Immobilization of enzymes on clay minerals for biocatalysts and biosensors

Applied Clay Science 114 (2015) 283–296

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Review article

Immobilization of enzymes on clay minerals for biocatalysts and biosensors Ning An a, Chun Hui Zhou a,b,c,d,⁎, Xiao Yu Zhuang a, Dong Shen Tong a, Wei Hua Yu a,b a Research Group for Advanced Materials & Sustainable Catalysis (AMSC), State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China b Zhejiang Changxing Sol–Gel Advanced Materials Co., Ltd, Changxing 313113, China c The Institute for Agriculture and the Environment, University of Southern Queensland, Toowoomba QLD 4350, Australia d Key Laboratory of Clay Minerals of Ministry of Land and Resources of The People's Republic of China, Zhejiang Institute of Geology and Mineral Resources, Hangzhou 310007, China

a r t i c l e

i n f o

Article history: Received 15 February 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online xxxx Keywords: Clay minerals Enzyme Immobilization Montmorillonite Catalyst Biosensor

a b s t r a c t Many studies suggest that naturally-occurring layered clay minerals can be used as a class of biocompatible solid supports for immobilizing enzymes. The corresponding clay mineral enzyme hybrids prove to have great potentials in catalysis and biosensing. This article reviews latest advances in using clay minerals as supports for the immobilization of enzymes. The immobilization of enzyme onto clay minerals can be made via non-covalent adsorption and covalent bonding. The non-covalent immobilization involves van der Waals forces, electrostatic interactions, hydrogen bonding, and hydrophobic interactions. For avoiding desorption of enzymes, immobilization can be conducted through direct covalent bonding between enzymes and clay minerals. Organic modification of clay minerals and addition of linking molecules are made to improve the immobilization so as to increase the loading, activity and stability of enzymes. Regarding the applications of enzyme immobilized on clay minerals, recent studies are made mainly in biocatalytic processes and in biosensors. For manufacturing biosensing electrodes, clay minerals with metal nanoparticles, graphene and carbon nanotubes prove to be more effective owing mainly to the enhanced electron transfer. Future work on clay mineral enzyme hybrids could lie in integrating more additional functional materials with clay mineral enzyme hybrids to build hierarchical structured catalysts and electrodes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Enzymes are a group of biological macromolecules, generally referred to as proteins with high catalytic activity, selectivity and specificity. In living organisms, enzymes act as extremely effective catalysts for thousands of specific reactions in the metabolic processes that sustain life (King et al., 1980; Atlow et al., 1984; Ansari and Husain, 2012). With the development of protein engineering, scientists can now create some new enzymes with novel properties, either through rationally designed synthesis or through in vitro evolution (Zhao et al., 2002; Ostermeier, 2009; Lutz, 2010). Some enzymes are successfully used in industry for the production of food (Akgöl et al., 2001; Pires-Cabral et al., 2010), chemicals and pharmaceuticals (D.T. Zhao et al., 2011; G.H. Zhao et al., 2011). Enzymes can also be used in environmental engineering (Kim et al., 2006; Yücel et al., 2011). The application of enzymes, however, is often hampered owing to their lack of stability in

⁎ Corresponding author at: Research Group for Advanced Materials & Sustainable Catalysis (AMSC), State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China. E-mail addresses: [email protected], [email protected] (C.H. Zhou).

http://dx.doi.org/10.1016/j.clay.2015.05.029 0169-1317/© 2015 Elsevier B.V. All rights reserved.

organic solvents and at a bit elevated temperatures. In addition, in a liquid system, the recovery and reuse of enzymes are difficult (Anderson et al., 1998; Lopez-Gallego et al., 2005; Polizzi et al., 2007; Tran and Balkus, 2011). To tackle these issues, many researchers have suggested that an alternative way would be to immobilize enzymes onto solid materials (Mozhaev et al., 1990; Cao, 2005; Laurent et al., 2008; Tang et al., 2014). A variety of solid materials, either organic (Martinek et al., 1977; Klibanov, 1979; Klibanov, 1982; Chen et al., 2000; Rebroš et al., 2007; Kim et al., 2009; Shu et al., 2011; Chauhan, 2014) or inorganic (Wang and Caruso, 2005; Lammirato et al., 2010; Feng and Ji, 2011; Moreno-Pirajàn and Giraldo, 2011; Yu et al., 2013a, 2013b, 2013c, 2015), have been employed as supports for enzyme immobilization. Studies have demonstrated that immobilization of enzymes onto organic materials, for example polymer microspheres (Oh and Kim, 2000) and organogels (Zoumpanioti et al., 2010), maintains a high activity of the enzymes. However, it has also been observed that organic materials themselves have unsatisfactory thermal and chemical stability. Moreover, some organic materials have a relatively high toxicity to enzymes (de Fuentes et al., 2001; Corma et al., 2002; Rahman et al., 2005). In this context, inorganic solid materials, such as one-dimensional carbon nanotube (Holzinger et al., 2012), two-dimensional layered clay

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minerals (Mousty, 2010; Zhou et al., 2011; Yu et al., 2013a, 2013b, 2013c, 2015) and three-dimensional porous silica (Siso et al., 1990; Sonnet et al., 1994; Mody et al., 1999; Soleimani et al., 2011; Zhou and Hartmann, 2013) have advantages over organic solid materials. As summarized and discussed on some good reviews, immobilization of enzyme on solid materials, which include zeolites, activated carbon (Datta et al., 2013) and ordered mesoporous silicas (Zhou and Hartmann, 2013), may result in improved activity, stability and recyclability of the enzyme in a broader working pH and temperature range than the native enzymes. Though in the field of inorganic solid materials used for enzyme immobilization, clay minerals have captured particular attention over the past few decades (Table 1), there are rarely reviews focusing on this topic published in the scientific journals. Different from zeolites, activated carbon and ordered mesoporous silicas, layered clay minerals have several physical and chemical peculiarities. Firstly, clay minerals are a class of hydrous aluminum or magnesium phyllosilicates abundant in nature. Secondly, clay minerals have twodimensional layered structure on a nanometer scale. For example, each layer of montmorillonite (Mt) is built of one or two tetrahedral silicate (Si\\O) sheets (Bergaya and Lagaly, 2013) (Fig. 1a) sandwiching one octahedral metal oxide (Fig. 1b) or hydroxide (M\\O or M\\OH; M_Mg, Al, etc.) sheet (Fig. 1c). Such structure allows the clay minerals to possess unique physicochemical properties such as large surface area, swelling, and ion exchangeability (Zhou et al., 2011; Yu et al., 2013a, 2013b, 2013c; Zhou and Keeling, 2013). Moreover, the large surface of clay minerals provides functional groups such as \\Si\\O\\Si\\, \\Si\\O\\OH and Al (M)\\OH. Hydrophobic\\Si\\O\\Si\\ groups on the tetrahedral silicate (Si\\O) sheets can contribute to the interaction

between clay minerals and organic enzyme molecules by hydrophobicity. Hydroxyl groups at broken-edges of tetrahedral and octahedral sheets are suitable to form hydrogen bonding with enzyme molecules. For smectites, the exchangeable cations such as Na+ or Ca2 + located in the interlayer space can be replaced by positively-charged enzyme molecules by a cation exchange reaction. These features enable clay minerals to serve as a class of excellent supports for immobilizing enzymes. In addition, the compositional and structural characteristics of layered clay minerals allow a variety of modification of clay minerals by organic, polymeric or biological molecules (Chang and Juang, 2007; Liu et al., 2015). In this way, modified clay minerals possess additional functional groups, increased hydrophobicity, adhesion area and lowered steric hindrance. Consequently, modified clay minerals have improved properties for enzyme immobilization, including increasing the loading amount, the thermal stability the pH durability, and the biocompatibility of immobilized enzymes (Chang and Juang, 2005; Mbouguen et al., 2006; Zhou et al., 2012; Zhou and Keeling, 2013). It has also been found that layered clay minerals can prevent the enzymes from microbial contaminations (Fishman et al., 2002; Bornscheuer, 2003). Accordingly, the uses of enzymes immobilized onto clay minerals have been expanded over the past decades. For example, recent years have witnessed that enzymes immobilized onto clay minerals have been increasingly directed and applied to biosensors for chemical, environmental and clinical detection (Carrasco et al., 1995; Naidja and Huang, 1996; Naidja et al., 1997; Fuentes et al., 2001). This article attempts to present the state-of-the-art studies and technologies in using clay minerals as supports for immobilizing enzymes. The immobilization methods are introduced first by highlighting the

Table 1 Typical clay minerals and their binding site, modification and interactions with enzymes for immobilization of enzymes. Enzyme

Pristine clay minerals and modification

Binding site

Interaction

References

Lipases

Mt

External surface Edges External surface External surface External surface Internal surface External surface External surface External surface Edges External surface External surface Internal surface External surface Edges External surface Internal surface External surface Internal surface External surface Internal surface Internal surface Internal surface Internal surface External surface External surface Internal surface Internal surface Internal surface Internal surface External surface External surface Internal surface Internal surface Internal surface Internal surface

Van der Waals Ion exchange Covalent bond Covalent bond Cross-linking Ion exchange Hydrophobic force Hydrophobic force Covalent bond Hydrogen bonding Hydrophobic force Cross-linking Ion exchange Van der Waals Hydrogen bonding Ion exchange Ion exchange Covalent bond Ion exchange Covalent bond Ion exchange Covalent bond Ion exchange Ion exchange Hydrophobic force Cross-linking Ion exchange Covalent bond Ion exchange Covalent bond Covalent bound Covalent bound Ionic binding Ion exchange Covalent bond Ionic binding

Reshmi and Sugunan (2013)

Mt-GA Bent-CTMAB Pal-3-APTES-G Sep

Tyrosinase

Pal-3-APTES-GA Pal-3-APTES-GA Ca-Mt

β-gluasidase

Mt-chitosan-GA Mt

Catalase

Bent

Invertase

Mt-K10

Alkaline phosphatase Glucoamylase

Acid-Mt (K-10) Acid-Mt (K-10)-GA Bent-HDTMA Pillared Bent Sep Sep-CTMAB Acid-Mt

α-amylase Urease

Acid-Mt (K-10) Acid-Mt (K-10)-GA Pal nanofibrillar-Fe3O4-3-APTES-GA Kaol-Fe3O4-3-APTES-GA Hal nanotubes Acid-Mt (K-10) Acid-Mt (K-10)-GA Hal nanotubes

Ghiaci et al. (2009a, 2009b) You et al. (2013) Isidoro et al. (2001) Huang et al. (2008) Huang et al. (2009) Miranda et al. (2011)

Chang et al. (2008) Serefoglou et al. (2008)

Öztürk et al. (2008) Sanjay and Sugunan (2005a, 2005b) Sanjay and Sugunan (2007) Andjelković et al. (2015) Sedaghat et al. (2009) Sanjay and Sugunan (2007) Sanjay and Sugunan (2007) Zhao et al. (2011a), Zhao et al. (2011b) Zhai et al. (2010) Sanjay and Sugunan (2007) Zhai et al. (2010)

3-APTES: 3-amino propyl triethoxy silane; Bent: bentonite; CTMAB: cetyltrimethyl ammonium bromide; GA: glutaraldehyde; Hal: halloysite; HDTMA: hexadecyl trimethylammonium; Kaol: kaolinite; Mt: montmorillonite; Pal: palygorskite; Sep: sepiolite.

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(b) Octahedral (O) sheet

(a) Tetrahedral (T) sheet

(c) 2:1-type (or TOT-type) clay minerals O

285

OH

(d) 1:1 type (or TO-type) clay minerals Al or Mg

Si

Fig. 1. Schematically drawings showing the structures of (a) the tetrahedral sheet, (b) the octahedral sheet, (c) 2:1-type clay mineral and (d) 1:1-type clay mineral. (Dependent upon species in the family of clay minerals, a small fraction of the tetrahedral Si atoms is isomorphically substituted by Al and/or a fraction of the octahedral atoms (Al or Mg) is substituted by atoms of lower oxidation number).

differences in the interactions between enzymes and clay minerals. Dependent upon of the interactions, available immobilization methods are divided into two categories: non-covalent immobilization and covalent conjugation. The two immobilization strategies also involve the modification of clay minerals and the uses of additional linking molecules. The uses of enzymes immobilized on clay minerals in biocatalysis or biosensing are then summarized and commented. Finally, the challenges and, the pressing need for efforts to develop advanced clay mineral enzyme hybrids are briefly remarked. 2. Immobilization Adsorption and binding of enzyme molecules by clay minerals entail a variety of physical and chemical interactions. They may result from van der Waals forces, electrostatic interaction, hydrogen bonding, hydrophobic interaction and covalent bonding between enzyme molecules and clay minerals. Inherently, the interactions depend upon the type of clay minerals and enzymes. Moreover, for a given clay mineral, different surfaces and sites on it also behave differently towards adsorbing enzyme molecules. For example, kaolinite (Kaol) is a 1:1type clay mineral which is composed of a single tetrahedral sheet condensed with a single octahedral sheet into one unit layer. Ideally, the chemical formula can be written as Al2O3·2SiO2·2H2O. As indicated by the chemical formula, Kaol minerals are almost electrically neutral

with little isomorphous substitution in the lattice (Bergaya and Lagaly, 2013) (Fig. 1d). Intercalation of enzyme into its interlayer space has proved to be extremely difficult (Fiorito et al., 2008), in comparison to smectites. Immobilization of enzymes on Kaol is merely restricted to the external crystal surfaces and the edges of Kaol (Yu et al., 2013a, 2013b, 2013c) (Fig. 2b). Under such a circumstance, hydrophobic force and hydrogen bonding are major interactions leading to enzyme immobilization onto Kaol. By contrast, 2:1-type clay minerals, in particular Mt, are more often used as supports for enzyme immobilization because the 2:1-type clay minerals provide extra adsorption and intercalation in their interlayer space (Fig. 2a). Moreover, the interactions can be tuned by using modified clay minerals or additional linking molecules (Table 1). According to the nature of the interactions, the immobilization of enzyme onto clay minerals can be divided into two categories: non-covalent immobilization and covalent immobilization. In general, non-covalent allows the conformational structure of the immobilized enzymes to be preserved well whereas covalent immobilization can lead to much durable attachment of enzyme to clay minerals.

2.1. Non-covalent immobilization In the context of using clay minerals as the support, the most used enzymes are lipases, tyrosinase, β-gluasidase, cellulase and catalase.

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(a)

(b)

(c)

(d) O

OH

Al or Mg

Si

Fig. 2. Surfaces and sites on 2:1-type clay minerals (a) and (c) and 1:1-type clay minerals (b) and (d) for immobilization of enzymes. (a) and (b):surface and edge position; (c) and (d): hydrophobic and hydrophilic sites.

They have been reportedly immobilized onto clay minerals through socalled non-covalent methods (Lozzi et al., 2001; Kelleher et al., 2003; Noinville et al., 2004). The non-covalent immobilization can be realized by direct physical or chemical adsorption of enzymes on clay minerals (Szabo et al., 2007). In other words, for such immobilization, a major operation is just to impregnate clay minerals in a solution containing enzymes and then to dry clay mineral enzyme hybrids under mild conditions (Quiquampoix et al., 1993; Benetoli et al., 2007; Cai et al., 2008; Wu et al., 2012). Usually, no additional coupling reagents, surface treatment and protein modification are required (Ozturk et al., 2007; Betancor and Luckarift, 2008; Valdes-Solis et al., 2009). As for frequently-used 2:1-type smectites, the electronic charges on the layers play a leading role in the electrostatic attraction between clay minerals and enzymes. For these clay minerals, isomorphous substitution in the framework results in permanent negative charges on the layers, balanced by exchangeable cations in the interlayer space. The exchangeable cations (e.g., Na+ or Ca2+ in the interlayer space of Mt) can be replaced by positive enzyme molecules. As a result, positive-charged enzyme molecules, such as catalase (Alkan et al., 2005) and βglucosidase (Serefoglou et al., 2008) are introduced into the interlayer space by cation exchange reactions, when the pH of enzyme solution is lower than isoelectric point of enzyme. The introduction of enzyme molecules into the interlayer space of clay minerals by cation exchange reactions are usually confirmed by X-ray diffraction (XRD) patterns in many studies (Lozzi et al., 2001; Sanjay and Sugunan, 2007; Johnston et al., 2011). In the XRD patterns, a reflection appears at lower 2θ = 8.9° and its intensity increases with the amount of enzyme adsorbed. These observations are often used to prove that the enzyme molecules are intercalated into the interlayer space of clay minerals.

Immobilization of enzyme by a cation exchange reaction helps to stabilize the immobilized enzyme against thermal denaturation (Lozzi et al., 2001; Sanjay and Sugunan, 2008; Wang et al., 2013). Compared with sepiolite (Sep) and palygorskite (Pal), smectites with remarkable cation exchange capacity prove more favorable for enzyme immobilization (Table 1). For instance, glucoamylase is immobilized on Mt or modified Mt usually by an ion exchange reaction. However, it is immobilized on Sep by hydrophobic force and cross-linking instead. In principle, a higher degree of isomorphous substitution in the clay minerals leads to a higher layer charge density and a higher cation exchange capacity. When Mt has a higher cation exchange capacity, the absorption capacity for enzymes is increased. Nonetheless, such a conclusion is believed to be merely applicable to some swelling clay minerals. Moreover, it should be noted that a much higher layer charge density may in turn lead to a much stronger attraction between the neighboring layers of clay minerals and thus prevent enzyme molecules from entering the interlayer space (de Oliveira et al., 2005a, 2005b). In addition, deprotonation of hydroxyl groups on the surface of clay minerals leads to variable negatively charged sites, for example \\Si\\O\\Al\\O− at the edges of clay minerals. On such sites, the positive enzymes are also readily adsorbed and fixed mainly by the electrostatic interactions (de Fuentes et al., 2001). The amount of negativelycharged sites is related to the deprotonation of surface hydroxyl at the broken edges of clay minerals (Fig. 2), so it can be adjusted by changing the pH of the media used for the impregnation process. Besides, it can be controlled by choosing clay minerals of different particle sizes. In this aspect, clay mineral nanoparticles are worth being judiciously chosen for enzyme immobilization. This choice of clay mineral nanoparticles is particularly important to the adsorption of enzyme onto the broken edge of

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clay minerals (Tang et al., 1993; Gopinath and Sugunan, 2005, 2007; Serefoglou et al., 2008; Tzialla and Pavlidis, 2010; Daniel et al., 2011). Additionally, some researcher suggested that so-called ionic adsorption binding was formed between enzymes and clay minerals. The ionic adsorption can provide high resistance to the change of temperature and strong storage stability (Zhai et al., 2010). In comparison, 1:1-type clay minerals have little isomorphous substitution and accordingly, in the interlayer space there are no exchangeable cations. Also, there are hydrogen bondings between H and OH groups of their adjacent layers. As a result, it is difficult to introduce enzyme molecules into their interlayer space. Nevertheless, it is possible to immobilize enzymes onto such 1:1-type clay minerals because the adsorption of enzymes can still occur at their edges and on their external surfaces (Fig. 2b). In addition to electrostatic interactions, hydrophobic interactions can also lead to the adsorption of enzymes onto the surfaces of clay minerals (Quiquampoix et al., 1993; Chen et al., 2000; Bajpai and Sachdeva, 2002). Remarkably, the external Si\\O\\Si plane of each tetrahedral sheet of clay minerals provides relatively large hydrophobic surface (Fig. 2, c and d). Hence, the adsorption of enzymes onto clay minerals through hydrophobic interactions is essential. Particularly, such surface immobilization is conducive to keep configuration of enzyme in an active state and thus maintains the biological activity of the enzymes (Gitlesen et al., 1997; Knezevic et al., 1998; Betancor and Luckarift, 2008). For example, the hydrophobic surface of Mt somehow resembles the interface of lipases. Such surfaces not only provide the platform for hosting lipases but also can direct enzyme molecules towards a particular orientation (Wannerberger et al., 1997). In addition, the hydrophilic nature of the parent clay minerals can be converted into hydrophobic one by replacing the exchangeable cations in the interlayer space with cationic organic molecules to form the so-called ‘organoclays’. Such hydrophobic modification of the clay mineral internal surface allows many hydrophobic guest molecules to be readily intercalated (Bastida et al., 1998; Kim et al., 2001). Noticeably, the hydrophobic modification can also be applied primarily to external surfaces of a clay mineral, rather than the interlayer space of a clay mineral. For example, Wicklein et al. (2011) demonstrated that the assembly of lipid bilayer phosphatidylcholine onto the external surface of natural Sep fibers yielded biomimetic interfaces for the stable immobilization of the urease and cholesterol oxidase enzymes. Layer packing density, hydrophilicity/hydrophobicity, and surface charge were investigated for enzyme immobilization and stabilization of their biological activity. The resulting bio-organoclay enzyme hybrids exhibited good preservation of bioactivity which was ascribed to the accommodation of the enzymatic system within the biomimetic lipid interface on Sep. Layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. It is worth noting that immobilization onto clay minerals by a direct cation exchange reaction is only suitable to small enzyme molecules, which are several nanometers or less in diameter, such as lysozyme (Johnston et al., 2011), β-glucuronidase (Fiorito et al., 2008) and cellulose (Sinegani et al., 2005), because of the limitation of the interlayer space height of clay minerals. To overcome the limitation, one alternative method is to pre-treat clay mineral by replacing inorganic cations in the interlayer space with bulky organic cations as mentioned above in the hydrophobic modification of the clay mineral internal surface (Pérez-Santano et al., 2005; de Paiva et al., 2008; Fiorito et al., 2008; Bernd et al., 2011; Fujimori et al., 2014). In addition to the fact that the hydrophobic properties of clay minerals are substantially improved and hydrophobic enzyme molecules tare more readily adsorbed on the surface of clay minerals, such hydrophobic modification of the clay mineral also allows bulky enzymes to be intercalated in their interlayer space (Fig. 3). More recently, Andjelković et al. (2015) have investigated the use of beidellite (Bd) for invertase immobilization. Bd was first modified by surfactant– hexadecyl trimethylammonium cation (HDTMA), pillaring with Al/Fe

287

∼2.0-7.0 nm

Mt

Organic cations

Enzyme

Fig. 3. Hydrophobic modification in the interlayer space of clay minerals for immobilizing enzymes by intercalation and adsorption. (Depentent upon the size of the intercalated organic cations, the height of the gallery varies in the range of ~2.0-7.0 nm).

containing polyhydroxy cations and acid modification of Naenriched and pillared clay minerals. Obviously, different chemical modifications drastically influenced the enzyme loading capacity of clay minerals. Besides electrostatic interactions and hydrophobic interaction, the formation of hydrogen bonding and salt bridges (Joshi et al., 2000; Sanjay and Sugunan, 2005a, 2005b) can also be used in binding enzymes onto clay minerals. For example, H+ in the acid-activated Mt can serve as centers for binding enzymes through the formation of hydrogen bonding though the interaction between H+ and the \\NH2 group of enzymes (Fig. 4). In addition, silanol groups at edge of each layer of clay minerals can immobilize enzyme owing to the formation hydrogen bondings between\\Si\\OH and enzyme molecules. Besides, non-polar portion of larger biological molecules can efficiently be bound to the surfaces of clay minerals through van der Waals force. Yet van der Waals force is generally too weak to keep the enzyme fixed on the supports under industrial conditions, for example high concentration of reactants and products and a high ionic strength (Adélia et al., 2003; Botella et al., 2004). Finally, it should be pointed out that although immobilizing enzymes onto clay minerals by non-covalent adsorption is a simple and green way, the interactions such as electrostatic interactions (Liang et al., 2007), hydrogen bonding, van der Waals forces, and hydrophobic interactions are weak compared with covalent bonds (Chang and Juang, 2005; Sanjay and Sugunan, 2008). Moreover, the stability of adsorbed enzymes by non-covalent binding is considerably affected by environmental conditions (pH, temperature, ionic strength and biomolecule concentration, etc. (Ding and Henrichs, 2002)). Therefore, the clay mineral enzyme hybrids made from non-covalent immobilization have the problem of the desorption of enzymes from clay minerals. Accordingly, such leaching of enzymes in a liquid-phase system gives rise to a rapid loss of the activity of clay mineral enzyme composites as catalysts or biosensors. 2.2. Covalent bonding When strong interaction between enzymes and clay minerals is required, immobilization by covalent bonding is a preferred choice. Several immobilization protocols using covalent binding have already been developed so far (Mateo et al., 2007; Cowan and Fernandez-Lafuente, 2011; Hernandez and Fernandez-Lafuente, 2011; Barbosa et al., 2013). First of all, covalent immobilization of enzymes onto clay minerals can be achieved by the formation of covalent bonds directly between the functional groups on the surface of clay minerals and functional groups on the enzyme molecules. For example, Lozzi et al. (2001) immobilized enzyme horseradish peroxidase (HRP) on Na-Mt and Cheng et al.

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Mt

H+

Enzyme

Fig. 4. Immobilization of enzymes onto clay minerals by hydrogen bonding.

(2006) immobilized HRP on aluminum-pillared interlayered clay mineral (Al-PILC). Some experimental data has been use to claim that there might be intermolecular interaction between enzyme and some specific sites of clay minerals. An examples is that after immobilization of HRP on Al-PILC, absorption band at 3631 cm− 1 correlated with stretching vibration of free hydroxyl groups disappeared (Cheng et al., 2006). Similar evidence was shown when invertase was immobilized on Bd (Andjelković et al., 2015). The covalent bonds can also be created with the assistance of linking molecules (Table 1). Several studies have proved that the covalent immobilization can improve reusability, and storage stability of enzymes (Gopinath and Sugunan, 2008; Tzialla and Pavlidis, 2010; Bayramoglu et al., 2013). The improvement is reasonably attributed to a substantial reduction in the rate of leaching of the enzyme (Occelli et al., 2000; Bayramoglu et al., 2003). It has been well-documented that the activation process produces and exposes more active sites for linking enzymes on the surface of the clay minerals (Komadel and Madejová, 2006; Sanjay and Sugunan, 2008). A typical example procedure is that investigated by Sanjay and Sugunan (2007), in which calcined Mt K-10 was mixed with 10% (v/v) solution of 3-amino propyl triethoxy silane in acetone and then treated with a 10% aqueous glutaraldehyde (GA) solution. The resultant modified clay minerals were used for glucoamylase immobilization by covalent bonds. As a result, covalently glucoamylase showed better reusability, storage stability and resistivity to leaching than adsorbed glucoamylase. Such activation of clay minerals for linking enzymes is usually needed and beneficial to make them form covalent bonds with enzymes (Bautista et al., 1998). In other words, more enzymes can be covalently linked to the activated clay minerals than pristine clay minerals. When the surface of clay minerals is grafted with specific functional groups, these groups can link enzymes by forming covalent bonds between clay minerals and enzymes. So far, functional agents such as GA, chitosan and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) (Table 1) have proved effective. Nevertheless, it should be pointed out that the benefits from surface modification of clay minerals are multiple. For example, GA-grafted clay minerals can provide varied hydrophilicity, porosity and adhesion area, and reduced mass transfer resistance for the immobilization of enzymes (Chang et al., 2008; Huang et al., 2008; Reshmi and Sugunan, 2013; You et al., 2013). In addition, chitosan molecules possess hydroxyl (OH) and amino (NH2) groups, which can be linked to enzymes easily (Fig. 5) (Srere and Uyeda, 1976; Chang and Juang, 2007). Moreover, immobilized enzymes on clay mineral chitosan nanocomposites could well preserve tertiary structure of an enzyme and avoid its conformational changes. Some researchers have revealed that such immobilized enzymes maintain a high activity in a broader pH range than free enzymes. For example, Chang and Juang (2005, 2007) investigated the activity and stability of enzymes immobilized on clay mineral chitosan nanocomposites. The stability of enzymes (such as α-amylase and glucoamylase)

immobilized on the clay mineral chitosan nanocomposite matrix used at low pH were found to be much improved. The increased durability can be ascribed partly to the presence of amino/amine groups of chitosan which can accommodate protons, thereby resulting in a decrease of local concentration of proton near enzyme molecule (Gupta and Ravikumar, 2000; Cetinus and Oztop, 2003). In addition, the surface modification and covalent bonding with two or more bulky multifunctional crosslinking agents is found conducive to forming insoluble 3D reticular structure on the surface of clay minerals for enzyme immobilization. Earlier, Rorrer et al. (1993) demonstrated that clay minerals were modified with chitosan which was then crosslinked with GA for covalent immobilization (Fig. 5). Enzyme was then successfully immobilized on the surface of clay minerals by the interaction between enzyme and GA. Gopinath and Sugunan (2007, 2008) prepared hybrids consisting of Mt K-10 (particle size b 0.2um) functionalized with 3-amino propyl triethoxy silane and GA for immobilization of invertase. It was revealed that the functionalization of clay minerals with silane groups and GA molecules led to entirely immobilization of invertase with Mt by entirely covalent binding. Another important advantage of keeping cross-linking reticular structure is that it can suppress the dissolution and leaching of enzyme even in much acidic solutions (pH b 2) (Rorrer et al., 1993; Servagent-Noinville et al., 2000; Gougeon et al., 2002). Clearly, the covalent immobilization is confined to clay mineral surface. All surface modifications are designed and conducted centering on promoting the fixation of enzymes on clay mineral surface while maintaining the bioactivity of the enzymes. As for the detection, for the formation of covalent bonds, IR and NMR are often used. As for the detection of the occurrence of immobilization on the surface or in the interlayer space, XRD data can be convincing evidence. Typically, if the immobilization is simply adsorbed and fixed to the external surface of

Fig. 5. A schematic drawing showing activation of clay minerals for their covalently immobilized enzyme.

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clay minerals, in the XRD patterns of clay mineral enzyme nanocomposite, no much change in the (001) reflection can indicate no much change in the basal spacing (Reshmi and Sugunan, 2013). By contrast, in the case of enzymes are introduced and immobilized in the interlayer space of clay minerals, the (001) reflection will shift further to occur at a lower 2θ angles. The results imply that undoubtedly, the interlayer space of the clay minerals is expanded, arising from the intercalation of the enzyme (Sanjay and Sugunan, 2007). 3. Applications of clay mineral enzyme hybrids 3.1. Biocatalytic engineering For clay mineral enzyme hybrids as an industrial biocatalysts, immobilization of enzyme on clay minerals should maintain higher activity and less sensitive to reaction conditions (temperature and pH) (Sanjay and Sugunan, 2006; Huang et al., 2009; Kim et al., 2012). Separation and reuse of enzyme is also the prime concern besides its catalytic activity. The immobilization of enzymes on clay minerals has long been considered as an alternative to produce a class of recoverable biocatalysts so that the biocatalysts have enhanced the stability of enzymes and can be easily separated from the liquid-phase reaction mixture particularly in a batch reactor in industry (Zhou, 2011; Yu et al., 2015). In the context of the separation of the catalysts after reactions, over the past decade, breakthroughs have been made in introducing magnetic particles into clay mineral enzyme hybrids as catalysts. Several kinds of clay minerals such as, Mt, Pal and Kaol have been successfully modified with magnetic materials (Bourlinos et al., 2000; Oliveiraa et al., 2003; Szabo et al., 2007; Daniel et al., 2011). For example, starch hydrolysis (Eq. (1)), residual activity of the immobilized enzymes on modified clay minerals in an acetate buffer was 71.86% for the Fe3O4@Kaol (Fe3O4 nanoparticles anchored on Kaol) and 64.96% for the Fe3O4@ Pal (Fe3O4 nanoparticles were anchored on Pal) after the 10th reuse (D.T. Zhao et al., 2011; G.H. Zhao et al., 2011). What is more, enzymes immobilized clay mineral modified with magnetic materials as the biocatalyst enable them to be magnetically recoverable, and thus effectively simplify the operation and reduce the cost of catalyst separation in industry.

ð1Þ In addition to conventional batch bioreactor, recent research progress also indicates that clay mineral enzyme hybrids as catalysts can be used in either packed or fluidized bed reactors (Tertre et al., 2013; Andjelković et al., 2015) (Fig. 6). For example, Sanjay and Sugunan

(a)

289

(2005a, 2005b) immobilized glucoamylase on calcined Mt K-10 via two methods, adsorption and covalent binding, and used the catalysts in a continuously fixed bed reactor. The covalently bound glucoamylase/Mt K-10 catalysts were able to be used in starch hydrolysis without any loss in activity for 100 h whereas those in the adsorbed form lost 5% activity after 84 h. In addition, they also found that immobilization of enzymes on clay minerals also helped to maintain the stability of enzymes in low pH and at high temperatures. Such enhanced operational stability and adaptability with a continuous reaction system are crucial and attractive for a large-scale process in industry. Similar positive results were also observed by Sanjay and Sugunan (2008) using immobilized a-amylase, glucoamylase and invertase on acid activated Mt K-10. The catalysts could be reused for more than 15 cycles for about 96 hours in the packed bed reactor without any loss in activity. Tzialla et al. (2009) immobilized Candida antarctica lipase B (CaLB) on organic-modified clay minerals for the epoxidation of terpenes (Eq. (2)). The use of immobilized CaLB led to up to 4 times higher product concentration than that of free enzyme, retaining up to 60% of its activity after 96 h of total operation.

ð2Þ

Besides the uses of the clay mineral enzyme hybrids as biocatalysts for the production of fine chemicals, recently more efforts have also been made to use the biocatalyts in environmental engineering (Cheng et al., 2006; Pirillo et al., 2010; Kim et al., 2012) and even and to the fabrication of biofuel cells (Zebda et al., 2011; Holzinger et al., 2012). For example, HRP had been successfully immobilized on the Alpillared layered clay minerals (PILC) via interaction between the functional groups of the PILC support and the enzyme (Cheng et al., 2006). Compared with free enzyme, the immobilized enzyme functioned in a broader range of pH from 4.5 to 9.3 and had better storage stability. In the presence of H2O2, the immobilized HRP exhibited good phenol (Eqs. (3), (4) and (5)) removal over a wide range of phenol concentrations between tens and hundreds of milligrams per liter. Modified clay minerals with crosslinking agent improved the efficiency of enzyme for the removal of phenolic compounds from wastewater. HRP was also immobilized on fulvic acid activated Mt by Kim et al. (2012) Such immobilized HRP also demonstrated noticeably increased enzyme stability under conditions with relatively large variations in pH, temperature, and ionic strength, long-term storage, and repetitive use. Dinçer et al. (2012) immobilized tyrosinase on GA crosslinked clay mineral chitosan nanocomposite beads and used for phenol removal. After seven

(b)

Fig. 6. Clay mineral enzyme hybrids as catalysts used in different bioreactors. (a) In a fixed bed reactor; (b) in a fluidized bed reactor.

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times of repeated tests with each run over 150 min, the efficiency of phenol removal using the same immobilized tyrosinase beads as catalysts were merely decreased to 43%. HRP þ H2 O2 →Compound  I þ H2 O

ð3Þ

Compound  I þ Phenol→ Compound  II þ Phenol Compound  II þ Phenol→HRP þ Ph

ð4Þ ð5Þ

Zhai et al. (2013) synthesized halloysite (Hal) chitosan hybridnanotubes for HRP immobilization through aid cross-linking by GA. Enzyme immobilized on Hal chitosan hybrid-nanotubes has a better stability and was active in a wide pH range. The catalyst, moreover, showed remarkable efficiency for phenol removal (Eqs. (3), (4) and (5)). About 98.8% for 0.5 mM, 94.3% for 1 mM, and 78.0% for 2 mM of total phenol were degraded in the initial 30 min. The overall high removal efficiency and the ability working in a wide range of conditions is of great significance for the treatment of wastewater because the pollutant contents in the wastewater varies considerably in the industrial processes using phenolic compounds or any other organic pigments and dyes. More recently, Chang et al. (2015) showed that laccase immobilized on modified clay mineral effectively removes hydrophobic organic compounds from soil/groundwater. Nonionic surfactantmodified clay mineral is claimed to be able to protect the laccase against denaturation and preserve its activity. 3.2. Biosensing Over the past decade, using clay mineral enzyme hybrids in biosensors have captured more attention than using them in biocatalytic engineering. Enzyme-coupled electrochemical biosensors (EEB) function on the basis of the detection of an electric signal induced by an electroactive species either produced or depleted by an enzymatic reaction (Shyu and Wang, 1998; Wang, 2008; Ronkainen et al., 2010). The development of the clay mineral enzyme hybrids for biosensing technology is also encouraged by an example in which that amperometric enzymebased bioelectrodes have already been successfully developed for in vivo monitoring of blood glucose. Now researchers are expecting that the applications of enzyme-based bioelectrodes can be extended to other chemical detections for quick clinical analyses (Merkoçi, 2010; Srivastava et al., 2013), food analysis (Pérez-López and Merkoçi, 2011; Monosik et al., 2012). As shown in Fig. 7, the evolution of electrode, a core part in the enzyme-coupled electrochemical biosensor, has undergone a progress in the simplification with the enhancement of the signal transduction pathway. The biosensor using enzyme immobilized on clay minerals can overcome some disadvantages in first- or second-generation biosensors, including enzyme inactivity, little reusability, high oxygen consumption, low electron transfer and easy electrode pollution from mediators

Substrate (e. g. Starch)

GOX ðFADÞ þ Glucose → GOX ðFADH2 Þ þ Gluconic acid

ð6Þ

GOX ðFADH2 Þ þ Medox → GOX ðFADÞ þ Medred

ð7Þ

Interestingly, the use of clay minerals contributes much to the detection range. For example, amperometric glucose biosensor based on the immobilization of GOx on a ferrocene@ NaY zeolite (ferrocene was encapsulated in a NaY zeolite), with a linear range for 8 × 10− 7 to 4 × 10−3, and detection limit of 2 × 10− 7 M (Dong et al., 2011). As shown in the study of Luo et al. (2013) a glucose biosensor based on enzyme immobilized on ZnO-based supports had an analytical range from 1 × 10−5 to 160 × 10−5 and a detection limit of 1 × 10−6 M (Liu et al., 2009). By contrast, a glucose biosensor based on enzyme immobilized on clay minerals had a broader analytical range from 5 × 10−5 to 370 × 10−5 M and a lower detection limit of 6.24 × 10−10 M (Table 2). Because of the high polarizing voltage applied (Eapp ≈ 0.6–0.8 V) to the electrode, interference such as ascorbic acid and uric acid, which are commonly present in biological fluids, can also be oxidized, leading to non-specific signals. Attempts have thus been made to improve the clay mineral-supported enzyme system to overcome this problem. A strategy is to use clay mineral-semipermeable polymer nanocomposites for electrodes to reduce the permeability to organic interfering compounds (Zen and Lo, 1996; Poyard et al., 1998, 1999; Shan et al., 2007; Mousty et al., 2009). Alternatively, some researcher suggested using redox mediators to reduce the electrode potential (Putzbach and Ronkainen, 2013). In this context, when clay mineral-supported enzymes are used, the clay minerals can play an additional role in stabilizing the mediator. Moreover, the large surface and porosity of the clay minerals provide a sufficient space for the mass transport of mediator. In addition, the interaction of functional groups on clay minerals with synthetic mediators such as poly(butylviologen) (Qian et al., 2002) and poly(ophenylenediamine) (Luo et al., 2013) can inhibit the

Substrate Product (e. g. Starch) (e. g. Glucose) Enzyme (e. g. Diastase) Substrate Product (e. g. Starch) (e. g. Glucose)

Enzyme (e. g. Diastase)

Electrode

e– The first generation electrode H 2O 2

(Mousty, 2004; Shan et al., 2009a, 2009b; Wu et al., 2011; Zapp et al., 2011a, 2011b). In addition, an ideal enzyme-coupled electrochemical biosensor should have a high sensitivity, a low detection limit, a broad linear range for the detection of the analytes. Recent studies indicated that the detection range and the detection limit of enzymatic biosensors have been considerably improved as a result of incorporating clay minerals with enzymes and other functional molecules in the electrode (Yao et al., 2006; Sun et al., 2010; Songurtekin et al., 2013). An enzyme biosensor based on electrode with clay mineral enzyme nanocomposites in it is schematically showed in Fig. 8. In the biosensor for glucose detection, glucose is oxidized by converting flavin adenine dinucleotide (FAD) component of glucose oxidase (GOx) into FADH2. Mediatorox regenerates the FAD, with a simultaneous self-reduction. Then the mediatorred is regenerated at the electrode surface, producing an electric signal. The reactions are depicted as follows (Eqs. (6) and (7)):

O2

M red M Ox (e. g. Vinylimidazole) Electrode

e–

The second generation electrode

Product (e. g. Glucose)

Enzyme (e. g. Diastase)

Electrode

e–

The third generation electrode

Fig. 7. Advances in the enzyme-based electrode system as a biosensor. M: redox mediator.

N. An et al. / Applied Clay Science 114 (2015) 283–296

Fig. 8. A schematical drawing of enzyme biosensor based on electrode with clay mineral hybrids.

leaching of the mediators from the biosensor in in vivo use over time. For example, Mbouguen et al. (2007) developed a phenol biosensor by immobilizing polyphenol oxidase (PPO) onto trimethylpropylammonium (TMPA)-modified clay minerals (Table 2), with the use of ferrocene as mediator. The bioelectrode consisting of mediator and enzyme immobilized on clay minerals demonstrated a widened linear range for catechol detection (2 × 10−8 to 1.2 × 10−5 M) and a lower detection limit of (9 × 10−9 M). In the electrode, the organoclay mineral was found to provide a favorable environment to enzyme activity. Ferrocene as mediator, improved the electron transfer. As discussed in preceding section, clay minerals modified with organic functional molecules as supports can host an increased amount of enzymes because the presence of organic functional molecules in clay minerals enhances the interactions between enzymes and modified clay minerals. For example, Mbouguen et al. (2006) developed an amperometric biosensor, where GOx was immobilized on aminopropyl (AP)-modified clay minerals through GA and applied to a platinum

291

electrode. The presence of aminopropyl groups grafted onto the clay mineral particles enables the covalent binding of a significant amount of GOx linked to the aluminosilicate clay mineral particles via GA while no significant enzyme immobilization was likely to occur when using unmodified clay mineral samples. Similar improvement in the performances arising from the use of organically modified clay minerals has also been observed by many other researchers (Serefoglou et al., 2008; Zhao et al., 2008; Han et al., 2010). In addition to the wide linear range and the low detection limit, the chemical and mechanical stability, response time, repeatability and reproducibility and operability of a biosensor can also be improved to some extent when the organo-clay mineral host matrix is used in the electrode (De Oliveira et al., 2014). It is worth nothing that great advances have been made in the assembly of clay minerals with functional molecules to yield a variety of nanostructured clay-containing hybrid films by the solvent casting, the spin-coating, the layer-by-layer (LbL) assembly (Kotov et al., 1997; Szabo et al., 2007) and the Langmuir–Blodgett (LB) techniques (Szabó et al., 2010). These new strategies imply that instead of single clay minerals, hierarchically structured clay mineral nanocomposites could offer enhanced performances for supporting enzymes and then for constructing biosensors. For example, Sun et al. (2010) constructed an electrode from Hal nanotubes/chitosan and HRP. Therein Hal nanotubes with the chitosan as a binder effectively adhered on glassy carbon electrode to form a nanocomposite film. The biosensor for detecting H2O2 based on (Eqs. (8), (9) and (10)) showed a short response time less 5 s with a detection limit of 0.7 μM. H2 O2 þ HRP→H2 O þ Compound  I;

ð8Þ

Compound  I þ e− → Compound  II;

ð9Þ

Compound  II þ e− →HRP:

ð10Þ

On the negative side, enzymes immobilized on clay minerals can lead to a rather slow rate of heterogeneous electron transfer because the clay minerals are nonconductive and the redox active sites from enzymes are buried deep in the clay minerals (Yao et al., 1998; Shan et al.,

Table 2 Enzymes immobilized on clay minerals recently investigated for biosensors. Detection limits/M

Response Ref. time/s

– – 14 16.3 – – 365 – 2.5 – – 55.8 12.25 – 6.28 –

0.1 × 10−3 3.8 × 10−5 1.5 × 10−6 4 × 10−9 6.24 × 10−10 3.09 × 10−8 9 × 10−9 23.5 × 10−7 4.2 × 10−8 1.897 9.0 × 10−9 0.5 × 10−10 0.7 × 10−10 5 × 10−6 – 3.0 × 10−8

– – – – 6 – 20 – – 35 – 2 – – – –

Liang et al. (2003) Seleci et al. (2012) Mbouguen et al. (2006) Han et al. (2010) Luo et al. (2013) Zapp et al. (2011a, 2011b) Mbouguen et al. (2007) Zapp et al. (2011a, 2011b) Songurtekin et al.(2013) Chen and Jin (2010) Zhao et al. (2008) Wu et al. (2011) Sun et al. (2010) Chen et al. (2011) Maghear et al. (2013) Oliveira et al. (2012)

–

0.41

–

de Oliveira et al. (2014)

1640 – –

0.36 × 10−6 5 × 10−8 3.7 × 10−7

– – –

Soylemez et al.(2013) Brondani et al. (2012) Pusch et al. (2013)

Enzyme

Clay

Analyte

Functional molecule

Electrode Analytical ranges/M Sensitivity/mA M−1

GOx

Mt DM-Mt Clay Baba Clay Pal Mt Clay Baba Mt Mt Pal Mt Pal NanoHal Pal Mt Mt

Glucose Glucose Glucose Glucose Glucose Rutin Catechol Methomyl Catechol Phenol H2O2 H2O2 H2O2 H2O2 AC Glyphosate

Ru(bpy)2+ 3 /GA BSA/GA AP/GA Nano-Pt/amidoamine Po/A BMI·BF4/nano-Ir TMPA/ferrocene BMI·BF4/nano-Pt Histidine – – Chs/nanoAu – – PEI –

ITO GCE Pt GCE Pt CPE GCE CPE GCE GCE GCE GCE GCE GCE GCE CPE

0.1–10 × 10−3 0.05–10 × 10−3 0.1–3.5 × 10−4 1–16 × 10−5 0.5–37 × 10−4 9.17–310 × 10−6 0.002–1.2 × 10−5 9.8–90 × 10−7 1.0–10 × 10−9 0.0005–1 × 10−4 3.9–3.1 × 10−3 0.2–150 × 10−9 2–75 × 10−9 5–300 × 10−6 5.25–49.5 × 10−6 0.1–45.5 × 10−3

TBHQ

Carbon nanotube/nafion CPE

1.65–9.82 × 10−3

PPO-LAC PPO LAC Tyrosinase HRP

Atemoya peroxidase Ingá-cipó Sep peroxidase ChOx peroxidase Sep Hal Mt

Cholesterol GA Ca Pt-BMI·PF6 Polyphenol Pt–Pd-BMI·PF6

GE CPE CPE

0.0–40.0 × 10−6 4–1.887 × 10−7 2.7–22 × 10−6

AC: acetaminophen; AP: aminopropyl; BMI·BF4: 1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid; BMI·PF6: 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid; BSA: bovine serum albumin; Ca: catecholamine; Chs: chitosan; ChOx: cholesterol oxidase; CPE: carbon paste electrode; DM: dimethylamine; GA: glutaraldehyde; GCE: glassy carbon electrode; GE: graphite electrode; GOx: glucose oxidase; Hal: halloysite; HRP: horseradish peroxidase; ITO: indium tin oxide; LAC: laccase; Mt: montmorillonite; Pal: palygorskite; PEI: polyethyleneimine; PO: poly(o-phenylenediamine); PPO: polyphenol oxidase; Sep: sepiolite; TBHQ: tert-butylhydroquinone; TMPA: trimethylpropylammonium.

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2004; Cosnier et al., 2008). Accordingly, electron transfer decays exponentially with the increasing distance between clay mineral support and electrode surface. A reduction in the dimensions of the particles of clay minerals could improve the electron transfer by promoting their interactions with enzymes and by reducing diffusional limitations (Zhao et al., 2008). For example, a glucose biosensing electrode was constructed by entrapping GOx in nanostructured Pal film-modified glassy carbon (GC) electrode (Serefoglou et al., 2008). Voltammetric measurements indicated that direct electron transfer between the active center of immobilized GOx and electrode was achieved. The GOx on the nano-Pal modified electrode retained its bioelectrocatalytic activity for the oxidation of glucose with a good stability and was found to be free from electrochemical interferences of co-existing species (Bryjak, 2003; Knutson et al., 2005). In addition, to tackle this issue of electron transfer, another effective strategy is to integrate clay minerals along with carbon nanotubes (CNTs), graphene and metal nanoparticles (Fig. 9). Metal nanoparticles, graphene and CNTs can act as “wiring line” to be incorporated into clay minerals for a better electron transfer between the active site of the enzyme and the electrochemical transducer. Chen et al. (2011) constructed a biosensor for detecting H2O2 based on HRP immobilized on Pal (Table. 2). An analytical ranges from 5– 300 × 10−6 M and a detection limits of 5 × 10− 6 M were achieved. Wu et al. (2011) developed a biosensor based on HRP immobilized on Pal, with nano-Au as “wiring line”. The biosensor for detecting cellular reactive oxygen species, had a broader analytical ranges of 0.2– 150 × 10−9 M, and a lower detection limits of 5 × 10− 11 M. More Recently, De Oliveira et al. (2014) immobilized peroxidase on Sep and incorporated into a carbon paste electrode (CPE) containing multiwall CNTs and nafion for the determination of tert-butylhydroquinone (TBHQ). A linear response between 1.65 and 9.82 mg L−1 was observed. The improved performances can be attributed to the enhanced efficiency of the peroxidase immobilization onto the Sep and the addition of CNTs which provided a high surface area, excellent electrical conductivity, chemical stability and mechanical strength. Metal nanoparticles such as gold, silver, and platinum have also been introduced into the electrode with enzymes/clay minerals (Cai et al., 2006; Xu et al., 2006; Zhao et al., 2008; Feng et al., 2013). A recent example is that Zapp et al. (2011a, 2011b) developed a peroxide biosensor comprising iridium or platinum nanoparticles entrapped in 1-butyl3-methylimidazolium tetrafluoroborate ionic liquid (Ir-BMI.BF4) on Mt. Such iridium or platinum nanoparticles entrapped in BMI.BF4 facilitated electron transfer in the electrode. Under optimized conditions, such a biosensor worked well in a broad linear range for rutin concentrations from 9.17 × 10− 8 to 3.10 × 10− 6 M with a detection limit of 3.09 × 10−8 mol L−1, and a broad linear range for methomyl pesticide concentrations from 9.8 × 10− 7 to 9.0 × 10− 6 mol L− 1 with a detection limit of 2.35 × 10− 7 mol L− 1. More recently, Hal nanotubes were used as the support for the immobilization of peroxidase (PO) together with platinum nanoparticles dispersed in 1-butyl-3methylimidazolium hexafluorophosphate ionic liquid (Pt-BMI.PF6) (Brondani et al., 2012). The corresponding biosensor showed good stability and lifetime, adequate reproducibility and a low detection limit

for the determination of dopamine and other catecholamines. However, it should be pointed out that in the electrode consisting of metal nanoparticles, protective agents such as small organic molecules or polymers is often necessary so as to prevent metal nanoparticle aggregation (Han et al., 2010). Moreover the poison and deactivation of enzymes by the nanoparticles and additives should not be ignored. 4. Concluding remark Many studies have proved that natural clay minerals have good compatibility with biological enzyme molecules. The enzymes can be immobilized on clay minerals in the pristine or modified form through non-covalent adsorption and covalent conjugation. In non-covalent immobilization, enzymes can be less denatured. The intrinsic structure and properties of both enzymes and clay minerals are preserved. However, enzymes immobilized by non-covalent adsorption can desorb from the clay mineral supports upon the changes in temperature, pH and ionic strength in liquid media. Such leaching becomes particularly problematic when such clay mineral enzyme hybrids are used in a liquid-phase system. In contrast, covalent conjugation provides more endurable attachment, but the structure of the covalently bound enzyme may be more easily disrupted. Though the characteristics of layered clay minerals such as ion exchange, intercalation and surface modification offer many routes to fine tune the immobilization of enzymes on them, to preserve the activity of the enzymes with enhancement of the stability and recyclability are still challenging and need further investigation. Conventionally, clay mineral enzyme hybrids are predominantly designed and made as recoverable biocatalysts used in bioreactors for chemical production. Correspondingly, the major concerns therein are the catalytic activity, separation, recyclability and lifetime. These are also prime concerns if they are used in environment engineering for the treatment of solid waste and wastewater. Recently a significant number of studies aim at the use of the clay mineral enzyme hybrids in biosensors. Much different from biocatalytic engineering, the presence of the clay minerals in the biosensing electrode system should be considered to help widen the linear range and improve the detection limit. Meanwhile, the biosensor should have performances such as a high selectivity, fast response time, along with good repeatability of the determination. The progress in the past years has demonstrated a variety of strategies conducive to meeting such requirements. To this end, in addition to single clay mineral as the support for immobilizing enzymes, addition of other functional materials from either organic compounds or inorganic nanomaterials proves essential and effective. Such integrated system may enhance the capabilities of the electrode biosensing device containing clay mineral enzyme hybrids. Despite the impressive advances in the field, there are still challenges in designing and using clay mineral enzyme hybrids to manufacture tight, stable and reliable biosensors. In addition, the compounds, which can be measured by the biosensor with clay mineral enzyme hybrids, are only a few up to now. It could be right to say that significant efforts need be made in innovatively creating hierarchical and functional clay-containing materials such as biocompatible membranes and films for the enzyme-

Clay minerals Electrode

Analyte

Support

Metal Particles Graphene

Enzyme

Carbon Tubes

Fig. 9. Electrode using clay mineral enzyme hybrids which are integrated with metal particles and carbon nanomaterials.

N. An et al. / Applied Clay Science 114 (2015) 283–296

based biosensors. Meanwhile, these clay technologies should be further integrated with genetically engineered enzymes. Undoubtedly, in addition to the analysis in food and chemical industry, there is an increasing need for non-invasive in vitro biosensors. In response to these pressing needs, next breakthroughs in the science and technology in clay mineral enzyme hybrids are certainly expected. Acknowledgments The authors wish to acknowledge the financial support from the National Natural Scientific Foundation of China (21373185), the Distinguished Young Scholar Grants from the Natural Scientific Foundation of Zhejiang Province (ZJNSF, R4100436), ZJNSF (LQ12B03004), Zhejiang “151 Talents Project”, and the projects (2010C14013 and 2009R50020-12) from the Science and Technology Department of Zhejiang Provincial Government and the financial support by the open fund from State Key Laboratory Breeding Base of Green ChemistrySynthesis Technology (GCTKF2014006) and the open fund from Key Laboratory of Clay Minerals of the Ministry of Land and Resources, Zhejiang Institute of Geology and Mineral Resources, China (2014-K02). CHZ conceived the work. NA wrote the paper with substantial and substantive corrections and revisions by CHZ. Other co-authors provided assistance during the work. References Adélia, J.A., Tunega, D., Haberhauer, G., Gerzabek, M.H., Lischka, H., 2003. Adsorption of organic substances on broken clay surfaces: a quantum chemical study. Comput. Chem. 24, 1853–1863. Akgöl, S., Kaçar, Y., Denizli, A., Arca, M.Y., 2001. Hydrolysis of sucrose by invertase immobilized onto novel magnetic polyvinyl alcohol microspheres. Food Chem. 74, 281–288. Alkan, S., Ceylan, H., Arslan, O., 2005. Bentonite-supported catalase. J. Serb. Chem. Soc. 70, 721–726. Anderson, E.M., Larsson, K.M., Kirk, O., 1998. Biocatalyst-many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal. Biotransformation 16, 181–204. Andjelković, U., Milutinović-Nikolić, A., Jović-Jovičić, N., Banković, P., Bajt, T., Mojović, Z., Vujčić, Z., Jovanovic, D., 2015. Efficient stabilization of Saccharomyces cerevisiae external invertase by immobilisation on modified beidellite nanoclays. Food Chem. 168, 262–269. Ansari, S.A., Husain, Q., 2012. Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol. Adv. 30, 512–523. Atlow, S.C., Bonadonna-Apro, L., Klibanov, A.M., 1984. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase. Biotechnol. Bioeng. 26, 599–903. Bajpai, A.K., Sachdeva, R., 2002. Immobilization of diastase onto acid-treated bentonite clay surfaces. Colloid Polym. Sci. 280, 892–899. Barbosa, O., Torres, R., Ortiz, C., Berenguer-Murcia, A., Rodrigues, R.C., Fernandez-Lafuente, R., 2013. Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 14, 2433–2462. Bastida, A., Sabuquillo, P., Armisen, P., Fernandez-Lafuente, R., Huguet, J., Guisan, J.M., 1998. Single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol. Bioeng. 58, 486–493. Bautista, F.M., Bravo, M.C., Campelo, J.M., Garcia, A., Luna, D., Marinas, J.M., Romero, A.A., 1998. Covalent immobilization of porcine pancreatic lipase on amorphous AlPO4 and other inorganic supports. J. Chem. Technol. Biotechnol. 72, 249–254. Bayramoglu, G., Akgol, S., Bulut, A., Denizli, A., Arica, M.Y., 2003. Covalent immobilisation of invertase onto a reactive film composed of 2-hydroxyethyl methacrylate and glycidyl methacrylate: properties and application in a continuous flow system. Biochem. Eng. J. 14, 117–126. Bayramoglu, G., Senkal, B.F., Arica, M.Y., 2013. Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase. Appl. Clay Sci. 85, 88–95. Benetoli, L.O.B., Souza, C.M.D., Silva, K.L., Souza, I.G., Santana, H., Paesano, A.J., Costa, A.C.S., Zaia, C.T.B.V., Zaia, D.A.M., 2007. Amino acid interaction with and adsorption on clays: FT-IR and Mössbauer spectroscopy and X-ray diffractometry investigations. Orig. Life Evol. Biosph. 37, 479–493. Bergaya, F., Lagaly, G. (Eds.), 2013. Handbook of Clay Science, 2nd ed . Developments in Clay Science vol. 5. Elsevier, Amsterdam. Bernd, W., Margarita, D., Pilar, A., Eduardo, R.H., 2011. Phospholipid sepiolite biomimetic interfaces for the immobilization of enzymes. ACS Appl. Mater. Interfaces 3, 4339–4348. Betancor, L., Luckarift, H.R., 2008. Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol. 26, 566–572. Bornscheuer, U.T., 2003. Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. 42, 3336–3337.

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