*3.3. Reusability*

Research shows that hNFs remain active for multiple reaction cycles before they lose activity regarding reusability. In addition, research has shown that they can be recycled by adding fresh enzymes. Yn et al. rebloomed the hNFs they produced using the following method (Figure 7). First, 0.2 mL of acetic acid or phosphoric acid was added to dissolve the original hNFs. After that, the reaction mixture was heated for 10 min at 100 ◦C to denature all of the enzymes. The denatured enzymes were then removed via filtration or centrifugation. Then, the solution pH was adjusted to 6.7 using Ca(OH)2. A rebloom of the hNFs occurred when fresh enzymes were added to the solution, and co-crystallization occurred with Ca(PO4)2. The reaction was kept at 4 ◦C for 24 h. Then, the nanoflowers were separated and used as the original hNFs. The researchers examined both the activity of the dual cycle hNFs and the recovery of Ca(PO4)2 for six different enzyme models. The results showed no noticeable differences between the original hNFs and the dual-cycle hNFs, which suggested that certain molecules, such as amino acids, do not affect the catalysis of the dual-cycle hybrid nanoflowers. While examining the recovery rate percentage of Ca(PO4)2 by checking their weight while dry and after recrystallization, the result showed up to a 99% recovery of Ca(PO4)2 for six enzymes-hNFs. In summary, the activity of the enzymes and the recovery of Ca(PO4)2 before and after the dual cycle were nearly constant for all of the tested enzyme model hNFs [37].

**Figure 7.** Dual cycle process for enzyme immobilization. Reprinted from [37] with permission from the Royal Society of Chemistry under the Creative Commons Attribution 3.0 Unported Licence.

Memon et al. synthesized hNFs using alcalase and Ca ions. When examining the reusability of the hNFs, the results showed that hNFs maintained 85.4% of their activity during seven cycles [40]. A research group investigated the reusability of hNFs produced from papain enzymes and Zn ions. The findings thus showed that the enzyme could maintain 88.8% of its original activity for ten cycles, but there was a steady decrease in its activity as the cycle number increased [41].

#### **4. Types and Synthesis of Organic–Inorganic Hybrid Nanoflowers**

As discussed earlier, hybrid nanoflowers are formed between a protein (organic component) and a metal ion (inorganic component). When proteins have a metal-binding site they can form complexes with ions through coordination interactions. For example, enzymes with nitrogen atoms in their amine and amide groups can form complexes with different metals through coordination interactions, i.e., the beginning of hybrid nanoflower synthesis [20,33,42]. Hybrid nanoflowers can be categorized based on the assembly of the particles, the type of protein/enzyme (organic component), and the metal ion (inorganic component) used. Table 1 shows the different metals and proteins used for hybrid nanoflower synthesis. The preparation of different hNFs based on the use of different metals is discussed next.




**Table 1.** *Cont.*


**Table 1.** *Cont.*

#### *4.1. Copper-Based Hybrid Nanoflowers*

For the first hNFs produced [20], an aqueous CuSO4 solution was added to phosphatebuffered saline (PBS) that contained bovine serum albumin (BSA). The reaction succeeded at room temperature and was incubated for 3 days. Scientists then confirmed the synthesis process of hNFs by replacing BSA with other proteins, including α-lactalbumin, carbonic anhydrase, laccase, and lipase (Figure 8). One of the experiments performed on laccasecopper phosphate nanoflowers showed that laccase hNFs had an activity that was 4.5–6.5 higher than that of free laccase for oxidizing catecholamine syringaldazine. Additionally, they showed a good stability and reusability. Another experiment showed that the activity of carbonic anhydrase-embedded hNFs was 2.6-fold higher than the free form activity in the hydration of CO2 [20].

**Figure 8.** SEM images of hybrid nanoflowers: (**<sup>a</sup>**–**l**) column 1, a-lactalbumin; column 2, laccase; column 3, carbonic anhydrase; column 4, lipase; at protein. Reprinted from [20] with permission from Springer Nature. Copyright © 2021, Nature Publishing Group. License Number: 5031780508604.

As the first discovered hybrid nanoflowers used protein-Cu ions, the majority of work was done on them. Another study showed the synthesis of hNFs from Turkish black radish peroxidase and copper ions. This study showed that hNFs had a better activity and stability in a wide range of pH values, as well as the ability to degrade 90% of Victoria blue dye [43]. As shown in Figure 9, hNFs are formed from glucose oxidase and copper ions embedded in amine-functionalized magnetic nanoparticle-labeled MNP-GOx NFs as antibacterial agents. The results showed that MNP-GOx NFs demonstrated an antibacterial activity with Gram-positive *S. aureus* and Gram-negative *E. coli* in a broad spectrum. This was done by disturbing the bacterial cells with the H2O2 produced by GOx [111]. Yang et al. produced hNFs from copper phosphate and horseradish peroxidase. These hNFs exhibited a linear detection from 100 nM to 100 μM H2O2. Additionally, they showed a good reusability and excellent storage stability [44]. Sun et al. [112] synthesized copper polyphosphate kinase 2 hNFs and formed an ADP regeneration approach from AMP using hNFs. The resulting hNFs had a better storage stability, in addition to a broader pH and temperature ranges. Additionally, it showed a better ADP production and retained 71.7% of its original activity after ten cycles, which showed good reusability.

**Figure 9.** Preparation of MNP-GOx NFs. Reprinted from [111] with permission from Elsevier. Copyright © 2021 Elsevier B.V. License Number: 5031790647430.

Li et al. [42] produced hybrid nanoflowers on a nanofiber membrane surface from copper ions and different proteins. This led to the production of a very biocompatible and multilevel surface. The produced copper hybrid nanoflowers displayed an improved stability compared with the free protein that could stem from the protein's protection from the inorganic crystals. Figures 10 and 11 show the mechanism of how this happens. The gained stability in the hybrid nanoflowers can lead to their introduction to the application, each as biodevices and biocatalysts. The researchers found that by changing the concentration of the protein (Figure 12), the incubation time, the composition of the nanofiber membrane, and the preparation of the mineralizing solutions, the composition and structure of the copper hNFs could be controlled on the nanofiber membrane. The results showed that the different proteins tested (papain, bovine serum albumin (BSA) laccase, and horseradish peroxidase) gave different hNF morphologies, which is supported by previous studies.

**Figure 10.** Formation of copper hNFs. Reprinted from [42] with permission from the American Chemical Society. Copyright © 2021 American Chemical Society.

**Figure 11.** The growing process copper−protein hNFs. Reprinted from [42] with permission from the American Chemical Society. Copyright © 2021 American Chemical Society.

**Figure 12.** SEM of Cu-BSA hNFs with different protein concentrations, with an incubation time of 6 h. Reprinted from [42] with permission from the American Chemical Society. Copyright © 2021 American Chemical Society.

#### *4.2. Calcium-Based Hybrid Nanoflowers*

Even though the most commonly used metal for the synthesis of hybrid nanoflowers is copper, another metal that is heavily used to produce hNFs is calcium. Wang et al. reported the synthesis of α-amylase-CaHPO4 hybrid nanomaterials, inspired by the allosteric effect. The work showed three different nanomaterial morphologies: nanoflowers, nanoplates, and parallel hexahedrons. While studying the enzymatic activity of α-amylase in the three different nanomaterial systems developed, and free α-amylase with and without calcium ions, the researchers credited two main factors that increased the enzymatic activity of α-amylase, namely: the allosteric effect of calcium ions with the amine group of the enzyme and the morphology of the nanomaterials [89]. Self-repairing hNFs were built from Ca3(PO4)2 and chloroperoxidase (CPO) with a sodium alginate (SA) coating. The results showed that the immobilized enzyme had similar K m and Kcat values, compared with the free enzyme. Additionally, it demonstrated the ability of the immobilized chloroperoxidase to work in acidic conditions, where it was able to maintain more than 85% of its activity after 12 cycles. Figure 13 shows the process of self-repairing SA-coated CPO-Ca3(PO4)2 hybrid nanoflowers [87]. Zhao et al. [92] synthesized calcium hNFs by combining Ca3(PO4)2 and α-acetolactate decarboxylase (ALDC) enzymes. These hNFs had a better activity than the free ALDC.

**Figure 13.** The process of self-repairing sodium alginate (SA)-coated CPO-Ca3(PO4)2 hybrid nanoflowers. Reprinted from [87] with permission from the Royal Society of Chemistry. Copyright © The Royal Society of Chemistry. License Number: 1105129-1.

#### *4.3. Manganese-Based Hybrid Nanoflowers*

Nearly all of the hybrid nanoflowers produced in the literature use copper or calcium ions. Nevertheless, several studies use different metal ions. One of these metals is manganese; specifically, manganese(II) phosphate is used because of its unique electrochemical properties [113–115]. Rai et al. [95] synthesized hNFs using manganese metal as the inorganic component and L-arabinose isomerase as the organic component. Recombinant L-arabinose isomerase with 474 amino acids was synthesized into E. coli from *Lactobacillus sakai*. Hybrid nanoflowers with a spherical hierarchical morphology were produced using purified recombinant isomerase.

Several studies were performed on the L-arabinose isomerase embedded in the hybrid nanoflowers, and a circular dichroism (CD) analysis recorded no change in the isomerase structure. Compared with free L-arabinose isomerase, hNF-embedded L-arabinose isomerase demonstrated better kinetic parameters. Interestingly, L-arabinose isomerase converted approximately 50% of D-galactose to D-tagatose, a rare type of sugar, without adding more manganese to the reaction, showing the possibility of commercial production of this sugar using manganese/L-arabinose isomerase hNFs. In addition, these hNFs show good reusability and reproducibility in multiple reaction cycles. Munyeman et al. [96] reported the synthesis of collagen/manganese phosphate hNFs in an environmentally friendly biomineralization method. In this study, collagen was used as the biotemplate agen<sup>t</sup> to produce hNFs. Additionally, it was used to bind the petals of the nanoflowers together. These hNFs had a grea<sup>t</sup> catalytic activity in relation to water oxidation. Zhang et al. [97] prepared hNFs from manganese(II) phosphate and bovine serum albumin (BSA). The results showed a good catalytic activity in the fuel cells.

#### *4.4. Zinc-Based Hybrid Nanoflowers*

Although the synthesis of copper hybrid nanoflowers is simple and easy, its three-day production process is one of its disadvantages. Thus, it is important to reduce the synthesis time to a more appropriate period. This can be done by choosing the right metal ions or by changing the synthetic method. When choosing metal ions, zinc displayed a faster reaction rate towards phosphate radicals than copper ions [35,41,100]. Zhang et al. showed that hNFs were prepared from zinc phosphate Zn3(PO4)2 and lipase. The formation time of Zn(PO4)2/lipase hNFs took less than three hours, and the formation time of hNFs using Cu(PO4)2 took three days. Zn(PO4)2/lipase hNFs showed a grea<sup>t</sup> operational stability compared with free enzymes [35]. In another study, hNFs were made using Zn(PO4)2 and papain, and the resulting hNFs showed a higher activity than free papain, in addition to a better thermal stability and storage life [41]. Zhang et al. [100] prepared hNFs using Zn and bovine serum albumin (BSA) at 25 ◦C. These hNFs had an average size of 2.3 μm with a surface area of 146.64 cm2/g. The hNFs were used for Cu (II) ion adsorption. The adsorption efficiency of the Zn hNFs towards copper ions was 86.33% at 5 min and 98.9% at 30 min. The highest adsorption capacity obtained with these hNFs was 6.85 mg/g. This study showed the ability of Zn(PO4)2/BSA hNFs to be used as a fast and efficient method for Cu2+ removal.

#### *4.5. Cobalt-Based Hybrid Nanoflowers*

Despite the various studies that investigated the synthesis of hybrid nanoflowers, only a few studies have shown the use of cobalt ions in the formation of hNFs. Kim et al. synthesized protein/cobalt hNFs using BSA and cobalt ions. The work illustrated that the BSA protein could be a template to interact with cobalt phosphate to produce protein-metal hNFs [104]. Kumar et al. [102] produced hNFs using cobalt ions and lipase enzymes. The immobilized lipase showed a 181% higher activity than the free enzyme. Additionally, it offered a better catalytic performance in harsh reaction conditions and higher temperatures.

#### *4.6. Iron-Based Hybrid Nanoflowers*

Studies that use iron ions to produce hNFs are limited. Ocsoy et al. [107] used Fe+<sup>2</sup> and horseradish peroxidase (HRP) to produce hNFs. The results showed an approximately 512% increase in the activity of HRP when stored at 4 ◦C and an approximately 710% increase when stored at room temperature compared with the free enzyme. Additionally, the immobilized HRP lost 2.9% and 10% of its initial activity after 30 days when stored at 4 ◦C and room temperature, respectively. However, the free HRP lost 68% of its activity when stored at 4 ◦C and 91% when stored at room temperature.

#### *4.7. Multi-Metal-Based Hybrid Nanoflowers*

As previously mentioned, there is an extensive range of research and studies in the literature on using different metals to produce hierarchical hybrid nanoflower structures, such as copper, calcium, manganese, zinc, and cobalt. However, few studies have attempted the production of multi-metal-based hybrid nanoflowers; here are a few of them. In 2019, Patel et al. produced hybrid nanoflowers based on copper and zinc ions. The novel multi-metal nanoflowers were synthesized using a laccase enzyme. The Cu/Zn-laccase showed a higher encapsulation yield percentage than the copper-laccase and zinc-laccase hNFs, which were 96.5%, 87.0%, and 90.2%, respectively. The multi-metal nanoflowers (Cu-/Zn-laccase) were 1.2-, 1.5-, and 2.6-fold higher than zinc-laccase, copper-laccase, and free laccase, respectively. Interestingly, the multi-metal nanoflowers showed a charge transfer resistance that was 2.1-fold lower than zinc-laccase hNFs, and when compared with copper-laccase hNFs it was 2.7-fold lower. For the degradation of bisphenol A, the remaining multi-metal nanoflower activity was 1.9-fold higher than that of zinc-laccase hNFs and 5.1-fold higher than that of copper-laccase hNFs [109].

#### *4.8. Non-Metal-Based Hybrid Nanoflowers*

As mentioned above, there is extensive work in the literature on certain metals, such as copper, calcium, manganese, zinc, cobalt, and iron. Nevertheless, few studies have tried to produce non-metal hybrid nanoflows. In 2018 [110], selenium (non-metal) hybrid nanoflows were synthesized. In recent years, selenium nanoparticles have been studied as drug carriers because they are nontoxic and have a good biological activity and bioavailability [116–121]. There have been attempts to produce selenium nanoparticles (SeNPs) with different structures, shapes, and morphologies, including nanoplates, nanotubes, and nanospheres [61–63]. The addition of a biopolymer that is functionalized with these nanoparticles has led to an increased stability and control over the shape and size [122,123]. hNFs were synthesized using pullulan/SeNPs, and pullulan was used as a substitute for the use of proteins [110]. SeNPs were then stabilized using folic acid-decorated cationic pullulan (FA-CP), and presented a flower-like structure. The produced nanoflowers showed an excellent drug adsorption for doxorubicin and had a 142.2 mg/g loading capacity. The study showed that doxorubicin's loading capacity is three times greater in pullulan/SeNP nanoflowers than in spherical SeNPs. Additionally, these hNFs showed a better activity towards cancer cells, and they were less toxic towards normal cells.

#### *4.9. Enhancing Hybrid Nanoflowers Synthesis*

One of the most significant drawbacks of hNFs is their size, which is usually in the nano- or micro-scale range. Thus, it is difficult to separate them from their reaction mixture. Some studies have been performed to improve the synthetic processes of hybrid nanoflowers. Recently, scientists developed supported hybrid nanoflower methods to overcome some of the drawbacks of the use of hNFs. Alginate gel beads were used to entrap α-acetolactate decarboxylase/calcium hNFs. These entrapped hNFs showed a better stability and recyclability than the free enzyme [92]. Another study by Zhu et al. used a cellulose acetate membrane for laccase/copper ion hNFs. The captured hNFs showed a high reproducibility and reusability for phenol detection [55]. Cao et al. [124] produced a glassy carbon electrode (GCE) surface on which bovine serum albumin (BSA)/Ag nanoflowers

were immobilized, coupled with a targeting lectin molecule for detecting human colon cancer cells. This sensor showed specificity for a cell expressing sialic acid. Therefore, it has a possible application for monitoring tumor cells.

#### *4.10. Morphology of Hybrid Nanoflowers*

hNFs commonly have a hierarchical structure and nanoplate petals that look like flower petals. These nanoplates/nanopetals are made of enzyme and metal phosphate. From the first hybrid nanoflower produced by J. Ge et al. in 2012, there have been numerous synthesis techniques to produce hNFs, which have resulted in different hNFs with different microstructures [125–127]. Table 2 shows the most common hNF microstructure morphologies, which are spherical, rosette, and rhombic, with their equivalent flowers in nature. Typically, hNFs have a diameter range between 1–30 μm. There are many factors and reaction conditions that control the shape and size of hNFs; these factors include (I) the type of enzyme used; (II) the type of metal ion; and (III) the reaction condition, which includes the pH value, the reaction temperature, and time [69]. Table 3 shows how morphology differs with changing the factors and conditions.


**Table 2.** Characteristic of hybrid nanoflowers.


**Table 2.** *Cont.*

60

#### *4.11. The Type of Enzyme Used*

As mentioned above, for hNFs to form, the presence of an enzyme is essential. Enzymes attach to the petals of various metal phosphate nanoplates. Numerous studies have shown that different enzymes lead to the formation of different hNF morphologies [21–24]. Chung et al. [52] developed hNFs using copper ions with various enzymes, including glucose oxidase (GOx), laccase, and catalase. Each enzyme gave a unique microstructure morphology of the hNFs, as illustrated in Table 3 (1, 2, and 3).

Zhang et al. [35] showed the use of lipase enzyme to form hNFs (Table 3 (4)), which has a completely different morphology from the three previously mentioned enzymes of glucose oxidase (GOx), laccase, and catalase. Another study used enzymes, laccase, papain, and horseradish peroxidase (HRP) to synthesize different shapes and sizes of hNFs [42]. Research explains that the diversity of hNF morphology arises from the various amide groups on the surface of the enzyme used. Thus, it presents different nucleation sites with varied geometries and densities for phosphate metal nanoplates to bind [20,33]. In addition, Lin et al. revealed that the employed enzyme's molecular weight has an impact on the hNF structure [36].

Additionally, it has been determined that the concentration of the enzyme plays a role in the final morphology of hNFs, where it can affect the size and density of the nanoplates [56]. Several studies have indicated that the lower the enzyme concentration used, the larger the size of the hNFs formed with a lightly constructed structure. In addition, an increase in the employed enzyme content leads to a denser packed structure, although the size will decrease if the enzyme concentration reaches a particular value [72,86,99]. The reason behind this is the subsequent increase in the number of nucleation sites [68,103]. Table 3 (5–12) shows the synthesis of two different hybrid nanoflowers using two different enzymes (lipase and papain) with the same metal ion (Zn). As seen from the table, the morphology of both hNFs dramatically changes with an increased amount of enzyme, from a rhombus shape to a square, oval shape. With the continuous addition of the enzyme (an excess amount), the shape changes into a dense cluster structure with cracks instead of a well-formed flower shape [35,41].

#### *4.12. The Type of Metal Ion Used*

In the formation of hNFs, metal ions play an essential role in the primary crystal nucleation step and metal-enzyme coordination to produce hNFs. Table 3 (13–15) shows a study where hNFs were synthesized using a chloroperoxidase enzyme with three different metals (copper, cadmium, and cobalt). It was observed that copper and cobalt hNFs had similar morphologies, while cadmium hNFs were completely different. Copper and cobalt hNFs had a spherical flower-like structure, whereas cadmium hNFs had a butterfly-like structure [29]. The effect of the metal ion concentration on the morphology of hNFs was also studied. The results show that with an increase in the metal ion concentration, the hNF morphology becomes much denser [38,85].

Rai et al. [95] produced hNFs using L-arabinose isomerase as the organic component, and 13 metal ions, including manganese, cobalt, magnesium calcium, potassium, sodium, nickel, and lithium, as the inorganic component. It was determined that manganese and cobalt ions can substantially improve the catalytic activity of the enzyme. In contrast, the magnesium calcium, potassium, sodium, nickel, and lithium ions had no impact on the enzyme activity.

#### *4.13. Reaction Condition (pH, Temperature, and Time)*

It is well known that the charge of the enzyme varies at different pH values. Thus, leading to the enzyme's different interaction capabilities in the formation of hNFs will influence the morphology of the hNFs [74]. Nadar et al. [39] examined the effect of changing the pH on hNFs synthesized using copper ions and glucoamylase enzymes. The study tested the pH range from 3.5 to 9.5. The net change in free glucoamylase enzymes is neutral, and it has an isoelectric point (pI) of approximately 6. The enzyme is expected

to have a positive charge below the isoelectric point, and a negative charge above the isoelectric threshold. No nanoflower formation was observed at pH values of 3.5, 4.5, and 5.5 (below the isoelectric point). This can be explained by the fact that the positively charged protonated glucoamylase enzyme had an extremely strong repulsion with the positive copper metal ions at a low pH. Hence, no nanoflowers were formed. At pH values higher than the pI point, the charge on the glucoamylase enzyme surface was negative because of deprotonation. Glucoamylase/Cu2+ ions hNFs were formed in this pH range. It was observed that at pH 7.5 the nanoflowers were less packed, which is attributed to the small rise in the negative charge on the enzyme. At pH 9.5, nanoflowers did not form. This is because of the increased repulsion between the negative charges that are highly dense on the surface of the glucoamylase enzyme. Therefore, Cu3(PO4)2·3H2O petals repel one another rather than attaching. Table 3 (16–19) shows the morphology of the synthesized lipase/copper hNFs at different pH values, where the enzyme concentration is 1.0 mg/mL. The pH values of phosphate-buffered saline (PBS) were adjusted to 6.0, 7.4, 8.0, and 9.0. It was noted that the density of the petals decreased as the pH value increased. However, the diameters and the size of the formed nanoflowers remained the same [69].

Another important reaction condition that affects the morphology of hNFs is temperature. Temperature can play a vital role by initiating the various diffusion activities of the enzyme at different applied temperatures. Thus, nanoflowers have different degrees of density in petals and alter their size and diameter [43]. Table 3 (20–23) shows the effect of temperature on the production of calcium ions/elastin-like polypeptide (ELP) hNf and copper ions/ELP hNFs. ELPs are a class of polypeptides derived from an amino acid sequence of naturally accusing elastin in humans. Their pentapeptide sequence is Val-Pro-Gly-X-Gly, where X is any amino acid, other than from Pro. ELPs have a key transition temperature (Tt), where ELPs are soluble in the solution below it. Nevertheless, above this temperature, ELPs will suffer a phase transition that is destructive to the polypeptide. As seen from the table, the morphology of the hNFs synthesized below Tt (4 ◦C) had larger, more expanded petals. However, the hNFs synthesized above Tt (37 ◦C) had a more closed structure [93]. Another study, shown in Table 3 (24–27), demonstrated that hNFs formed at 20 ◦C had a more oval spherical shape with cracks. However, when the temperature increased above 40 ◦C, the shape of the hNFs changed to a more sheet-like structure, and as the temperature further increased, the hNF sheet morphology increased [35]. Altinkaynak et al. [51] showed the production of hNFs from copper ions and lactoperoxidase (LPO), and it was concluded that at a lower temperature, the petals of the hNFs became more compacted. This result is shown in Table 3 (28–29). For this, the ideal temperature for the synthesis of the best hNF structure must be investigated.

An additional factor that can affect the hNF morphology is the reaction time. The reaction time of the hNFs depends on the method used to produce them. Since the discovery of hNFs, there have been different time intervals used when producing them. Some of these time intervals include 3 days [20,30,38,44,68,111], 24 h [34,39,109], 3 h [35], and 5 min [52,58]. Table 3 (30–32) shows the different stages of hNF formation. The first step, nucleation, which is the formation of primary copper phosphate crystals, occurs between 0 and 2 h. The second step, growth, is when the metal ion and the enzyme form large agglomerates, which are the primary petals. This occurs between 2 to 8 h. In the last step, complete hNFs are formed, which occurs between 8 to 24 h [39].

#### **5. Applications for Hybrid Nanoflowers**

The excellent catalytic properties of hNFs have provided a wide selection of applications. Some of these applications are in the fields of biosensors, biomedical, bioremediation, and industrial biocatalysts [45,128–130]. Different examples of these applications are illustrated in Figure 14.

**Figure 14.** Summary of different applications used for hNFs. (**A**) Fast detection of phenol using laccase/cooper phosphate hNFs integrated into a membrane. Reprinted from [55] with permission from John Wiley and Sons. Copyright © 2021 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim. License Number: 5031800087304. (**B**) Detection of ractopamine using a protein/manganese ion hNF electrochemical biosensor. Reprinted from [98] with permission from Elsevier. Copyright © 2021 Elsevier B.V. License Number: 5031800271904. (**C**) Selective separation of cadmium and lead in water, cigarette, and hair samples using BSA/copper ions hNFs. Reprinted from [131] with permission from Elsevier. Copyright © 2021 Elsevier B.V. License Number: 5031800434687. (**D**) Degradation of bisphenol A using laccase/cooper phosphate hNFs. Reprinted from [34] with permission from Elsevier. Copyright © 2021 Elsevier B.V. License Number: 5031800600650. (**E**) Epoxidation of alkenes using dual enzyme system GOx and lipase/copper ions hNFs. Reprinted from [77] with permission from Elsevier. Copyright © 2021 Elsevier Ltd. License Number: 5031800753597. (**F**) Transformation of D-Galactose to D-Tagatose using L-Arabinose Isomerase/manganese ions hNFs. Reprinted from [95] with permission from the American Chemical Society. Copyright © 2021 American Chemical Society.
