amino groups on the surface of Fe₃O₄ MNPs, which could generate electrostatic repulsion between nanoparticles to prevent agglomeration. Not only that, chitosan also could be used for immobilization of laccase by forming covalent bonds with laccase molecules. The immobilized laccase could preserve 75.2% of the initial activity after 4 weeks of storage at 4 °C, whereas the free laccase preserved only 40.2%. The oxidation results of chlorophenol showed that 91.4% and 75.5% of 2,4-dichlorophenol (2,4-DCP) and 4-chlorophenol (4-CP) could be converted by Laccase-Fe₃O₄@CTS at room temperature for 12 h, respectively. Furthermore, Laccase-Fe₃O₄@CTS could still achieve 75.8% and 57.4% conversion rates after 10 cycles.

Figure 3. The oxidation of 2,4-dichloro-phenol and 4-chloro-phenol catalyzed by Laccase-Fe₃O₄@CTS.

Liu et al. [34] used multiwalled carbon nanotubes (MWNTs) and Fe₃O₄ MNPs to form magnetic multiwalled carbon nanotubes (M-MWNTs), and then immobilized the porcine pancreas lipase (PPL) on the surface of M-MWNTs (PPL-M-MWNTs) through hydrophobic interaction, which was used for the synthesis of 2H-chromenes (Figure 4). Lipase was adsorbed on the hydrophobic surface, and the “lid" of the enzyme could be opened through interfacial activation mechanism, so the active center was fully exposed thus improving the catalytic performance. The synthesis of 2H-chromenes by using enzymes could solve the shortcomings of chemical processes, such as low yield, long reaction time and complex reactions, and it is suitable for a variety of substrates. Nevertheless, the enzymes were immobilized on M-MWNTs through physical absorption, which is why they were prone to leakage. These results indicate that the yield of 2H-chromenes was significantly reduced after 5 cycles. Therefore, M-MWNTs can form multi-point covalent connection with PPL in the future to improve the fixation capacity and reduce the enzyme leakage.

Figure 4. The synthesis of 2H-chromenes. Reprinted with permission from [34].

Saikia et al. [36] co-immobilized laccase and versatile peroxidase in magnetic silica microspheres (MSMS) to depolymerize waste lignin into an important flavoring agent vanillin (Figure 5). Laccase had the ability to depolymerize the phenolic compounds of lignin and versatile peroxidase could oxidize phenolic and non-phenolic compounds, which could be obtained in high yield by the synergistic action of two enzymes. Under the optimal conditions (pH = 6, 6 h, and 30 °C), the yield of vanillin was improved up to 27.1%, and was higher than individual laccase and peroxidase system. Based on the synergistic effect of the two enzymes, lignin can be converted into important chemical raw materials to realize the efficient utilization, which provides a facile access to the recycling of a large amount of waste lignin.

Figure 5. The synthesis of vanillin.

Gold nanoparticles

Gold nanoparticles (Au NPs) are suitable for electrochemical detection due to their unique high conductivity. Their application has been widely studied in the fields of environmental pollutants and veterinary drug residues detection.

Zhang et al. [37] developed a novel laccase based electrochemical biosensor for the detection of catechol. They created a laccase-based biosensor by a new nanocomposite that self-assembled of AuNPs and MoS₂ nanosheets, and Nafion, an effective electrochemical binder, was used for immobilization of the laccase. MoS₂ is a two-dimensional (2 D) layered nanomaterial with some properties of good biocompatibility and mechanical strength, high specific surface area, easy recyclability but poor conductivity. AuNPs not only could immobilize enzymes, but also have excellent conductivity which could effectively improve the sensitivity of the electrochemical sensor and reduce the detection limit. The combination of the two materials can make up for their respective shortcomings. The detection range of the electrochemical sensor was 2–2000 μmol/L and the limit of detection (LOD) was 2 μmol/L (S/N = 3). After repeating the test 10 times, the relative standard deviation (RSD) of the detection results was 1.2%, indicating a good reproducibility of results. With the help of Nafion, the biosensor could still retain 90% of its initial value after storage at 4 °C for one month, indicating that the stability had been enhanced effectively.

Luo et al. [38] immobilized horseradish peroxidase (HRP) on Au NPs for the detection of chloramphenicol (CAP) in food (Figure 6). The concentration of CAP in samples was rapidly detected by using flow injection chemiluminescence immunoassay (FI-CLIA) with a syringe pump. The detection range was 0.001–10 ng/mL (R²= 0.9961) and the LOD was 0.33 pg/mL. Compared with other detection methods with large equipment, such as GC-MS with a detection limit of 0.05 ng/mL, this method had a lower LOD and was more convenient. The recovery rates of the standard addition method in shrimp and honey samples were in the range of 83.5%–99.6%, indicating that this method could be used for accurate detection of real samples.

Figure 6. The detection of chloramphenicol (CAP). Reprinted with permission from Ref. [38].

Mesoporous silica

Mesoporous silica has the advantages of large specific surface area, easy surface modification, excellent chemical inertia and biocompatibility, as well as certain mechanical strength. The micropores on the surface are suitable for the transfer of substrates, and further improve the stability of enzymes [59].

Jo et al. [60] used a template-free interfacial condensation method to synthesize the hollow silica nanocapsules to embed enzymes. The efficiency of coupled enzymatic reactions could be enhanced by constructing multi-enzymes into hollow silica nanocapsules together. The SiO₂ membrane could protect the enzymes from the harsh environment and reduce leakage. It did not limit the mass transfer of substrates, which ensured the catalytic efficiency of enzymes.

Mukundan et al. [61] used spray drying method to synthesize silica microcapsules by silica nanoparticles, and at the same time entrapped the S. lactis cells in them. The cells expressed β-galactosidase activity that could catalyse the hydrolysis of lactose into glucose and galactose. The immobilized cells had the higher activity of lactose hydrolysis than free cells (K_M=8.33 mmol/L and 4.16 mmol/L, V_max= 71.43 μmol/L min and 125 μmol/L min for free cells and immobilized cells, respectively). This may be because part of the enzyme was released during the encapsulation process, and the catalytic activity was improved.

Elias et al. [43] prepared nanocellulose (NC) and nano-sized SiO₂ by the sustainable biomaterials-oil palm leaves (OPL). Then a nanofiller was synthesized by using NC modified SiO₂ NPs to immobilize Candida rugosa lipase (CRL). Finally, the nanofiller has combined with polyethersulfone (PES) to form a membrane material (NC-SiO₂-PES/CRL) with stronger mechanical stability which was used for the synthesis of pentyl valerate (PeVa) (Figure 7a). The results showed that the yield of PeVa could reach 91.3% within 3 h, which had great advantages in terms of cost and energy consumption compared to previously developed biocatalysts (reaction need 17 h or even 7–8 days).

Figure 7. The synthesis of pentyl valerate (a) and ethyl levulinate (b).

The advantages of mesoporous SiO₂ nanomaterials have also been reflected by the research of Jia et al [44]. They prepared mesoporous silica nanoflowers by a reverse microemulsion method to immobilize lipase and used them for the synthesis of ethyl levulinate (Figure 7b). The yield of ethyl levulinate achieved by immobilized lipase was 99.5% at 40 °C for 8 h, which was much higher than that by free lipase (67.9%). The yield could be preserved (68%) after 7 cycles, while the free enzymes or inorganic acid cannot be recycled.

Wu et al. [45] reported the immobilized phospholipase D (PLD) using macroporous SiO₂ NPs modified by the epoxy resin-based cationic polymer polyethylenimine (PEI) as a carrier. Phosphatidylserine (PS) could be synthesized from phosphatidylcholine (PC) by using the PLD (Figure 8). The cationic and amphiphilic PEI coating had a good affinity not only to negatively charged PLD molecules but also to water-insoluble PC, which increased the stability of enzymes and the concentration of substrates around enzymes. A great impact of pH was observed during the immobilization of enzymes. The isoelectric point of PLD was about 5.39 and when pH = 5.39 ∼ 7.00, it could be well adsorbed with PEI coating. When pH > 7.00, the negative charges on the surface of PLD would generate electrostatic repulsion between the two enzyme molecules. The yield of PS catalyzed by immobilized PLD reached 96.20%, which was much higher than the free PLD (68.10%). More importantly, the yield could still achieve 79.30% after 12 cycles.

Figure 8. The synthesys of phosphatidylserine (PS).

Metal–organic frameworks (MOFs)

Metal-organic frameworks (MOFs) are a type of porous materials that are composed of metal nodes and organic frameworks [62]. Compared with traditional porous materials, MOFs have much higher porosity and specific surface area [63]. Furthermore, they can be synthesized by a variety of metals and organic substances. There are two types of enzyme immobilization methods with MOFs: (1) to immobilize enzymes on the surface of MOFs by physical adsorption or covalent crosslinking, (2) to encapsulate enzymes in the cavities of MOFs.

Yuan et al. [49] prepared UiO-66 with zirconium chloride and terephthalic acid, then used it to immobilize the Pseudomonas fluorescence lipase (PFL) that was coated with polyethylene glycol (PFL-PEG-UiO-66). The immobilized PFL had been used for the kinetic resolution of 2-(4-hydroxyphenyl) propionic acid ethyl ester enantiomers (2-HPPAEE) and 4-methoxymandelic acid enantiomers (4-MMA) (Figure 9). PFL was successfully immobilized on the surface of UiO-66 by the covalent cross-linking action and this prevented the enzymes from aggregation. Because the diameter of PFL was larger than the micropores on the surface of UiO-66, the PFL molecules could not enter the interior of UiO-66. The results of the kinetic study showed that PFL-PEG-UiO-66 had a higher affinity for the substrates than free PFL (K_M=69.07 mmol/L vs. 117.43 mmol/L). This might be due to the changes in the structure of enzymes during the immobilization process, making the active center more accessible. The conversion rates could achieve 47% and 49% for 2-HPPAEE (ee > 99%) and 4-MMA (ee > 99%), respectively. However, the enzymes immobilized on the surface of MOFs were exposed to the external environment that would cause enzymes leakage or inactivation. The data showed that the loss of 2-HPPAEE conversion rate was only 2.5%–5% in the first four cycles of the enzymes, but rapidly decreased to 18.5% in the next two cycles. Similarly, the conversion rate of 4-MMA quickly dropped to 13% in the fifth cycle.

Figure 9. The kinetic resolution of 2-(4-hydroxyphenyl) propionic acid ethyl ester (2-HPPAEE) enantiomers and 4-methoxymandelic acid(4-MMA) enantiomers.

The second method is designed to embed enzymes in the internal cavities of the MOFs to maintain the stability of the enzymes and stop the leakage. However, this method is not suitable for the enzymes whose diameters are larger than the micropores diameter on the surface of MOFs.

Li et al. [52] prepared single-crystal ordered macroporous ZIF-8 (SOM-ZIF-8) by adding 3 D-polystyrene sphere (PS) template during the synthetic preparation of ZIF-8. The diameter of micropores on the surface of ZIF-8 was expanded, while the inner micropores were retained. Therefore, HRP could directly enter the interior of SOM-ZIF-8 through macropores on the surface and be trapped in the interior cavities. The immobilized HRP was used for biodegradation of hazardous dyes (methyl orange, Congo red, rhodamine B and rhodamine 6 G) and could retain 85% of its initial activity after 5 cycles. Compared with enzymes immobilized by covalent cross-linking, the reusability of enzymes, in this case, was improved effectively.

There was a new type of method defined as in-situ growth, in which enzymes were encapsulated in the MOFs in situ during the formation process of MOFs. And the micropores of MOFs could provide ample channels for the mass transfer of substrates [53].

Huang et al. [54] encapsulated glucose oxidase (GOx) within Fe-ZIF-8(GOx@Fe-ZIF-8) by this method. The Fe-ZIF-8 had peroxidase-like activity and could be used to detect glucose in combination with GOx (Figure 10). The addition of Fe²⁺ had three main functions: (1) to expand the pore size of ZIF-8, (2) to generate the Fe-GOx complex and increase the enzyme load, (3) to accelerate the formation of ZIF-8 and make it with peroxidase-like activity. The cascade reactions catalyzed by GOx and Fe-ZIF-8 were completed in the same space, and the catalytic efficiency was synergistically enhanced. The glucose concentrations in multiple samples were measured by GOx@Fe-ZIF-8 and a glucometer, and the results from both were similar (RSD = 2.4%–17.4%), indicating that it was suitable for the detection of real samples.

Figure 10. The detection mechanism of glucose concentration by Fe-ZIF-8 combined with glucose oxidase (GOx).

Nanozymes

Immobilization of enzymes on nanomaterials can partly solve the shortcomings of natural enzymes, such as reusability and stability. However, the inherent protein structure of natural enzymes cannot exist stably under extreme conditions and can easily be decomposed by proteases. At the same time, the costs of separation and purification are relatively high. To some extent, their large-scale applications have been limited by the drawbacks above. With the development of nanotechnology, the inherent enzyme-like activities of nanomaterials have attracted a lot of attention. As shown in Table 3, nanozymes have been applied in many fields such as tumor therapy, chiral catalysis, cell protection, antibacterial activity, biosensor, environmental protection [64, 76, 87, 101–103]. In the following part, the research progress and applications of nanozymes in the past two years will be described according to the classification of materials.

Table 3. Nanomaterials with enzyme-like activities.

Nanomaterial^a	Enzyme	Application^b	Ref.
Au-silica nanohybrid	Glucose-oxidase	Glucose oxidation	[19]
Au-Ag@HA NPs	Peroxidase	Enhanced cancer therapy	[64]
Au-Pt@SF NPs	Oxidase and peroxidase	Enhanced cancer therapy	[65]
Pt-C	Oxidase and peroxidase	Photodynamic and catalytic synergistic tumor therapy	[66]
Au-Pt/SiO2	Peroxidase	Immunosorbent assay for aflatoxin B1	[67]
Pt-Ni@CA hollow nanospheres	Peroxidase	Human serum albumin (HSA) detection	[68]
Au@MagSiO2	Peroxidase	Glucose detection in human whole blood	[69]
Au–Pt core/shell nanorod	Peroxidase	Measles virus diagnosis	[70]
Au-Pt	Peroxidase	Surface enhanced Raman scattering	[71]
Au-Pt NPs	Peroxidase	Ag^+ detection	[72]
Fe-Cu-DA/NC	Cytochrome c oxidase	Complete oxygen reduction reaction	[73]
Cu- polydopamine	Peroxidase	Biosensing and catalytic reduction of organic dyes	[74]
Ru NPs-MWCNTs	Peroxidase	Glucose detection	[75]
CuxO nanoparticle clusters	Peroxidase, superoxide dismutase, catalase, and glutathione peroxidase	Ameliorating Parkinson’s disease	[76]
Au-Pt-Co@DMSN	Peroxidase and catalase	Decontamination of two kinds of wastewater	[77]
Fe3O4 NPs	Peroxidase	Enantioselective catalysis	[78]
Fe3O4-C nanowires	Peroxidase	Detection of platelet-derived growth factor BB	[79]
FeWOX nanosheets	Peroxidase	Sensing cancer via photoacoustic imaging	[80]
CeO2 nanorod	Oxidative	Tumor therapy	[81]
CeO2 NPs	Peroxidase	Simultaneous phosphoprotein isolation and detection	[82]
CoFe2O4 NPs	Peroxidase	Kanamycin detection	[83]
MnO2 nanosheets	Peroxidase	On-site monitoring of oxalate	[84]
MnO2-BSA	Peroxidase	Real-time imaging and enhanced phototherapy of esophageal cancer	[85]
MnO2-mesoporous silica	Catalase	Enhancing ROS‐mediated breast cancer therapy	[86]
EPC	Oxidase and peroxidase	Biosensor for acid phosphatase and H2O2	[29]
Nanodiamonds	Peroxidase	Against periodontal bacterial infection	[87]
Rosette-shaped graphitic carbon nitride	Peroxidase	Glucose detection	[88]
G-C3N4	Peroxidase and glucose-oxidase	H2O2 and glucose detection	[89]
G-C3N4/Pt^2+	Peroxidase	H2O2 and glucose detection	[90]
G-C3N4/CeO2	Peroxidase	Mercury (II) detection	[91]
MoS2/rGO	Peroxidase	Smart bacterial killing	[92]
Nitrogen-doped porous carbon nanospheres	Oxidase, peroxidase, catalase and superoxide dismutase	ROS regulation for tumor catalytic therapy	[93]
GOQD	Peroxidase	Photocatalytic degradation of organic dyes	[94]
GQDs	Peroxidase	Enhanced cancer therapy	[95]
NEQC-340	Peroxidase	Glutathione determination	[96]
MIL-88	Peroxidase	Detection of aflatoxin B1	[97]
MOF-818	Catechol oxidase	Catalysis of catechol oxidation	[98]
MIL-88/Pt/MIL-88	Peroxidase	Ultrasensitive exosomal miRNA analysis	[99]
Prussian blue NPs	Peroxidase	Detect glycated albumin	[100]

a HA: hyaluronic acid. SF: silk fibroin. CA: citric acid. MagSiO₂: magnetic SiO₂. DA/NC: three-dimensional porous N-doped carbon. DMSN: dendrimer-like macroporous silica nanoparticles. BSA: bovine serum albumin. EPC: enteromorpha prolifera-based hierarchical porous carbon. rGO: reduced graphene oxide. GOQD: graphene oxide quantum dots. GQDs: graphene quantum dots. NEQC-340: nanosized electroactive quasicoral-340.

b ROS: reactive oxygen species.

Metal based

Compared with single-metal complexes, multi metal complexes could exhibit synergistic catalytic activity and enhance the catalytic efficiency. For example, Au-Pt NPs showed much higher peroxidase-like activity than PtNPs or AuNPs alone [104]. AuNPs have been widely used as a sensitizer in tumor radiation therapy due to their extensive biocompatibility, stability and strong photoelectric absorption ability. However, their non-specificity in cells may cause damage to normal cells.

In 2020, Chong et al. [64] prepared Au-Ag NPs modified with hyaluronic acid (Au-Ag@HA NPs) for enhancing the therapeutic effect of tumor radiotherapy. Through the surface modification of hyaluronic acid (HA), the CD44 receptor that was overexpressed on the surface of 4T1 breast cancer cells was targeted by Au-Ag NPs, while the concentration of Au-Ag NPs in tumor cells was increased and their damage to normal cells was reduced.

Au-Ag NPs showed peroxidase (POD) activity in the acidic microenvironment of tumor cells. When Au-Ag NPs got into the tumor cells, abundant ROS (·OH) could be generated by the reduction of H₂O₂ via the Fenton-like reaction and X-ray irradiation. The highly reactive ROS could not only kill tumor cells, but also further facilitate the generation of toxic Ag⁺ from Au-Ag NPs, resulting in synergistically enhanced antitumor efficacy (Figure 11). When without Au-Ag NPs, the tumor therapeutic was limited and almost depends on the inefficient decomposition of H₂O₂ to ·OH by X-ray irradiation. More importantly, Au-Ag NPs displayed superoxide dismutase (SOD)-like activity in the neutral environment of normal cells, which could scavenge ROS and protect normal cells. There are numerous metal-metal and metal-nonmetal complexes that have been reported toward excellent therapeutic effects on tumors, such as Au-Pt NPs and Pt-C NPs [65, 66].

Figure 11. Schematic illustration shows synergetic multimodal for tumor therapy by Au-Ag@HA nanoparticles. Reprinted with permission from [64].

Natural enzymes used in enzyme-linked immunosorbent assay (ELISA) have some drawbacks such as poor stability and difficult storage. Therefore, nanozyme-linked immunosorbent assay (NLISA) was developed. It uses nanozymes to replace natural enzymes [67].

The noble metal Pt has excellent peroxidase-like activity and is used for the detection of a variety of substrates. However, the large-scale application of Pt is limited by its high costs. Thus, the application of Pt can be reduced by mixing Pt with other cheap metals to form multi-metal complexes. Gupta et al. [68] developed novel citric acid-functionalized Pt-Ni hollow nanospheres (CA@Pt-Ni hNS) to replace HRP for the ELISA of human serum albumin (HSA). Citric acid (CA) not only stabilized Pt/Ni hNS, but also provided a negative surface charge of hNS for the attraction of the chromogenic substrate TMB. CA@Pt-Ni hNS showed stronger affinity on both TMB and H₂O₂ than HRP (K_M=0.18, 2.5 mmol/L vs. 0.43, 3.0 mmol/L for CA@Pt-Ni hNS and HRP, respectively), and the NLISA showed better detection performance and stability than ELISA. In other reports, nanozymes applied in detection almost all showed superior performance and stability than natural enzymes. For example, Au-Pt NPs for the detection of aflatoxin B₁[72], Au-Mag SiO₂ for the detection of glucose in human whole blood [69], Au-Pt NPs for the detection of Ag⁺ [72].

In addition to noble metals, some nanozymes composed of cheap metals such as copper and iron have also been reported. Du et al. [73] prepared a nanozyme by embedding Fe-Cu dual atomic sites in three-dimensional porous N-doped carbon (Fe Cu-DA/NC), which has cytochrome c oxidase (CcO) activity and can be used to accelerate the oxygen reduction reaction (ORR). ORR is a chemical reaction occurring in some batteries and its slow kinetics limits the performance of batteries. The electrocatalytic ORR activity of Fe Cu-DA/NC is comparable to or even better than that of commercial Pt/C. In addition, the Fe Cu-DA/NC showed better long-term stability and superior resistance to methanol poisoning than Pt/C in both alkaline and acidic environments.

Metallic oxide based

Substrate specificity is one of the most important properties of natural enzymes, which means enzymes can catalyze only one of a variety of substances. This is one of the most common differences between nanozymes and natural enzymes, because pure nanomaterials exhibit little substrate specificity. Chiral separation of racemates and asymmetric catalysis of substrates are usually crucial in the process industrial pharmaceutical-production. Therefore, to narrow the gap between nanozymes and natural enzymes, methods intend to improve the substrate specificity, especially the enantioselectivity of nanozymes [101], such as modifying chemical compounds, amino acids or DNA on the surface of nanozymes.

Zhou et al. [78] prepared a series of chiral nanozymes (Fe₃O₄@Poly(AA)) by using Fe₃O₄ NPs as a catalytic center and adopting different kinds of amino acids-appended chiral polymers as a chiral selector. The surface of Fe₃O₄ was covered with a layer of SiO₂, and then the chiral amino acid polymer was linked to the surface of SiO₂, finally the SiO₂ layer was removed in a high-concentration NaOH solution to form the yolk-shell structure Fe₃O₄@Poly(AA) NPs. The advantage of this structure was that a lot of chiral ligands could be introduced into the shell to improve the chiral selectivity. Moreover, the chiral ligands and Fe₃O₄ NPs were not directly linked through covalent bonds, which avoided shielding the catalytic sites of Fe₃O₄ NPs. Thus, peroxidase-like activity was similar to that of pure Fe₃O₄ NPs. After introducing D/L-tryptophan (D/L-Trp) to the surface of Fe₃O₄ NPs yolk (Fe₃O₄@Poly(D/L-Trp)), D/L-tyrosinol could be selectively transformed into the corresponding D/L-dityrosinol (ee > 99%) (Figure 12). The k_cat/K_M values showed that Fe₃O₄@Poly(D-Trp) was 5.38-fold more active to D-tyrosinol than to L-tyrosinol. And the selectivity of Fe₃O₄@Poly(D-Trp) was higher than HRP (4.77-fold).

Figure 12. Enantioselective synthesis of tyrosinol catalyzed by Fe₃O₄@Poly(L-/D-Trp).

Nanozymes are suitable for the detection of substrates due to their properties such as high catalytic activity and stability. There have been many reports on the application of metallic oxide based nanozymes in the fields of detection. Zhang et al. [79] prepared Fe₃O₄@C nanowires (Fe₃O₄@C NWs) with peroxidase-like activity. After combining with a biotin-labeled aptamer, they could be used for the ultra-sensitive detection of platelet-derived growth factor BB (PDGF-BB). The LOD could be as low as 50 amol/L, which was much lower than the detection limit of PDGF-BB in 50% human serum (100 fmol/L) [79].

Yıldırım et al. [82] prepared the monodisperse-porous CeO₂ NPs with peroxidase-like activity by the staged sol-gel templating protocol. The resulting product could be used as the adsorbent in metal oxide affinity chromatography (MOAC), which could achieve the separation and enrichment of phosphoprotein in carious samples. Moreover, the CeO₂ NPs with peroxidase-like activity could be used to detect the concentration of phosphoprotein.

Based on the intrinsic oxidase-like activity of MnO₂ nanosheets, a “lab in a tube” platform was developed for the determination of oxalate (C₂O₄^2-) in samples [84]. The chromogen substrate TMB (3,3′,5,5′-tetramethylbenzidine) could be oxidized to oxTMB (blue) by MnO₂. In the presence of oxalate, the MnO₂ nanosheets would be decomposed to Mn²⁺ and the color reaction would be suppressed (Figure 13). The color difference of the solutions could be detected and used for the quantitative determination of oxalate concentration. In order to ensure the stability of MnO₂ in complex samples, sodium alginate (SA) hydrogel was used to load MnO₂ nanosheets. The porous structure of the SA hydrogel could resist the interference of complex environments to a certain extent, and at the same time would not impede the mass transfer of substrates, which was conducive to the long-term storage of samples.

Figure 13. Schematic illustration for the detection of oxalate. The chromogen reagent TMB will not be oxidized to oxTMB with Mn²⁺, thus resulting in the color difference.

The rapid detection process was completed in a micro-tube and the kit could be stored for seven days with almost no change. There was a good linear relationship between the blue light intensity and the concentration of oxalate in the range of 0.8–800 μmol/L. Furthermore, as detected by the results of standard addition method, the recovery rates of the urine samples were in the range of 98.69–103.60% with the RSDs from 4.16% to 8.54%, indicating this kit was able to analyze the concentration of oxalate in human urine quantitatively.

Metallic oxide nanozymes possessing superoxide dismutase (SOD)-like activity are used for promoting the cleavage of ROS. Moderate ROS concentration is necessary for normal cells to maintain intracellular functions such as the defense against bacteria and viruses. However, it may lead to cell damage if the ROS concentration is too high. One of the main pathological mechanisms of Parkinson’s disease (PD) is mitochondrial dysfunction mediated by ROS [76]. Hao et al. [76] prepared novel chiral molecule-mediated porous Cu_xO nanoparticle clusters (Cu_xO NCs) with four kinds of enzyme-like activities (SOD, peroxidase, catalase and glutathione peroxidase). L-phenylalanine (L-Phe) was selected as the structure guiding agent, and other chiral amino acids, L-tyrosine (L-Tyr) and L-aspartic acid (L-asp) could not form the uniform structure of NCs. The cell experiments have shown that porous Cu_xO NCs could alleviate the cell death caused by neurotoxin 1-methyl-4-phenylpyridinium (MPP+), suggesting that they had the ability to scavenge ROS in cells. At the same time, they showed an excellent ability of anti-oxidative stress in the PD mouse models and successfully realized cognitive recovery in mice. Cu_xO NCs had good stability and low toxicity in vivo, which was beneficial to the treatment of organisms.

Carbon based

Carbon based nanomaterials have always been the focus of attention because of their extensive chemical inertness, good biocompatibility and unique high hardness.

Fan et al. [93] developed the N-doped porous carbon nanospheres(N-PCNSs) with four enzyme-like activities (peroxidase, catalase, oxidase and SOD). With the help of ferritin, N-PCNSs were specific for tumor cells and could produce ROS in tumor cells, leading to the death of tumor cells. Ibarbia et al. [94] developed a 3-(triethoxysilyl)propyl methacrylate (MPS) modified graphene oxide quantum dot (GOQD-MPS) with significantly enhanced peroxidase-like activity. Irradiation with visible light, GOQD-MPS could degrade Rhodamine B (RHB) effectively, indicating the GOQD-MPS was suitable for the treatment of organic dye wastewater.

Fang et al. [87] developed oxygenated nanodiamonds (ONDs) with peroxidase-like activity against periodontal bacterial infection. Previous studies have confirmed that the carbonyl groups and carboxyl groups on the surface of a nanozyme with peroxidase-like activity were the catalytically active sites and the substrate binding sites, while the hydroxyl groups would inhibit the catalytic activity [105, 106]. Therefore, the researchers treated the ordinary nano-diamonds (NDs) in the mixed acid composed of nitric acid and sulfuric acid for different time (12 h and 24 h). Consequently, the hydroxyl groups on the surface of NDs would be oxidized to carbonyl and carboxyl groups; the ONDs with enhanced peroxidase activity were finally synthesized.

Antibacterial agents, such as β-lactams, have lots of restrictions, since they are allergenic in the process of use and lead to multiple drug resistance. Another antimicrobial agent, H₂O₂, would not cause effective damage to bacteria at low concentrations (0.33 mol/L − 1 mol/L), while may have adverse effects on healthy tissues at high concentrations [87]. The ONDs could generate abundant ROS (·OH) at low concentration of H₂O₂ to kill bacteria and avoid the potential damage of high H₂O₂ concentrations to the human body (genetic damage, oxidative damage). The experimental results showed that ONDs could effectively kill gram-negative and gram-positive bacteria under physiological conditions. The longer the treatment time of mixed acids, the better antibacterial performance produced. Compared with traditional antimicrobial agents, the nanozyme antibiotic hardly generated multi-drug resistance, which provided a new strategy to against bacterial infections.

Ren et al. [29] synthesized a kind of porous carbon with Enteromorpha prolifera (EPC) that has oxidase and peroxidase-like activities. E. prolifera is a renewable marine waste biomass. EPC exhibited oxidase-like activity in the presence of oxygen, while showing peroxidase-like properties in the presence of H₂O₂ and without oxygen.

Based on the oxidase-like activity of EPC, an acid phosphatase colorimetric sensor was developed with a linear range of 0.5–15 U/L and a LOD of 0.1 U/L. Based on the peroxidase-like activity of EPC, a H₂O₂ fluorescent sensor was developed with a linear range of 0.05–80 μmol/L and a LOD of 0.017 μmol/L. Compared with H₂O₂ bio-electrochemical sensor with HRP [31], EPC has more excellent performance in both detection range and LOD. Furthermore, EPC could be used to detect the glucose by combining with GOx, having a linear range of 0.05–10 mmol/L and a LOD of 30 μmol/L. The use of marine waste biomass as the raw material could reduce waste and costs, and the generated EPC has excellent sensing performance.

Others

In addition to the above nanozymes, there are other nanomaterials possessing enzyme-like activities, such as MOFs, Prussian blue (PB) NPs and so on.

Li et al. [98] synthesized MOF-818 nanozyme with trinuclear copper centers by mimicking the binuclear copper centers structure of natural catechol oxidase. MOF-818 has catechol oxidase-like activity instead of peroxidase-like activity, so it could be used to oxidize O-diphenol to the corresponding O-quinone in the presence of oxygen (Figure 14).

Figure 14. The oxidation of 3,5-Di-tert-butylcatechol catalyzed by MOF-818.

Son et al. [100] synthesized a novel 3-aminophenylboronic acid (APBA) modified Prussian blue NPs (PBBA), which were applied to detect glycated albumin (GA) through colorimetric and electrochemical methods without affecting the peroxidase-like activity. PBBA exhibited a superior peroxidase-like activity with low detection limits (7.32 μg/mL and 3.47 μg/mL for colorimetric and electrochemical immunoassay, respectively), which could be used as a substitute for natural enzymes in the quantitative analysis of GA.

Conclusions and outlook

The advantage of enzyme catalysis lies in its high catalytic activity and greener reaction process, while the disadvantage lies in the lack of stability and difficult recycling. In fact, the immobilization of enzymes could overcome the above shortcomings. The immobilization carrier and immobilization methods are two most important factors by interfering the immobilization process. Nanomaterials are used to immobilize enzymes requiring the following criteria: mild synthesis process, high enzyme load, stability under extreme conditions (pH or temperature), large surface area and high mass transfer efficiency, which could improve the catalytic efficiency and increase the recycle times of immobilized enzymes.

As natural enzyme mimics, nanozyme undoubtedly occupies an advantage in terms of stability and reusability. However, current research on nanozymes is mainly concentrated on exploring the enzyme-like activity of some existing materials and less on the mechanism of the catalytic process. In addition, scientists recognize that nanozymes could have potential biotoxicity. Although nanostructured biocatalysts have been studied extensively, few products have been applied to large-scale industrial production actually. Recently, the application fields of nanozymes are still relatively few they mainly include sensors and biomedicine. Continuous efforts about nanozyme could be made in the following aspects: (1) profound study on the nanozyme catalytic mechanism to enable the design and synthesis of nanomaterials with specific enzyme activities; (2) development toward a nanozyme switch which could selectively turn on/off specific catalytic activity; (3) control over the substrate specificity of nanozymes through surface modification or the design of their materials; (4) expanded scope on the enzymatic activities and the application fields.

In this review, the development of nanomaterial-enzyme complexes and nanozyme were reviewed, and their advantages and disadvantages were compared. However, the two forms of nanostructured biocatalysts are not antagonistic. As we mentioned in the section about metal–organic frameworks (MOFs), a cascade reaction could be performed simultaneously by combining nanomaterial-enzyme complexes and nanozyme. It is a promising strategy to carry out joint research in the two fields. In addition, it is necessary to develop unified standards on the catalytic capacity and biological toxicity of nanostructured biocatalysts. With the development of nanotechnology and novel enzymes, we believe that great opportunities will be provided by nanostructured biocatalysis in the fields of biopharmaceutical, chemical engineering and environmental protection.

Acknowledgments

We are grateful for the National Natural Science Foundation of China (Project 21808205), Zhejiang Provincial Science and Technology Plan Project-Academician Special Project (No. 2019-ZJ-JS-03) and the Zhejiang Provincial Key R&D Project (No. 2020C03006)) for financial aids to this work.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Disclosure statement

The authors declare no conflict of interest.