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Review
Graphene-based nanobiocatalytic systems: recent advances and future prospects Ioannis V. Pavlidis1,2, Michaela Patila1, Uwe T. Bornscheuer2, Dimitrios Gournis3, and Haralambos Stamatis1 1
Laboratory of Biotechnology, Department of Biological Applications and Technologies, University of Ioannina, 45110, Ioannina, Greece 2 Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, D-17487, Greifswald, Germany 3 Department of Material Sciences and Engineering, University of Ioannina, 45110, Ioannina, Greece
Graphene-based nanomaterials are particularly useful nanostructured materials that show great promise in biotechnology and biomedicine. Owing to their unique structural features, exceptional chemical, electrical, and mechanical properties, and their ability to affect the microenvironment of biomolecules, graphene-based nanomaterials are suitable for use in various applications, such as immobilization of enzymes. We present the current advances in research on graphene-based nanomaterials used as novel scaffolds to build robust nanobiocatalytic systems. Their catalytic behavior is affected by the nature of enzyme–nanomaterial interactions and, thus, the availability of methods to couple enzymes with nanomaterials is an important issue. We discuss the implications of such interactions along with future prospects and possible challenges in this rapidly developing area. Enzyme immobilization onto graphene-based nanomaterials During the past 10 years, the synergistic interactions between nanotechnology and biotechnology have resulted in innovative functional biological nanosystems with potential applications in biotechnology, biosensing, and biomedical areas. The development of effective nanobiocatalysts, in which enzymes are immobilized onto robust nanostructured materials with tailor-made properties, is a typical example [1–4]. Enzyme immobilization, a well-established and mature technology, leads to enhanced stability, facilitates the reuse and easy separation from the reaction medium, allows the modulation of the catalytic properties of immobilized enzymes, and thus creates robust biocatalysts suitable for the development of commercial biocatalytic processes [5–9].
Corresponding author: Stamatis, H. ([email protected]). Keywords: graphene-based nanomaterials; enzyme immobilization; enzyme–nanomaterial interactions; nanobiocatalysis; biofuel cells. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.04.004
Several approaches have been developed in the recent past for immobilization of enzymes [4,6,10]. Among the immobilization carriers used to date, nanostructured composite and hybrid materials, including nanoparticles, nanofibers, and carbon-based nanomaterials, are now under the focus of intense fundamental and applied research [1–4,7]. One of the most advantageous features of nanostructured materials is their potential for manipulating the environment of the biomolecules and thus of their biological function and stability [4,7]. The unique properties of nanostructured materials as immobilization supports, together with other desirable properties, such as conductivity and magnetism, offer particularly exciting opportunities for the preparation of effective nanobiocatalysts and the development of unique enzyme applications [2,11]. Carbon-based layered materials, such as graphene and graphene oxide (GO), owing to their high specific surface area and their exceptional physicochemical properties are increasingly used for applications in various fields such as catalysis, sensing, environmental remediation, and energy storage [12–16]. These layered materials are 2D nanosystems that consist of platelets weakly stacked to form 3D structures. These 2D materials are defined as solids with strong in-plane chemical bonds but weak out-of-plane, van der Waals bonds [17]. Graphene, a one-atom thick layer of sp2 hybridized carbon atoms in a crystalline hexagonal arrangement, represents an archetypical layered material. Immediately after its isolation in 2004, this 2D material triggered scientific interest owing to its extraordinary properties, which led to the Nobel Prize for Physics in 2010 [18]. Theoretically, defect-free isolated graphene sheets have a vast and easily modified surface area, very good mechanical and thermal stability, chemical inertness, and excellent electronic properties (Box 1). Thus, these materials comprise ideal systems for applications in biotechnology and biomedicine such as in gene and drug delivery, bioimaging and biosensing, bioelectronics, tissue engineering, and as antibacterial agents, as documented in recent review articles [14–17,19]. The large surface area of graphene-based nanomaterials creates an ideal immobilization support for various Trends in Biotechnology xx (2014) 1–9
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Box 1. Engineering of graphene-based nanomaterials Isolated graphenes are layers of monoatomic thickness. The direct use of their unique surface properties is inherently prohibited by underlying physical–chemical constraints because, owing to their aromatic p-systems, these structures coalesce immediately. This obstacle can be overcome by chemical functionalization. The chemical functionalization of graphene is a well-established technique for grafting suitable functional groups onto its surface to obtain derivatives with desired properties [23,24]. The surface chemistry of the functionalized nanomaterials can affect their dispersibility and interactions with other materials or molecules [83], while it keeps graphene layers detached. Functionalization of the pristine graphene sheet is generally difficult owing to its poor solubility. However, several methods have been proposed for surface functionalization of conjugated graphene sheets via noncovalent p–p stacking or covalent C–C coupling reactions [23,24]. GO is an oxygen-rich derivative of graphite created by strong oxidation, decorated with hydroxyl, epoxy, and carboxyl groups [84]. These oxygen-containing groups are distributed randomly on the basal planes and edges of the GO sheets. Owing to the existence of such hydrophilic moieties, GO is highly dispersible in water and other polar solvents, and it shows important swelling and intercalation properties. Under proper conditions, GO can be exfoliated in water forming colloidal suspensions of single sheets [85]. GO can easily be
produced in large quantities and the number and type of the oxygencontaining groups depends on the synthetic method that was used [86,87]. GO under reducing conditions, like high-temperature thermal treatment and chemical treatment with reducing agents, loses its carboxylic, epoxy, and hydroxyl groups and resembles graphene, owing to excessive exfoliation [23,24]. Reduced GO regains its conductivity, whereas its oxygen content, surface charge, and water dispersibility are reduced [88]. GO nanoplatelets have chemically reactive sites, such as the carboxylic acid groups at their edges, the epoxy and hydroxyl groups on the basal planes, as well as their p-conjugated system. As a result, chemical functionalization of GO includes four different types of reactions (see Figure 1 in main text) [86]: (i) covalent attachment at the carboxylic acid groups, which are located usually at the edges of the graphene sheets, using nucleophiles such as amine or hydroxyl groups; (ii) covalent attachment on the epoxy groups at the basal planes of the sheet, via ring opening reactions of amines; (iii) noncovalent functionalization that includes van der Waals interactions, with polymers, surfactants, and other small molecules, or p–p interactions, with polyaromatic hydrocarbon derivatives; and (iv) functionalization of reduced GO (e.g., cycloaddition, diazonium reactions, etc.).
biomolecules including enzymes [3,20–22]. The surface chemistry of these nanomaterials can influence their interactions with biomolecules and thus affect the adsorption as well as the conformation and biological function of conjugated proteins [21,22]. Graphene-based nanomaterials can be engineered for grafting desirable functional groups (such as epoxide, carboxylic, and hydroxyl groups) onto their surface providing functionalized nanomaterials with tailor-made properties and improved suitability for their application as
nanoscaffolds (Figure 1; Box 1) [19,23,24]. Furthermore, the engineering of graphene-based nanomaterials offers the possibility for introduction of functionalities that enhance fine-tuned actions of the immobilized enzymes such as protection from enzymatic cleavage [19], enhancement of transportation capability in living cells [25,26], switchable activities responding to external signals [27], facilitation of electron transfer to the protein [28–30], and the incorporation of enzymes in microdevices and microchip bioreactors [31,32].
Reacons with GO At the carboxylic acid group
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Figure 1. Different approaches for the functionalization of GO. Abbreviations: DCC, N,N0 -dicyclohexylcarbodiimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; GO, graphene oxide; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; PAHs, polyaromatic hydrocarbons; SOCl2, thionyl chloride.
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Review We present an overview of the current advances in the field of enzyme immobilization onto graphene-based nanomaterials to build robust nanobiocatalytic systems. Recent advances towards the preservation, optimization, and enhancement of enzyme activity and stability on graphene surfaces will be covered. Finally, recent applications of graphene-based nanobiocatalytic systems in various fields will be summarized. Understanding enzyme–nanomaterial interactions for improved immobilization efficiency and catalytic behavior The interactions of biomolecules with graphene-based nanomaterials are of extreme importance because they define the strength by which an enzyme is bound to an immobilization carrier and to what extent it affects the catalytic behavior [33]. Thus, their understanding is a crucial step for the design of effective nanobiocatalytic systems. Although several works have tried to identify the major interactions taking place with nonspecific immobilization [34–37], these results are often neglected. Graphene-based nanomaterials can interact with biomolecules mostly through electrostatic, van der Waals forces, p–p stacking, or hydrophobic interactions [34,38– 40]. GO, the most commonly used graphene derivative in immobilization studies, bears carboxyl, epoxy, and hydroxyl groups on its surface. These functional groups provide a negative surface charge to the materials; owing to their polarity, they allow weak interactions like hydrogen bonding. The unmodified areas of the surface maintain their free p-electrons, making any p–p interactions feasible. The extent of the interactions depends on the structure, surface chemistry, charge, and hydrophilicity of the nanomaterials [21,35,41,42], and they may affect the conformational state and thus the catalytic activity of the biomolecules [21,34]. Electrostatics play a significant role in enzyme–nanomaterial interactions, especially in the case of the negatively charged GO [40,43]. At pH values lower than the isoelectric point (pI) of horseradish peroxidase, the immobilization efficiency was enhanced compared to that observed at a pH over its pI, indicating the significance of the electrostatic interactions on the immobilization [40]. In a similar example, cytochrome c (cyt c) was immobilized onto GO derivatives [43]. The best loading efficiency was observed when cyt c was positively charged onto negatively charged nanomaterials. Interestingly, once GO was reduced (rGO), the pH of the solution had no more effect on the immobilization of the enzyme [37], whereas the enzyme loading decreased dramatically [43]. In this case, hydrophobic interactions play a major role, according to contact angle experiments [37] and the use of surfactants during immobilization [22,44]. Hydrophobic interactions are the major interactions upon immobilization onto graphene or non-functionalized carbon nanotubes (CNTs), and their strength is affected by the curvature of the nanomaterial [42]. The strong interactions can be avoided by the curvature of the carbon-based nanomaterial: in a recent study, we showed that the curvature of the CNTs seems beneficial both for the structure and activity of immobilized hydrolases compared to the respective plane GO derivatives [35]. Moreover, the beneficial effect that PEGylated GO has on trypsin’s catalytic behavior is
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negated by the use of respective CNTs [21]. Similar results were drawn from immobilization of acetylcholinesterase on GO and fullerenes [45]. Because soluble proteins often do not have hydrophobic residues exposed on their surface, the structure of enzymes alters to facilitate the immobilization, at least in cases where hydrophobic interactions are the most prominent ones [37,40,45]. The immobilization procedure leads to depletion of a-helices and increases in b-sheets for glucose oxidase [39], catalase [46], and cyt c [43]. Similar results were also observed for the interaction of a Bacillus subtilis esterase with several functionalized graphene-based nanomaterials [35]. Significant structural changes often lead to lower activity [35,37,39,46], although the nature of the enzyme should be taken into account to evaluate the extent of the structural changes and the alteration of the catalytic behavior. Wei and Ge suggested that the high amount of reactive oxygen on the GO surface interferes with electron transfer in the active site of catalase, and thus leads to lower apparent activity [46]. By contrast, the catalytic behavior of lipases is enhanced upon immobilization onto carbon-based nanomaterials because they are adopted to act on interfaces, whereas esterases, which catalyze the same reaction, are significantly deactivated [22,35]. This is connected to the fact that the structure of lipases is not significantly altered upon immobilization [22]. However, another enzyme, acetylcholinesterase, retained its native conformation and most of its activity when immobilized onto GO [45]. By contrast, a lipase from Yarrowia lipolytica underwent some structural changes while its activity was reduced upon immobilization [47], indicating that the effect of graphene-based nanomaterials on the catalytic activity and structure of an enzyme is difficult to predict and depends on the nature of the enzyme. Reports of structural changes in cyt c upon immobilization on graphene-based materials are even more vague; some studies suggest that no significant structural changes could be observed [38,41], whereas others suggest loss of the a-helical structure [43,48]. However, all studies agree that the heme microenvironment is altered to a more accessible conformation, which leads to higher peroxidase activity [41,43,48]. When GO was functionalized [41] or reduced [43], the peroxidase activity of cyt c decreased, which is ascribed to a more compact protein and less accessible active site. Similar exposure of the flavin adenine dinucleotide (FAD) moiety was observed for glucose oxidase immobilized onto GO, accompanied with conformational changes [36]. Based on the above reported works, it can be concluded that the interaction of proteins onto the surface of graphene-based nanomaterials is a complex process that depends upon the nature of the protein and the physicochemical properties of the nanomaterials, such as surface chemistry and nanomaterial curvature. However, more studies on enzyme–nanomaterial interactions should be performed, in order to broaden the understanding of the effect on structure and catalytic behavior. Immobilization of enzymes on graphene-based materials Several immobilization approaches have been developed, however the optimum approach strongly relies on several 3
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highly reactive and can be directly used for the formation of a stable amide bond with a free amine group on the surface of the protein (Figure 2). However, the intermediate ester is unstable and easily hydrolyzed from water. For this reason, N-hydroxysuccinimide (NHS) or the more hydrophilic N-hydroxysulfosuccinimide is used during the process to produce a semi-stable amine reactive ester, which is later replaced by the protein. This approach was successfully applied for the immobilization of glucose oxidase [57], bovine serum albumin (BSA) [58], and trypsin [59]. Another typical crosslinker is glutaraldehyde, which is used for graphene-based nanomaterials that bear amine functional groups [22,60]. Manjunatha et al. functionalized graphene’s surface with BSA in order to provide free amine groups for crosslinking with cholesterol oxidase and cholesterol esterase using glutaraldehyde [61]. Zue and coworkers developed a more sophisticated process that combines both approaches at once: Fe3O4 nanoparticles functionalized with aminopropyltriethoxysilane were covalently bound on the surface of GO through EDC/NHS chemistry, and then glutaraldehyde was used to covalently immobilize hemoglobin to the silane derivative of GO [62]. A ‘hybrid’ immobilization approach, which combines both covalent and noncovalent interactions, is based on the use of 1-pyrenebutanoic acid succinimidyl ester. Its pyrene moiety interacts with the surface of the graphene by irreversible p–p stacking, while the protein substitutes the NHS moiety through nucleophilic attack, resulting in the formation of an amide bond [63]. This approach has been used successfully for the immobilization of glucose oxidase and glutamic acid dehydrogenase [64]. Several affinity immobilization approaches have been developed, the reversible character of which could facilitate the regeneration of the nanomaterial or the enzyme
factors such as the enzyme involved, but also practical and commercial applicability [2,29]. To date, nonspecific binding via physical adsorption is the prevailing immobilization procedure [37,48–51], which relies on protein– nanomaterial interactions. Adsorption is usually preferred as an immobilization technique because it is a simple and chemical-free enzyme binding process [2]. The number of graphene layers does not have a significant effect on enzyme immobilization, so a total exfoliation of graphite is not necessary for an efficient immobilization [49]. However, surface chemistry of the graphene-based nanomaterials is a crucial factor because it can affect enzyme–nanomaterial interactions and thus the catalytic behavior of the immobilized enzymes [21,41,52]. Apart from covalent chemical functionalization of the nanomaterials, graphene sheets can be decorated with calcium ions [53] or ionic liquids [54]. These approaches enhance the immobilization efficiency without disrupting the graphene surface, as chemical functionalization does. However, the major shortcoming of noncovalent immobilization is protein leakage from the surface of the nanomaterial [50,55]. The aforementioned drawback can be addressed by covalent immobilization, which, in most cases, leads to higher stability owing to the increased robustness [56]. The progress in chemical functionalization of nanomaterials provides novel materials with a vast diversity of functional groups, and thus facilitates the development of any possible covalent linkage approach. The most commonly used approach is the use of a suitable crosslinker, depending on the functional groups present on the surface of nanomaterials. Carbodiimides such as 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) are used for carboxylated materials [55]. EDC attacks the carboxyl group of the nanomaterial to form O-acylisourea. This is
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Figure 2. Immobilization of trypsin on graphene oxide (GO) using poly-L-lysine and polyethylene glycol (PEG)–diglycolic acid [59]. In every step, the 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction is used. It should be noted that for simplification of the scheme, only the monomers are shown.
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resulting in nanobiocatalytic systems with two different enzyme populations [68]. All these imply that there is no rational design and optimization of the immobilization process, which limits the potentials of the developed nanobiocatalysts. The use of mathematical tools, such as response surface methodology, would benefit the field of enzyme immobilization [69].
used. In a typical process, graphene-based nanomaterials are functionalized with antibodies that recognize either the protein of interest directly, or another antibody that is fused to the protein. For instance, graphene functionalized with rabbit anti-human IgG antibody used for recognition and selective immobilization of human IgG has been reported [65]. In another approach, GO functionalized with avidin was used to immobilize a biotin-modified aptamer in order to prepare a thrombin detector [66]. To underline the potential of this approach in various biotechnological applications, the immobilization of biotinylated purple membranes containing bacteriorhodopsin onto a GO–avidin complex has recently been reported [67]. Several applications could be developed with these conjugates, such as the development of biosensors and motion- and photo-detectors, owing to the photoelectric properties of bacteriorhodopsin. In order to further investigate the efficiency of the immobilization approaches, more studies are needed. Loo and coworkers performed an interesting comparative study of all immobilization approaches and showed that although affinity-based immobilization was more stable than physical absorption, it led to lower selectivity, questioning the accuracy of the affinity interactions [66]. At the same time, most published studies lack the verification of the formation of a covalent bond between the nanomaterial and the immobilized enzyme. X-ray photoelectron spectroscopy was used from our group to verify the covalent immobilization of hydrolases onto functionalized GO nanomaterials [22]. In addition, in most studies of covalent immobilization, the physical adsorption is not hindered,
Applications of graphene-immobilized enzymes As discussed previously, the use of graphene-based nanomaterials as scaffolds for enzyme immobilization offers the possibility of manipulation of the nanoscale environment of an enzyme, increasing enzyme operational stability and catalytic activity, enhancing enzyme loading, improving protein electron transfer, and introducing functionalities that enhance the fine-tuned actions of an immobilized enzyme. It is therefore anticipated that the use of graphene-based nanobiocatalytic systems could expand practical applications of enzymes. In this section, we discuss in detail some of the potential applications of graphene-based nanobiocatalytic systems in various fields such as biocatalytic transformations, degradation of pollutants, development of biofuel cells, and microchip bioreactors (Figure 3). There has been significant progress in the field of biosensing (Box 2), which is not under the scope of the present review, owing to the extent of the literature on the field. Applications in biocatalysis Graphene-based materials are increasingly used as immobilization carriers for enzymes for biocatalytic applications. Their unique mechanical and electrochemical
Biocompability
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Figure 3. Immobilization of enzymes onto graphene-based nanomaterials combines the advantages of both, leading to a diverse field of applications.
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Review Box 2. Graphene-based enzyme biosensors Graphene is an extremely attractive material in the bioanalytical area for electrode design because it can combine properties of high surface-to-volume ratio (2630 m2/g), acceptable biocompatibility, chemical and electrochemical stability, and good electrical conductivity (1738 siemens per meter). Recently, graphene and its derivatives such as GO, rGO, and ultrathin multilayer graphene nanoplatelets have been used in several types of enzyme-based biosensors. Shan et al. first demonstrated that graphene could be used for the direct electrochemistry of glucose oxidase to construct glucose biosensors [89]. Since then, numerous graphene-based biosensors were developed, using various enzymes and proteins such as glucose oxidase, horseradish peroxidase [90–92], acetylcholinesterase, cholesterol oxidase, alcohol dehydrogenase, tyrosinase, cyt c [93,94], and hemoglobin. These biosensors were used for the electrochemical detection of various compounds such as glucose, H2O2, and O2, phenolic pollutants and organophosphates, catechol, ethanol, NADH, nitric oxide, and nitromethane. The development of graphene-based enzyme biosensors with good operational and storage stability, as well as high sensitivity, selectivity, and reproducibility, has been the subject of excellent review articles published over recent years [28,30,95]. To further improve the electrochemical properties of graphene, various graphene-based hybrid materials have been prepared and investigated for biosensing applications. The materials that have been employed for the hybridization of graphene and the preparation of graphene-based nanocomposites include noble metals (Au, Pt, Pd, Ag), semiconductor quantum dots, cyclodextrin, and conducting polymers, as summarized in recent review articles [28,30,96]. Based on several studies that took place in the past 5 years, it can be concluded that the incorporation of graphene in electrochemical biosensors could provide a platform for the construction of inexpensive and stable nanobiosensors and bioelectronics with excellent biocompatibility.
properties, along with the diversity of the functional groups that can be engineered on their surface, render these nanomaterials promising enzyme immobilization carriers. For instance, their hydrophobic surface is suitable for enzymatic activity in organic media. Recently a lipase was immobilized onto carboxylated GO for the enantioselective resolution of (R,S)-1-phenylethanol in heptane [47]. The immobilized lipase increased its selectivity after immobilization, whereas its catalytic efficiency was up to 1.6fold higher, indicating the potential GO has as immobilization support. Various graphene-based materials were employed for the development of immobilized enzyme systems that could be promising for the degradation of pollutants and wastewater treatment. Alkaline protease was immobilized onto GO for the hydrolysis of casein or waste-activated sludge to free amino acids [60] or horseradish peroxidase on the same nanomaterial for the degradation of various phenolic compounds [50], whereas hemoglobin was entrapped into GO hydrogels for the oxidation of phenols in organic solvents [70]. The reusability, storage stability, and thermostability of immobilized enzymes were improved compared to that of free enzyme, underlining their potential for applications in bioremediation of various organic pollutants. One of the most important tasks in bottom-up proteomic analyses is to develop efficient, rapid, recyclable, and automated protein digestion systems [71]. Various GObased nanocomposites were employed for in situ protein digestion using trypsin, in which denaturation and autolysis of the immobilized enzyme was minimized [34,71–73]. 6
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A protein digestion system based on trypsin immobilization onto functionalized GO with poly-L-lysine and polyethylene glycol (PEG)–diglycolic acid was also reported (Figure 2) [59]. The microwave-assisted on-plate proteolysis with immobilized trypsin followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) resulted in high efficiency. Several GO-based microchip bioreactors were developed and coupled with MALDI-TOF-MS analysis for digestion and peptide mapping [31,34,72,73]. A schematic representation of the construction and principle of use of such a bioreactor can be seen in Figure 4. These microchip bioreactors have been employed for the rapid digestion and identification of several standard proteins, including human serum proteins. The aforementioned studies indicate that GO nanocomposites, due to their large surface area, high hydrophilicity, and excellent microwave-absorption ability are promising protease supports for the development of effective nanobiocatalytic systems for protein digestion and peptide mapping. Recently, the preparation of a replaceable on-chip enzymatic microreactor platform for ultrasensitive organophosphorus pesticide detection was presented by utilizing GO magnetic nanocomposites as an acetylcholinesterase immobilization platform [32]. These magnetic bioconjugates can be easily packed with the help of an external magnetic field. This enzymatic microreactor exhibited high reproducibility and stability and provided a promising tool for the efficient and low-cost analysis of pesticides. GO was combined with magnetic nanomaterials for the immobilization of cellulase [20]. The immobilized cellulase efficiently catalyzed the hydrolysis of cellulose derivatives. By contrast, the incorporation of magnetic nanoparticles facilitates the recovery and reuse of the enzyme over multiple cycles. Graphene-based enzyme biofuel cells An enzyme-based biofuel cell (EBFC) utilizes biomassderived energy carriers, such as glucose, ethanol, and oils for the generation of electricity via electrochemical reactions catalyzed by enzymes. Various oxidoreductases, such as alcohol dehydrogenase, glucose oxidase, and glucose dehydrogenase, are used for the oxidation of fuels at the anode of an EBFC in order to generate protons, electrons, and other byproducts. At the cathode, oxygen-reducing enzymes, such as laccase or bilirubin oxidase, are used to catalyze the reaction of an oxidant (usually oxygen) with the produced protons, generating water. The two main application areas that are being considered for EBFCs are in vivo implantable power supplies for electronic medical devices, such as pacemakers, and ex vivo power supplies for small portable power devices [74]. Over the last decade, major improvements in EBFCs have actually been due to the use of carbon-based materials such as CNTs and graphene to fabricate enzymefunctionalized electrodes because they possess advantages such as high conductivity and high surface area for enzyme immobilization [75,76]. Graphene was used for the construction of membraneless EBFCs based on silica sol–gelimmobilized graphene sheets/enzyme composite electrodes [77]. Glucose oxidase was used as the anode enzyme and
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(A)
Glass fiber CTS, GO,...
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Figure 4. Schematic representation of the immobilization of trypsin and its use in microfluidic proteolysis (A) Layer-by-layer assembly of graphene oxide (GO) and chitosan (CTS) for the immobilization of trypsin. (B) The amplified in-channel fiber bioreactor. (C) The process of protein digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping. Reproduced, with permission, from [31].
bilirubin oxidase as the oxygen reduction catalyst in the cathode. This graphene-based EBFC yields a maximum power density threefold higher than that generated by single wall CNT-based EBFCs. The capability of graphene to form electrochemically functionalized multilayer nanostructures onto electrodes was demonstrated by Wang et al. [78]. By using graphene as the spacer, the multilayered nanostructures of graphene/methylene green and graphene/multi-wall CNTs were formed onto electrodes through layer-by-layer chemistry. The potential of such functionalized nanostructures as electronic transducers in EBFCs was demonstrated in a glucose/O2 EBFC using a glucose dehydrogenase-based bioanode and a laccase-based biocathode. An effective membraneless glucose/O2 EBFC has been developed by employing an rGO/multi-wall CNT-modified glassy carbon electrode as anode and a graphene–Pt composite modified glassy carbon electrode as cathode [79]. Recently, an Fe3O4 magnetic nanoparticle/rGO nanosheet modified glassy carbon electrode was proposed [80]. The Fe3O4/rGO hybrid combined with lactate dehydrogenase showed favorable electrochemical features. Graphene materials have recently found application in the development of microbial fuel cells [81]. Recently, encapsulation in an electrochemically-active GO hydrogel of Saccharomyces cerevisiae displaying glucose oxidase was reported [82]. GO was shown to be an effective conducting scaffold for glucose oxidase-displaying yeast enabling direct communication between the enzyme and the surface of the electrode, a feature that is very important for EBFC applications. The observed superior performance of graphene-based electrodes can be attributed to their larger surface area compared to the bulk immobilization carriers used to date,
which facilitates the immobilization of high amounts of enzymes and thus increases catalytic efficiency [77]. Equally significant for such electrochemical applications is the conductivity of these nanomaterials, which is easily modified by surface functionalization. Thus, the implementation of immobilized enzymes onto graphene-based nanomaterials is expected to provide much more efficient biofuel cells. Concluding remarks and future perspectives Graphene-based nanomaterials have rapidly become the most widely studied carbon-based materials owing to their unique structural features and exceptional chemical, electrical, and mechanical properties, as well as their capability for multivalent functionalization and efficient loading of biomacromolecules. The remarkable progress in synthesis and surface engineering of graphene nanomaterials has opened new avenues exploring their use as nanoscaffolds for the development of nanobiocatalytic systems. These novel bioconjugates differ from traditional immobilized enzymes in terms of catalytic efficiency, operational stability, and application potential. Although the progress in the field is very promising, some critical points still need to be addressed in order to expand the applications of graphene-based materials as immobilization matrices in nanobiotechnology. Further investigation is required to gain a deeper understanding of the effects of graphene-based materials on structure and function of enzymes and other proteins. For instance, studying the effect of the type of functionalization as well as the structure and physicochemical characteristics of engineered graphene nanomaterials on the immobilization efficiency and orientation of proteins will provide further understanding of the interactions of nanomaterials with 7
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Review protein molecules, which could lead to optimization of nanobiocatalytic systems. The development of simple and low-cost methods for the design and creation of new graphene-based materials with tailor-made physicochemical properties and surface functionalities, combined with suitable selection of the immobilization method, will certainly lead to the development of functional nanobiocatalytic systems. The creation of graphene-based nanomaterials with high biocompatibility, reduced toxicity, and negligible environmental effect is essential for creating assemblies and functional devices that will expand the application of graphene-based nanobiocatalytic systems in the field of biocatalytic transformations, enzyme engineering, biofuel and energy production, enzyme-based biosensing, and bioassays. References 1 Kim, J. et al. (2008) Nanobiocatalysis and its potential applications. Trends Biotechnol. 26, 639–646 2 Verma, M.L. et al. (2013) Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production. Appl. Microbiol. Biotechnol. 97, 23–39 3 Ge, J. et al. (2012) Nanobiocatalysis in organic media: opportunities for enzymes in nanostructures. Top. Catal. 55, 1070–1080 4 Ansari, S.A. and Husain, Q. (2012) Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol. Adv. 30, 512– 523 5 Talbert, J.N. and Goddard, J.M. (2012) Enzymes on material surfaces. Colloids Surf. B: Biointerfaces 93, 8–19 6 Mateo, C. et al. (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451–1463 7 Rana, S. et al. (2010) Engineering the nanoparticle–protein interface: applications and possibilities. Curr. Opin. Chem. Biol. 14, 828–834 8 Bornscheuer, U.T. (2003) Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. Engl. 42, 3336–3337 9 Cao, L. and Schmid, R.D. (2006) Carrier-bound Immobilized Enzymes: Principles, Application and Design, Wiley-VCH 10 Sassolas, A. et al. (2012) Immobilization strategies to develop enzymatic biosensors. Biotechnol. Adv. 30, 489–511 11 Johnson, P.A. et al. (2011) Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization. Methods Mol. Biol. 679, 183–191 12 Coleman, J.N. et al. (2011) Two-dimensional nanosheets produced by liquid exfoliation of layered materials. Science 331, 568–571 13 Nicolosi, V. et al. (2013) Liquid exfoliation of layered materials. Science 340, http://dx.doi.org/10.1126/science.1226419 14 Bitounis, D. et al. (2013) Prospects and challenges of graphene in biomedical applications. Adv. Mater. 25, 2258–2268 15 Du, D. et al. (2012) Graphene-based materials for biosensing and bioimaging. MRS Bull. 37, 1290–1296 16 Goenka, S. et al. (2014) Graphene-based nanomaterials for drug delivery and tissue engineering. J. Control. Release 173, 75–88 17 Krishna, K.V. et al. (2013) Graphene-based nanomaterials for nanobiotechnology and biomedical applications. Nanomedicine 8, 1669–1688 18 Geim, A.K. and Novoselov, K.S. (2007) The rise of graphene. Nat. Mater. 6, 183–191 19 Wang, Y. et al. (2011) Graphene and graphene oxide: biofunctionalization and applications in biotechnology. Trends Biotechnol. 29, 205–212 20 Gokhale, A.A. et al. (2013) Immobilization of cellulase on magnetoresponsive graphene nano-supports. J. Mol. Catal. B: Enzym. 90, 76–86 21 Jin, L. et al. (2012) Functionalized graphene oxide in enzyme engineering: a selective modulator for enzyme activity and thermostability. ACS Nano 6, 4864–4875 22 Pavlidis, I.V. et al. (2012) Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials. Bioresour. Technol. 115, 164–171 8
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Review
Graphene-based nanobiocatalytic systems: recent advances and future prospects Ioannis V. Pavlidis1,2, Michaela Patila1, Uwe T. Bornscheuer2, Dimitrios Gournis3, and Haralambos Stamatis1 1
Laboratory of Biotechnology, Department of Biological Applications and Technologies, University of Ioannina, 45110, Ioannina, Greece 2 Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, D-17487, Greifswald, Germany 3 Department of Material Sciences and Engineering, University of Ioannina, 45110, Ioannina, Greece
Graphene-based nanomaterials are particularly useful nanostructured materials that show great promise in biotechnology and biomedicine. Owing to their unique structural features, exceptional chemical, electrical, and mechanical properties, and their ability to affect the microenvironment of biomolecules, graphene-based nanomaterials are suitable for use in various applications, such as immobilization of enzymes. We present the current advances in research on graphene-based nanomaterials used as novel scaffolds to build robust nanobiocatalytic systems. Their catalytic behavior is affected by the nature of enzyme–nanomaterial interactions and, thus, the availability of methods to couple enzymes with nanomaterials is an important issue. We discuss the implications of such interactions along with future prospects and possible challenges in this rapidly developing area. Enzyme immobilization onto graphene-based nanomaterials During the past 10 years, the synergistic interactions between nanotechnology and biotechnology have resulted in innovative functional biological nanosystems with potential applications in biotechnology, biosensing, and biomedical areas. The development of effective nanobiocatalysts, in which enzymes are immobilized onto robust nanostructured materials with tailor-made properties, is a typical example [1–4]. Enzyme immobilization, a well-established and mature technology, leads to enhanced stability, facilitates the reuse and easy separation from the reaction medium, allows the modulation of the catalytic properties of immobilized enzymes, and thus creates robust biocatalysts suitable for the development of commercial biocatalytic processes [5–9].
Corresponding author: Stamatis, H. ([email protected]). Keywords: graphene-based nanomaterials; enzyme immobilization; enzyme–nanomaterial interactions; nanobiocatalysis; biofuel cells. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.04.004
Several approaches have been developed in the recent past for immobilization of enzymes [4,6,10]. Among the immobilization carriers used to date, nanostructured composite and hybrid materials, including nanoparticles, nanofibers, and carbon-based nanomaterials, are now under the focus of intense fundamental and applied research [1–4,7]. One of the most advantageous features of nanostructured materials is their potential for manipulating the environment of the biomolecules and thus of their biological function and stability [4,7]. The unique properties of nanostructured materials as immobilization supports, together with other desirable properties, such as conductivity and magnetism, offer particularly exciting opportunities for the preparation of effective nanobiocatalysts and the development of unique enzyme applications [2,11]. Carbon-based layered materials, such as graphene and graphene oxide (GO), owing to their high specific surface area and their exceptional physicochemical properties are increasingly used for applications in various fields such as catalysis, sensing, environmental remediation, and energy storage [12–16]. These layered materials are 2D nanosystems that consist of platelets weakly stacked to form 3D structures. These 2D materials are defined as solids with strong in-plane chemical bonds but weak out-of-plane, van der Waals bonds [17]. Graphene, a one-atom thick layer of sp2 hybridized carbon atoms in a crystalline hexagonal arrangement, represents an archetypical layered material. Immediately after its isolation in 2004, this 2D material triggered scientific interest owing to its extraordinary properties, which led to the Nobel Prize for Physics in 2010 [18]. Theoretically, defect-free isolated graphene sheets have a vast and easily modified surface area, very good mechanical and thermal stability, chemical inertness, and excellent electronic properties (Box 1). Thus, these materials comprise ideal systems for applications in biotechnology and biomedicine such as in gene and drug delivery, bioimaging and biosensing, bioelectronics, tissue engineering, and as antibacterial agents, as documented in recent review articles [14–17,19]. The large surface area of graphene-based nanomaterials creates an ideal immobilization support for various Trends in Biotechnology xx (2014) 1–9
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Box 1. Engineering of graphene-based nanomaterials Isolated graphenes are layers of monoatomic thickness. The direct use of their unique surface properties is inherently prohibited by underlying physical–chemical constraints because, owing to their aromatic p-systems, these structures coalesce immediately. This obstacle can be overcome by chemical functionalization. The chemical functionalization of graphene is a well-established technique for grafting suitable functional groups onto its surface to obtain derivatives with desired properties [23,24]. The surface chemistry of the functionalized nanomaterials can affect their dispersibility and interactions with other materials or molecules [83], while it keeps graphene layers detached. Functionalization of the pristine graphene sheet is generally difficult owing to its poor solubility. However, several methods have been proposed for surface functionalization of conjugated graphene sheets via noncovalent p–p stacking or covalent C–C coupling reactions [23,24]. GO is an oxygen-rich derivative of graphite created by strong oxidation, decorated with hydroxyl, epoxy, and carboxyl groups [84]. These oxygen-containing groups are distributed randomly on the basal planes and edges of the GO sheets. Owing to the existence of such hydrophilic moieties, GO is highly dispersible in water and other polar solvents, and it shows important swelling and intercalation properties. Under proper conditions, GO can be exfoliated in water forming colloidal suspensions of single sheets [85]. GO can easily be
produced in large quantities and the number and type of the oxygencontaining groups depends on the synthetic method that was used [86,87]. GO under reducing conditions, like high-temperature thermal treatment and chemical treatment with reducing agents, loses its carboxylic, epoxy, and hydroxyl groups and resembles graphene, owing to excessive exfoliation [23,24]. Reduced GO regains its conductivity, whereas its oxygen content, surface charge, and water dispersibility are reduced [88]. GO nanoplatelets have chemically reactive sites, such as the carboxylic acid groups at their edges, the epoxy and hydroxyl groups on the basal planes, as well as their p-conjugated system. As a result, chemical functionalization of GO includes four different types of reactions (see Figure 1 in main text) [86]: (i) covalent attachment at the carboxylic acid groups, which are located usually at the edges of the graphene sheets, using nucleophiles such as amine or hydroxyl groups; (ii) covalent attachment on the epoxy groups at the basal planes of the sheet, via ring opening reactions of amines; (iii) noncovalent functionalization that includes van der Waals interactions, with polymers, surfactants, and other small molecules, or p–p interactions, with polyaromatic hydrocarbon derivatives; and (iv) functionalization of reduced GO (e.g., cycloaddition, diazonium reactions, etc.).
biomolecules including enzymes [3,20–22]. The surface chemistry of these nanomaterials can influence their interactions with biomolecules and thus affect the adsorption as well as the conformation and biological function of conjugated proteins [21,22]. Graphene-based nanomaterials can be engineered for grafting desirable functional groups (such as epoxide, carboxylic, and hydroxyl groups) onto their surface providing functionalized nanomaterials with tailor-made properties and improved suitability for their application as
nanoscaffolds (Figure 1; Box 1) [19,23,24]. Furthermore, the engineering of graphene-based nanomaterials offers the possibility for introduction of functionalities that enhance fine-tuned actions of the immobilized enzymes such as protection from enzymatic cleavage [19], enhancement of transportation capability in living cells [25,26], switchable activities responding to external signals [27], facilitation of electron transfer to the protein [28–30], and the incorporation of enzymes in microdevices and microchip bioreactors [31,32].
Reacons with GO At the carboxylic acid group
At the epoxy groups
• Acvaon using SOCl2, EDC, DCC, HATU • Addion of nucleophilic species, such as amines or hydroxyls O
Ring-opening reacons (SN2 nucleophilic substuon) of amines
R NH OH
O
COOH
NHR
OH HOOC
O O
OH COOH
HOOC
Noncovalent funconalizaon π–π interacons of PAHs (anthracene, pyrene, etc.), van der Waals, etc. R
OH
O
OH
OH
Funconalizaon of reduced GO
e.g., 1, 3-dipolar cycloaddion
R1 N
R2
OH TRENDS in Biotechnology
Figure 1. Different approaches for the functionalization of GO. Abbreviations: DCC, N,N0 -dicyclohexylcarbodiimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; GO, graphene oxide; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; PAHs, polyaromatic hydrocarbons; SOCl2, thionyl chloride.
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Review We present an overview of the current advances in the field of enzyme immobilization onto graphene-based nanomaterials to build robust nanobiocatalytic systems. Recent advances towards the preservation, optimization, and enhancement of enzyme activity and stability on graphene surfaces will be covered. Finally, recent applications of graphene-based nanobiocatalytic systems in various fields will be summarized. Understanding enzyme–nanomaterial interactions for improved immobilization efficiency and catalytic behavior The interactions of biomolecules with graphene-based nanomaterials are of extreme importance because they define the strength by which an enzyme is bound to an immobilization carrier and to what extent it affects the catalytic behavior [33]. Thus, their understanding is a crucial step for the design of effective nanobiocatalytic systems. Although several works have tried to identify the major interactions taking place with nonspecific immobilization [34–37], these results are often neglected. Graphene-based nanomaterials can interact with biomolecules mostly through electrostatic, van der Waals forces, p–p stacking, or hydrophobic interactions [34,38– 40]. GO, the most commonly used graphene derivative in immobilization studies, bears carboxyl, epoxy, and hydroxyl groups on its surface. These functional groups provide a negative surface charge to the materials; owing to their polarity, they allow weak interactions like hydrogen bonding. The unmodified areas of the surface maintain their free p-electrons, making any p–p interactions feasible. The extent of the interactions depends on the structure, surface chemistry, charge, and hydrophilicity of the nanomaterials [21,35,41,42], and they may affect the conformational state and thus the catalytic activity of the biomolecules [21,34]. Electrostatics play a significant role in enzyme–nanomaterial interactions, especially in the case of the negatively charged GO [40,43]. At pH values lower than the isoelectric point (pI) of horseradish peroxidase, the immobilization efficiency was enhanced compared to that observed at a pH over its pI, indicating the significance of the electrostatic interactions on the immobilization [40]. In a similar example, cytochrome c (cyt c) was immobilized onto GO derivatives [43]. The best loading efficiency was observed when cyt c was positively charged onto negatively charged nanomaterials. Interestingly, once GO was reduced (rGO), the pH of the solution had no more effect on the immobilization of the enzyme [37], whereas the enzyme loading decreased dramatically [43]. In this case, hydrophobic interactions play a major role, according to contact angle experiments [37] and the use of surfactants during immobilization [22,44]. Hydrophobic interactions are the major interactions upon immobilization onto graphene or non-functionalized carbon nanotubes (CNTs), and their strength is affected by the curvature of the nanomaterial [42]. The strong interactions can be avoided by the curvature of the carbon-based nanomaterial: in a recent study, we showed that the curvature of the CNTs seems beneficial both for the structure and activity of immobilized hydrolases compared to the respective plane GO derivatives [35]. Moreover, the beneficial effect that PEGylated GO has on trypsin’s catalytic behavior is
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negated by the use of respective CNTs [21]. Similar results were drawn from immobilization of acetylcholinesterase on GO and fullerenes [45]. Because soluble proteins often do not have hydrophobic residues exposed on their surface, the structure of enzymes alters to facilitate the immobilization, at least in cases where hydrophobic interactions are the most prominent ones [37,40,45]. The immobilization procedure leads to depletion of a-helices and increases in b-sheets for glucose oxidase [39], catalase [46], and cyt c [43]. Similar results were also observed for the interaction of a Bacillus subtilis esterase with several functionalized graphene-based nanomaterials [35]. Significant structural changes often lead to lower activity [35,37,39,46], although the nature of the enzyme should be taken into account to evaluate the extent of the structural changes and the alteration of the catalytic behavior. Wei and Ge suggested that the high amount of reactive oxygen on the GO surface interferes with electron transfer in the active site of catalase, and thus leads to lower apparent activity [46]. By contrast, the catalytic behavior of lipases is enhanced upon immobilization onto carbon-based nanomaterials because they are adopted to act on interfaces, whereas esterases, which catalyze the same reaction, are significantly deactivated [22,35]. This is connected to the fact that the structure of lipases is not significantly altered upon immobilization [22]. However, another enzyme, acetylcholinesterase, retained its native conformation and most of its activity when immobilized onto GO [45]. By contrast, a lipase from Yarrowia lipolytica underwent some structural changes while its activity was reduced upon immobilization [47], indicating that the effect of graphene-based nanomaterials on the catalytic activity and structure of an enzyme is difficult to predict and depends on the nature of the enzyme. Reports of structural changes in cyt c upon immobilization on graphene-based materials are even more vague; some studies suggest that no significant structural changes could be observed [38,41], whereas others suggest loss of the a-helical structure [43,48]. However, all studies agree that the heme microenvironment is altered to a more accessible conformation, which leads to higher peroxidase activity [41,43,48]. When GO was functionalized [41] or reduced [43], the peroxidase activity of cyt c decreased, which is ascribed to a more compact protein and less accessible active site. Similar exposure of the flavin adenine dinucleotide (FAD) moiety was observed for glucose oxidase immobilized onto GO, accompanied with conformational changes [36]. Based on the above reported works, it can be concluded that the interaction of proteins onto the surface of graphene-based nanomaterials is a complex process that depends upon the nature of the protein and the physicochemical properties of the nanomaterials, such as surface chemistry and nanomaterial curvature. However, more studies on enzyme–nanomaterial interactions should be performed, in order to broaden the understanding of the effect on structure and catalytic behavior. Immobilization of enzymes on graphene-based materials Several immobilization approaches have been developed, however the optimum approach strongly relies on several 3
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highly reactive and can be directly used for the formation of a stable amide bond with a free amine group on the surface of the protein (Figure 2). However, the intermediate ester is unstable and easily hydrolyzed from water. For this reason, N-hydroxysuccinimide (NHS) or the more hydrophilic N-hydroxysulfosuccinimide is used during the process to produce a semi-stable amine reactive ester, which is later replaced by the protein. This approach was successfully applied for the immobilization of glucose oxidase [57], bovine serum albumin (BSA) [58], and trypsin [59]. Another typical crosslinker is glutaraldehyde, which is used for graphene-based nanomaterials that bear amine functional groups [22,60]. Manjunatha et al. functionalized graphene’s surface with BSA in order to provide free amine groups for crosslinking with cholesterol oxidase and cholesterol esterase using glutaraldehyde [61]. Zue and coworkers developed a more sophisticated process that combines both approaches at once: Fe3O4 nanoparticles functionalized with aminopropyltriethoxysilane were covalently bound on the surface of GO through EDC/NHS chemistry, and then glutaraldehyde was used to covalently immobilize hemoglobin to the silane derivative of GO [62]. A ‘hybrid’ immobilization approach, which combines both covalent and noncovalent interactions, is based on the use of 1-pyrenebutanoic acid succinimidyl ester. Its pyrene moiety interacts with the surface of the graphene by irreversible p–p stacking, while the protein substitutes the NHS moiety through nucleophilic attack, resulting in the formation of an amide bond [63]. This approach has been used successfully for the immobilization of glucose oxidase and glutamic acid dehydrogenase [64]. Several affinity immobilization approaches have been developed, the reversible character of which could facilitate the regeneration of the nanomaterial or the enzyme
factors such as the enzyme involved, but also practical and commercial applicability [2,29]. To date, nonspecific binding via physical adsorption is the prevailing immobilization procedure [37,48–51], which relies on protein– nanomaterial interactions. Adsorption is usually preferred as an immobilization technique because it is a simple and chemical-free enzyme binding process [2]. The number of graphene layers does not have a significant effect on enzyme immobilization, so a total exfoliation of graphite is not necessary for an efficient immobilization [49]. However, surface chemistry of the graphene-based nanomaterials is a crucial factor because it can affect enzyme–nanomaterial interactions and thus the catalytic behavior of the immobilized enzymes [21,41,52]. Apart from covalent chemical functionalization of the nanomaterials, graphene sheets can be decorated with calcium ions [53] or ionic liquids [54]. These approaches enhance the immobilization efficiency without disrupting the graphene surface, as chemical functionalization does. However, the major shortcoming of noncovalent immobilization is protein leakage from the surface of the nanomaterial [50,55]. The aforementioned drawback can be addressed by covalent immobilization, which, in most cases, leads to higher stability owing to the increased robustness [56]. The progress in chemical functionalization of nanomaterials provides novel materials with a vast diversity of functional groups, and thus facilitates the development of any possible covalent linkage approach. The most commonly used approach is the use of a suitable crosslinker, depending on the functional groups present on the surface of nanomaterials. Carbodiimides such as 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) are used for carboxylated materials [55]. EDC attacks the carboxyl group of the nanomaterial to form O-acylisourea. This is
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Figure 2. Immobilization of trypsin on graphene oxide (GO) using poly-L-lysine and polyethylene glycol (PEG)–diglycolic acid [59]. In every step, the 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction is used. It should be noted that for simplification of the scheme, only the monomers are shown.
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resulting in nanobiocatalytic systems with two different enzyme populations [68]. All these imply that there is no rational design and optimization of the immobilization process, which limits the potentials of the developed nanobiocatalysts. The use of mathematical tools, such as response surface methodology, would benefit the field of enzyme immobilization [69].
used. In a typical process, graphene-based nanomaterials are functionalized with antibodies that recognize either the protein of interest directly, or another antibody that is fused to the protein. For instance, graphene functionalized with rabbit anti-human IgG antibody used for recognition and selective immobilization of human IgG has been reported [65]. In another approach, GO functionalized with avidin was used to immobilize a biotin-modified aptamer in order to prepare a thrombin detector [66]. To underline the potential of this approach in various biotechnological applications, the immobilization of biotinylated purple membranes containing bacteriorhodopsin onto a GO–avidin complex has recently been reported [67]. Several applications could be developed with these conjugates, such as the development of biosensors and motion- and photo-detectors, owing to the photoelectric properties of bacteriorhodopsin. In order to further investigate the efficiency of the immobilization approaches, more studies are needed. Loo and coworkers performed an interesting comparative study of all immobilization approaches and showed that although affinity-based immobilization was more stable than physical absorption, it led to lower selectivity, questioning the accuracy of the affinity interactions [66]. At the same time, most published studies lack the verification of the formation of a covalent bond between the nanomaterial and the immobilized enzyme. X-ray photoelectron spectroscopy was used from our group to verify the covalent immobilization of hydrolases onto functionalized GO nanomaterials [22]. In addition, in most studies of covalent immobilization, the physical adsorption is not hindered,
Applications of graphene-immobilized enzymes As discussed previously, the use of graphene-based nanomaterials as scaffolds for enzyme immobilization offers the possibility of manipulation of the nanoscale environment of an enzyme, increasing enzyme operational stability and catalytic activity, enhancing enzyme loading, improving protein electron transfer, and introducing functionalities that enhance the fine-tuned actions of an immobilized enzyme. It is therefore anticipated that the use of graphene-based nanobiocatalytic systems could expand practical applications of enzymes. In this section, we discuss in detail some of the potential applications of graphene-based nanobiocatalytic systems in various fields such as biocatalytic transformations, degradation of pollutants, development of biofuel cells, and microchip bioreactors (Figure 3). There has been significant progress in the field of biosensing (Box 2), which is not under the scope of the present review, owing to the extent of the literature on the field. Applications in biocatalysis Graphene-based materials are increasingly used as immobilization carriers for enzymes for biocatalytic applications. Their unique mechanical and electrochemical
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Figure 3. Immobilization of enzymes onto graphene-based nanomaterials combines the advantages of both, leading to a diverse field of applications.
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Review Box 2. Graphene-based enzyme biosensors Graphene is an extremely attractive material in the bioanalytical area for electrode design because it can combine properties of high surface-to-volume ratio (2630 m2/g), acceptable biocompatibility, chemical and electrochemical stability, and good electrical conductivity (1738 siemens per meter). Recently, graphene and its derivatives such as GO, rGO, and ultrathin multilayer graphene nanoplatelets have been used in several types of enzyme-based biosensors. Shan et al. first demonstrated that graphene could be used for the direct electrochemistry of glucose oxidase to construct glucose biosensors [89]. Since then, numerous graphene-based biosensors were developed, using various enzymes and proteins such as glucose oxidase, horseradish peroxidase [90–92], acetylcholinesterase, cholesterol oxidase, alcohol dehydrogenase, tyrosinase, cyt c [93,94], and hemoglobin. These biosensors were used for the electrochemical detection of various compounds such as glucose, H2O2, and O2, phenolic pollutants and organophosphates, catechol, ethanol, NADH, nitric oxide, and nitromethane. The development of graphene-based enzyme biosensors with good operational and storage stability, as well as high sensitivity, selectivity, and reproducibility, has been the subject of excellent review articles published over recent years [28,30,95]. To further improve the electrochemical properties of graphene, various graphene-based hybrid materials have been prepared and investigated for biosensing applications. The materials that have been employed for the hybridization of graphene and the preparation of graphene-based nanocomposites include noble metals (Au, Pt, Pd, Ag), semiconductor quantum dots, cyclodextrin, and conducting polymers, as summarized in recent review articles [28,30,96]. Based on several studies that took place in the past 5 years, it can be concluded that the incorporation of graphene in electrochemical biosensors could provide a platform for the construction of inexpensive and stable nanobiosensors and bioelectronics with excellent biocompatibility.
properties, along with the diversity of the functional groups that can be engineered on their surface, render these nanomaterials promising enzyme immobilization carriers. For instance, their hydrophobic surface is suitable for enzymatic activity in organic media. Recently a lipase was immobilized onto carboxylated GO for the enantioselective resolution of (R,S)-1-phenylethanol in heptane [47]. The immobilized lipase increased its selectivity after immobilization, whereas its catalytic efficiency was up to 1.6fold higher, indicating the potential GO has as immobilization support. Various graphene-based materials were employed for the development of immobilized enzyme systems that could be promising for the degradation of pollutants and wastewater treatment. Alkaline protease was immobilized onto GO for the hydrolysis of casein or waste-activated sludge to free amino acids [60] or horseradish peroxidase on the same nanomaterial for the degradation of various phenolic compounds [50], whereas hemoglobin was entrapped into GO hydrogels for the oxidation of phenols in organic solvents [70]. The reusability, storage stability, and thermostability of immobilized enzymes were improved compared to that of free enzyme, underlining their potential for applications in bioremediation of various organic pollutants. One of the most important tasks in bottom-up proteomic analyses is to develop efficient, rapid, recyclable, and automated protein digestion systems [71]. Various GObased nanocomposites were employed for in situ protein digestion using trypsin, in which denaturation and autolysis of the immobilized enzyme was minimized [34,71–73]. 6
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A protein digestion system based on trypsin immobilization onto functionalized GO with poly-L-lysine and polyethylene glycol (PEG)–diglycolic acid was also reported (Figure 2) [59]. The microwave-assisted on-plate proteolysis with immobilized trypsin followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) resulted in high efficiency. Several GO-based microchip bioreactors were developed and coupled with MALDI-TOF-MS analysis for digestion and peptide mapping [31,34,72,73]. A schematic representation of the construction and principle of use of such a bioreactor can be seen in Figure 4. These microchip bioreactors have been employed for the rapid digestion and identification of several standard proteins, including human serum proteins. The aforementioned studies indicate that GO nanocomposites, due to their large surface area, high hydrophilicity, and excellent microwave-absorption ability are promising protease supports for the development of effective nanobiocatalytic systems for protein digestion and peptide mapping. Recently, the preparation of a replaceable on-chip enzymatic microreactor platform for ultrasensitive organophosphorus pesticide detection was presented by utilizing GO magnetic nanocomposites as an acetylcholinesterase immobilization platform [32]. These magnetic bioconjugates can be easily packed with the help of an external magnetic field. This enzymatic microreactor exhibited high reproducibility and stability and provided a promising tool for the efficient and low-cost analysis of pesticides. GO was combined with magnetic nanomaterials for the immobilization of cellulase [20]. The immobilized cellulase efficiently catalyzed the hydrolysis of cellulose derivatives. By contrast, the incorporation of magnetic nanoparticles facilitates the recovery and reuse of the enzyme over multiple cycles. Graphene-based enzyme biofuel cells An enzyme-based biofuel cell (EBFC) utilizes biomassderived energy carriers, such as glucose, ethanol, and oils for the generation of electricity via electrochemical reactions catalyzed by enzymes. Various oxidoreductases, such as alcohol dehydrogenase, glucose oxidase, and glucose dehydrogenase, are used for the oxidation of fuels at the anode of an EBFC in order to generate protons, electrons, and other byproducts. At the cathode, oxygen-reducing enzymes, such as laccase or bilirubin oxidase, are used to catalyze the reaction of an oxidant (usually oxygen) with the produced protons, generating water. The two main application areas that are being considered for EBFCs are in vivo implantable power supplies for electronic medical devices, such as pacemakers, and ex vivo power supplies for small portable power devices [74]. Over the last decade, major improvements in EBFCs have actually been due to the use of carbon-based materials such as CNTs and graphene to fabricate enzymefunctionalized electrodes because they possess advantages such as high conductivity and high surface area for enzyme immobilization [75,76]. Graphene was used for the construction of membraneless EBFCs based on silica sol–gelimmobilized graphene sheets/enzyme composite electrodes [77]. Glucose oxidase was used as the anode enzyme and
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Figure 4. Schematic representation of the immobilization of trypsin and its use in microfluidic proteolysis (A) Layer-by-layer assembly of graphene oxide (GO) and chitosan (CTS) for the immobilization of trypsin. (B) The amplified in-channel fiber bioreactor. (C) The process of protein digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping. Reproduced, with permission, from [31].
bilirubin oxidase as the oxygen reduction catalyst in the cathode. This graphene-based EBFC yields a maximum power density threefold higher than that generated by single wall CNT-based EBFCs. The capability of graphene to form electrochemically functionalized multilayer nanostructures onto electrodes was demonstrated by Wang et al. [78]. By using graphene as the spacer, the multilayered nanostructures of graphene/methylene green and graphene/multi-wall CNTs were formed onto electrodes through layer-by-layer chemistry. The potential of such functionalized nanostructures as electronic transducers in EBFCs was demonstrated in a glucose/O2 EBFC using a glucose dehydrogenase-based bioanode and a laccase-based biocathode. An effective membraneless glucose/O2 EBFC has been developed by employing an rGO/multi-wall CNT-modified glassy carbon electrode as anode and a graphene–Pt composite modified glassy carbon electrode as cathode [79]. Recently, an Fe3O4 magnetic nanoparticle/rGO nanosheet modified glassy carbon electrode was proposed [80]. The Fe3O4/rGO hybrid combined with lactate dehydrogenase showed favorable electrochemical features. Graphene materials have recently found application in the development of microbial fuel cells [81]. Recently, encapsulation in an electrochemically-active GO hydrogel of Saccharomyces cerevisiae displaying glucose oxidase was reported [82]. GO was shown to be an effective conducting scaffold for glucose oxidase-displaying yeast enabling direct communication between the enzyme and the surface of the electrode, a feature that is very important for EBFC applications. The observed superior performance of graphene-based electrodes can be attributed to their larger surface area compared to the bulk immobilization carriers used to date,
which facilitates the immobilization of high amounts of enzymes and thus increases catalytic efficiency [77]. Equally significant for such electrochemical applications is the conductivity of these nanomaterials, which is easily modified by surface functionalization. Thus, the implementation of immobilized enzymes onto graphene-based nanomaterials is expected to provide much more efficient biofuel cells. Concluding remarks and future perspectives Graphene-based nanomaterials have rapidly become the most widely studied carbon-based materials owing to their unique structural features and exceptional chemical, electrical, and mechanical properties, as well as their capability for multivalent functionalization and efficient loading of biomacromolecules. The remarkable progress in synthesis and surface engineering of graphene nanomaterials has opened new avenues exploring their use as nanoscaffolds for the development of nanobiocatalytic systems. These novel bioconjugates differ from traditional immobilized enzymes in terms of catalytic efficiency, operational stability, and application potential. Although the progress in the field is very promising, some critical points still need to be addressed in order to expand the applications of graphene-based materials as immobilization matrices in nanobiotechnology. Further investigation is required to gain a deeper understanding of the effects of graphene-based materials on structure and function of enzymes and other proteins. For instance, studying the effect of the type of functionalization as well as the structure and physicochemical characteristics of engineered graphene nanomaterials on the immobilization efficiency and orientation of proteins will provide further understanding of the interactions of nanomaterials with 7
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Review protein molecules, which could lead to optimization of nanobiocatalytic systems. The development of simple and low-cost methods for the design and creation of new graphene-based materials with tailor-made physicochemical properties and surface functionalities, combined with suitable selection of the immobilization method, will certainly lead to the development of functional nanobiocatalytic systems. The creation of graphene-based nanomaterials with high biocompatibility, reduced toxicity, and negligible environmental effect is essential for creating assemblies and functional devices that will expand the application of graphene-based nanobiocatalytic systems in the field of biocatalytic transformations, enzyme engineering, biofuel and energy production, enzyme-based biosensing, and bioassays. References 1 Kim, J. et al. (2008) Nanobiocatalysis and its potential applications. Trends Biotechnol. 26, 639–646 2 Verma, M.L. et al. (2013) Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production. Appl. Microbiol. Biotechnol. 97, 23–39 3 Ge, J. et al. (2012) Nanobiocatalysis in organic media: opportunities for enzymes in nanostructures. Top. Catal. 55, 1070–1080 4 Ansari, S.A. and Husain, Q. (2012) Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol. Adv. 30, 512– 523 5 Talbert, J.N. and Goddard, J.M. (2012) Enzymes on material surfaces. Colloids Surf. B: Biointerfaces 93, 8–19 6 Mateo, C. et al. (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451–1463 7 Rana, S. et al. (2010) Engineering the nanoparticle–protein interface: applications and possibilities. Curr. Opin. Chem. Biol. 14, 828–834 8 Bornscheuer, U.T. (2003) Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. Engl. 42, 3336–3337 9 Cao, L. and Schmid, R.D. (2006) Carrier-bound Immobilized Enzymes: Principles, Application and Design, Wiley-VCH 10 Sassolas, A. et al. (2012) Immobilization strategies to develop enzymatic biosensors. Biotechnol. Adv. 30, 489–511 11 Johnson, P.A. et al. (2011) Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization. Methods Mol. Biol. 679, 183–191 12 Coleman, J.N. et al. (2011) Two-dimensional nanosheets produced by liquid exfoliation of layered materials. Science 331, 568–571 13 Nicolosi, V. et al. (2013) Liquid exfoliation of layered materials. Science 340, http://dx.doi.org/10.1126/science.1226419 14 Bitounis, D. et al. (2013) Prospects and challenges of graphene in biomedical applications. Adv. Mater. 25, 2258–2268 15 Du, D. et al. (2012) Graphene-based materials for biosensing and bioimaging. MRS Bull. 37, 1290–1296 16 Goenka, S. et al. (2014) Graphene-based nanomaterials for drug delivery and tissue engineering. J. Control. Release 173, 75–88 17 Krishna, K.V. et al. (2013) Graphene-based nanomaterials for nanobiotechnology and biomedical applications. Nanomedicine 8, 1669–1688 18 Geim, A.K. and Novoselov, K.S. (2007) The rise of graphene. Nat. Mater. 6, 183–191 19 Wang, Y. et al. (2011) Graphene and graphene oxide: biofunctionalization and applications in biotechnology. Trends Biotechnol. 29, 205–212 20 Gokhale, A.A. et al. (2013) Immobilization of cellulase on magnetoresponsive graphene nano-supports. J. Mol. Catal. B: Enzym. 90, 76–86 21 Jin, L. et al. (2012) Functionalized graphene oxide in enzyme engineering: a selective modulator for enzyme activity and thermostability. ACS Nano 6, 4864–4875 22 Pavlidis, I.V. et al. (2012) Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials. Bioresour. Technol. 115, 164–171 8
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