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Ugi Four Component Assembly Process: An Efficient Approach for One-pot Multi-functionalization of Nano Graphene Oxide in Water and Their Application in Lipase Immobilization Aram Rezaei, Omid Akhavan, Ehsan Hashemi, and Mehdi Shamsara Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b00099 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016

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Ugi Four-Component Assembly Process: An Efficient Approach for One-pot Multi-functionalization of Nano Graphene Oxide in Water and Their Application in Lipase Immobilization Aram Rezaei*†‡, Omid Akhavan*†¥, Ehsan Hashemi‡, and Mehdi Shamsara‡ †Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran ¥Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box 14588-89694, Tehran, Iran ‡National Research Center for Transgenic Mouse & Animal Biotechnology Division, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965-161, Tehran, Iran ABSTRACT: Graphene-based materials are revealing the leading edge of advanced technology for their exceptional physical and chemical properties. Chemical manipulation on graphene surface to tailor its unique properties and modify atomic structures is being actively pursued. Therefore, the discovery of robust and general protocols to anchor active functionality on graphene basal plane is still of great interest. Multicomponent reactions promise an enormous level of interest due to addressing both diversity and complexity in combinatorial synthesis, in which more than two starting compounds react to form a product derived from entire inputs. In this article, we present the first covalent functionalization route beginning with carboxylated-graphene oxide through Ugi four-component assembly process (Ugi 4-CAP), in which amine, aldehyde, isocyanide and acid components come together in a one-pot reaction to generate hydrophobic-, hydrophilic- or amiphiphilic multi-functionalized graphene composites. Investigation on the covalent immobilization and bio-catalytic activity of Bacillus thermocatenulatus lipase (BTL) on graphene surface showed the efficiency and competency of Ugi 4-CAP. The success of the multicomponent-coupling approach was confirmed by AFM, Raman, UV-Vis, FT-IR, 1H NMR, EDS, SEM, TGA and XPS.

INTRODUCTION Graphene has emerged as a flat monolayer of carbon atoms tightly packed into a two-dimensional (2D) honeycomb lattice with wide range of unique physical and chemical properties, such as high surface area, excellent conductivity, high carrier mobility, half-integer quantum, Hall effect at room temperature, ease of functionalization and production.13 Graphene and its derivatives promise an enormous level of interest for use in nanoelectronic devices,4 nanocomposite materials,5 energy conversion,6,7 biotechnology,8-10 and chemical transformations.11 Moreover, in an effort to tailor its properties and interfacial characteristics, chemical functionalization on the graphene surface creates a new window to manipulate its physical and chemical properties and modify its atomic structures for a variety of next-generation technologies.12 It is well known that modification of the carbon network by grafting atoms or molecules is important in designing graphene-based nanomaterials since it may provide a means to enrich graphene’s properties.13 To date, several methods have been reported in the literature for the chemical covalent functionalization of graphene, which are multi-step in nature and are restricted for producing metastable, chemically inhomogeneous, and spatially disordered GO structures.12,13 Normally, most of the general methods involve addition of free radicals to sp2 carbon atoms of graphene,14 the aryl diazonium-based reaction,15 click chemistry,16 1,3 dipolar cycloaddition,17 nitrene addition,18 oxidation and reduction reaction,19 esterification and amidation reactions,20,21 EDC coupling,22 polymerization,23,24 and

others under harsh chemical environments during modification protocols and multi-step reactions with difficult work-up procedures. As another drawback, none of these methods are capable of acting as an operationally, versatile and one-step multi-functionalization method, especially from readily available compounds. On the other hand, it would be highly desirable to develop synthetic methods to achieve multifunctional hybrid materials that take advantage of superior properties of both graphene and functionalizing materials. Isocyanide-based multicomponent reactions (IMCRs) are programmable chemical transformations, in which starting materials react in a sequence of elementary steps based on a plan to access a high level of diversity and complexity in combinatorial chemistry.25 These reactions offer a number of advantages, such as remarkable atom economic, high E factors (mass ratio of waste to desired product), time-saving, versatile and convergent procedure. Besides, IMCRs as diversity-oriented synthesis (DOS) allow more than two simple and flexible building blocks to be combined with high bond-forming-index (BFI) in one synthetic transformation.26 Thus, MCR represents a network of mobile equilibria, in which each input is assembled according to a cascade of chemical reactions to achieve a wide range of complexity.27 Most IMCRs are based on the classical reactions of Passerini and Ugi. In 1921, Passerini developed a three-component synthesis of α-acyloxycarboxamide by reaction of a carboxylic acid, an aldehyde, and an isocyanide.28 The Ugi reaction is defined as four-component condensation of an amine, oxocompound, carboxylic acid and an isocyanide, which followed by a Mumm rearrangement reaction to afford α-

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aminoacyl amide derivatives with water as the only byproduct.29 Especially, the Ugi adducts have generated tremendous interest due to remarkable functional group tolerant that can be used in subsequent synthesis and provides an extraordinary improvement in many fields of organic chemistry such us combinatorial chemistry,30 medicinal chemistry,31,32 and peptide chemistry.33 The presence of oxygenated aliphatic regions (sp3 carbon atoms) containing hydroxyl, epoxy, carbonyl, and carboxyl functional groups in graphene oxide (GO) renders it strongly hydrophilic and water soluble and also provides a handle for the chemical modification of graphene.1-3 Considering the ability of Ugi four-component assembly process (Ugi 4CAP) for obtaining structurally diverse scaffolds through a range of functional groups that could participate in these convergent syntheses, and the exceptional chemistry of graphene because of various oxide-containing species, IMCR is the interesting reaction to test on GO. This paper presents a fundamentally novel approach to prepare a multi-functionalized graphene composites by Ugi 4-CAP between oxo-compounds, amine, cyclohexylisocyanide on the surface of carboxylated-GO in water, as a green solvent at ambient temperature. In addition, for examining the efficiency and competency of Ugi 4-CAP as a comprehensive method, the lipase enzyme was immobilized on carboxylated-GO by the proposed methodology. This covalently immobilized biocatalyst on GO surface was then utilized for the practical biocatalytic application to perform hydrolysis short- and long-chain triacylglycerols. As a brief conclusion, our results show that Ugi 4-CAP is a powerful tool to prepare multi-functionalized graphene composites, which can be used to achieve unique physical and chemical properties for the creation of new hybrid materials. EXPERIMENTAL SECTION Materials and characterization. Starting materials and solvents were obtained from Merck (Germany) and Fluka (Switzerland) and were used without further purification. Samples were prepared for the AFM measurements by depositing the corresponding suspension of products on the mica, drying it in vacuum, and investigating it using a SPM (AFM mode, DME-95-50 E) equipped with a Si tip (with tip radius of 10 nm) in tapping mode (with frequency of 320 kHz). A UV-Vis spectrophotometer (Perkin Elmer UV-VisNIR Model Lambda 950) was used to study the optical characteristics of the diluted suspension of graphene oxide and products at room temperature. Raman spectra were obtained using an Almega Thermo Nicolet Dispersive Raman Spectrometer equipped with an Nd-YLF excitation source operating at wavelength of 532 nm. The XPS measurements were performed by using a hemispherical analyzer with an Al Kα X-ray source (hv = 1486.6 eV). The Fourier transform infrared (FTIR) spectra of the samples were obtained using the KBr pellet method by BRUKER VERTEX 70/70v FTIR spectrometers. 1H NMR spectra was measured (DMSO-d6 solution) with a BRUKER DRX-250 AVANCE spectrometer at 250.0 MHz. SEM and EDX analysis was performed on a scanning electron microscope (TESCAN Vega Model) equipped with an energy dispersive X-ray spectroscopy measurement system. Thermal gravimetric analysis (TGA) was conducted using a TGA-7 Perkin Elmer calorimeter under N2 flow at a heating rate of 10 °C min-1.

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Preparation of graphene oxide (GO). Graphene oxide was synthesized from graphite flakes using the Tour method with slight modification.34 Briefly, flake graphite (1 g, Merck) and KMnO4 (7g) were added to a 9:1 mixture of concentrated H2SO4/H3PO4 (120:13.3 mL). The reaction was sonicated for 20 min and then stirred for 10 h at 50 °C. The mixture poured onto ice (100 ml), and reacted with a 30% H2O2 (2 mL) to complete the oxidation. For workup, the mixture was centrifuged (6000 rpm for 1 h), and the supernatant was decanted away. The solid was reprotonated with HCl (100 mL, 25 %) and stirred for 1 h at 40 °C to remove inorganic anion and other impurities. The remaining solid materials was washed with 300 mL water and 300 mL of ethanol (2×) and was then filtered over a PTFE membrane with a 0.45 µm pore size. The solid obtained on the filter was dispersed in deionized water (DI) via sonication to give yellow suspension of GO. Carboxylation of GO. For carboxylation, an aqueous suspension of GO (5 mL, 1 mg/mL) was sonicated for 10 min to obtain a clear solution. KOH (0.5 g) and chloroacetic acid (1.0 g) were added to the GO suspension and the mixture was sonicated for 2 h and then stirred for 10 h at 60 °C to transform –OH groups to -COOH via conjugation of acetic acid moieties. The resulting G-COOH solution was neutralized with dilute HCl, and was washed with 150 mL DI and 100 mL ethanol (70%, 2×). The resulting powder was suspended in DI using ultrasonication to obtain stable GCOOH suspension. Ugi four-component reaction on G-COOH. To a magnetically stirred solution of amine (5 mmol) and 626 µL of cyclohexylisocyanide were added to a solution of G-COOH suspension (2 mL, 0.5 mg/mL) and aldehyde (5 mmol) in water at 25 °C. The mixture was stirred for 2 h and was then filtered with a Millipore membrane with a 0.45 µm pore size, and the solid was washed thoroughly with water and ethanol (70%). The product was dispersed in 20 mL of water by mild sonication. Enzyme immobilization on G-COOH. An aqueous solution of formaldehyde (1 mmol) and 2 drops of cyclohexylisocyanide were added to a solution of lipase (0.5 mg, 15 nmol) and G-COOH (300 µL, 0.5 mg/mL) in 0.1 M phosphate buffer (3 mL, pH 7.4) at 25 °C. The mixture was set aside for 30 min and shaken occasionally, centrifuged, and dialyzed with 0.01 M phosphate buffer pH 7.4 and stored at 4 °C. Leaching experiments were performed by incubation of the immobilized derivatives in 1 M NaCl solution. The lack of the free lipase in the solution confirmed the covalent nature of the linkage. Enzymatic activity assay and stability. The activities of the soluble lipase and its immobilized preparations were analyzed in a pH-STAT using a variety of tributyrin (C4), tricaprin (C6), tricaprylin (C8), tricaprin (C10), trilaurin (C12), trimyristin (C14), tripalmitin (C16), and olive oil (C18) as substrates. 20 ml of substrate solution was adjusted to pH 8.5 at 55 °C, subsequently immobilized lipase on graphene was added to the substrate solution and the liberated fatty acids were titrated automatically with 0.05 M NaOH. Lipase activity was calculated by the amount of NaOH needed to maintain pH at 8.5. One unit was defined as the amount of enzyme that released 1.0 µmol of fatty acid per minute.35 The effect of temperature on lipase activity was studied by carrying out the enzyme reaction at different temperatures in

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the range of 45–65 °C in a pH-stat by using tricaprylin (C8) as substrate (pH 8.5). The time stability of free and immobilized lipases was determined by measuring the residual activity after incubation in a buffer solution at 55 °C for a certain period of time by using tricaprylin (C8) as substrate. The effect of pH on the activity of the free and immobilized enzyme were determined by pre-incubating at room temperature in phosphate buffer (10 mM) at pH ranging from 5 to 10, followed by determination of enzymatic activity in a pH-stat by using tricaprylin (C8) as substrate (pH 8.5). The residual activity of the immobilized lipase was normalized to the initial value determined at 55 °C, pH 8.5 (the initial activity was defined as 100%). Reusability of the immobilized lipases was measured through repeated uses in the hydrolysis reaction at 55 °C and pH 8.5. The immobilized lipases were recovered with centrifuge at 8000 rpm for 10 min and washed extensively with a phosphate buffer solution (pH 7.4) prior to be used in the next reaction batch. RESULTS AND DISCUSSION After a decade of discovering the amazing properties of graphene as a two-dimensional (2D) hexagonal carbon lattice and seeking its applications in various fields of science, scholars have recently focused on adding another aspect to the tremendous complex world of graphene by attaching functional groups to the graphene building blocks for a variety of cutting edge technologies.36 The GO sheets possess oxygenated aliphatic regions containing hydroxyl, epoxy, carbonyl, and carboxyl functional groups. The basal plane is covered with epoxy and hydroxyl groups and the carboxylic groups are usually present at the edge of the GO sheet. Given that carboxylic acid is one of the participants in the multicomponent reactions, and therefore a deficiency in the density of carboxyl group on the basal plane of graphene sheet could be compensated with a carboxylation process in which hydroxyl and epoxy groups were converted to carboxylic acid by chloroacetic acid under strongly basic conditions (Scheme 1).37

Scheme 1. Illustration of carboxylation process to convert epoxy and hydroxyl groups to carboxylic acid by chloroacetic acid

The reaction sequence that was developed for the first isocyanide multi-functionalization of GO is depicted in Scheme 2. By Ugi 4-CAP, here, we developed a fundamentally synthetic methodology, in which amine, aldehyde, isonitrile and carboxylic acid components came together in a single reaction vessel to form a new hybrid material (see experimental section and supporting Information). To implement the proposed method, first, an aqueous suspension of GO was prepared by using an improved Tour’ method since it provided a greater amount of hydrophilic oxidized region compared to Hummers’ method.34 The GO suspension was oxidized by chloroacetic acid under basic condition for the increased density of carboxyl group on the basal plane of graphene sheets. The obtained carboxylatedGO (G-COOH) had higher solubility and stability in aqueous solutions. Second, to explore Ugi 4-CAP on graphene surfaces, formaldehyde, benzyl amine, and cyclohexylisocyanide were used as components to test the Ugi 4-CAP on GCOOH in water as green solvent to generate hydrophobic multi-functionalized graphene composite (HydrophobicMFGC) (Scheme 3). In stark contrast to the GO and GCOOH, our Hydrophobic-MFGC was not dispersed in water at all. However, it swelled and, after a brief ultrasonic treatment, readily formed long-term stable and homogeneous dispersions in organic solvents such as DMF, Nmethylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), methylene chloride, and toluene. To assess the efficiency and diversity of the Ugi 4-CAP on G-COOH, 1-(3-aminopropyl)-3-methylimidazolium chloride (IL-NH2) and lipase enzyme were examined as amine component and formaldehyde and 4-chlorobenzaldehyde as oxocompounds in the presences of cyclohexylisocyanide to prepare multi-functionalized graphene materials with unique features (Scheme 3).

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Scheme 2. Schematic representation of Ugi 4-CAP on graphene surface to generate multi-functionalized graphene composites

Imidazolium-based ionic liquids (ILs) have gained wide popularity over the last few years, owing to their remarkable characteristics such as negligible vapor pressure, nonflammability, excellent mechanical and thermal stability, high ionic conductivity, low toxicity, controllable hydrophobicity and broad biochemical windows.38 Initially, ILs have emerged as environmentally friendly solvents and reagents in nanostructures synthesis,39,40 catalysis,41 and microextraction techniques.42 Currently, ILs are widely used as water purification agents, safe energetic materials,43 supercapacitors,44 and heat-transfer fluids.45 In addition, ILs offer great potential for applications in the life science such as designing IL-containing ion conductive DNA film to serve as a biomass,46,47 enzymes immobilization and separation,48,49 extraction of trace amounts of DNA or cytochrome c,50,51 dissolution and regeneration of proteins,52 and DNA preservation and stabilization.53 At the present time, a burst of research activities has been centered on the development of hybrid nanocomposite materials based on graphene and ionic liquid,54 triggering the creation of new and future prominent applications in electrocatalyst,55 and lubricant for low friction and wear.56

In the continuation of this study, we investigated the possibility of attaching IL-NH2 to G-COOH via Ugi 4-CAP. Cyclohexylisocyanide, formaldehyde, IL-NH2 and carboxylic acid reacted smoothly in water to construct hydrophilic multi-functionalized graphene composite (HydrophilicMFGC). It was found that the resulting hybrid graphene could be well dispersed into the polar solvent. The structures and properties of multi-functional graphene based ionicliquid backbone could be tuned by selecting the appropriate combination of organic cations and anions. With a unique multicomponent assembly architecture, tunable graphene was obtained using 4-chlorobenzaldehyde instead of formaldehyde in Ugi 4-CAP. In other words, a four-component condensation between 4-chlorobenzaldehyde, IL-NH2, cyclohexylisocyanide in the presence of G-COOH introduced amphiphilic multi-functionalized graphene composite (Amphiphilic-MFGC). The amphiphilic character of these composites was due to the hydrophobic nature of graphene sheet and 4-chlorobenzaldehyde combined with the hydrophilic moieties of the ILs backbone, which consequently facilitated the dispersion of Amphiphilic-MFGC in both organic low polar and water-miscible high polar solvents (Scheme 3). A mechanistic rationalization for this reaction is provided in Scheme 4. It is conceivable that the initial event is the condensation of the aldehyde 1, amine 2, and G-COOH 3 entities to an intermediate iminium ion 6. Nucleophilic addition of the cyclohexylisocyanide 4 to the intermediate iminium ion 6 leads to nitrilium intermediate 7. This intermediate might be attacked by conjugate base of the acid 3 to form 1:1:1 adduct 8. This adduct might undergo Mumm rearrangement reaction to afford the multi-functionalized graphene 5.25

Scheme 3. Carboxylated graphene sheets functionalized with small molecules via four-component condensation to afford Hydrophobic-MFGC (a), Hydrophilic-MFGC (b), and Amphiphilic-MFGC (c)

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Scheme 4. Possible mechanism for the formation of multifunctionalized graphene composite via Ugi 4-CAP

Advanced characterization of hybrid graphene The multi-functionalized graphene sheets were characterized by various techniques such as Fourier transform infrared spectroscopy (FT-IR), UV-Visible Spectroscopy (UV-Vis), Raman spectroscopy, Atomic force microscopy (AFM), XRay photoelectron spectroscopy (XPS), Energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), thermo gravimetric analysis (TGA) and Proton nuclear magnetic resonance (1H NMR). AFM is a most direct method to establish the physical properties of hybrid graphene such as thickness, degree of exfoliation and surface roughness. Figure 1 demonstrates the AFM images of the functionalized graphene with different lateral dimensions and the corresponding line-scanning indicates that the average thickness of Hydrophobic-MFGC, Hydrophilic-MFGC and Amphiphilic-MFGC were ca. 1.45, 1.12, 1.20 nm respectively. Comparing with well exfoliated G-COOH sheets, with a thickness of 0.98 nm, the distance between hybrid graphene was greater, as would be expected. This result could be attributed to the attached organic molecules on both sides of the graphene sheets. For further investigation of the selectivity of the multicomponent reactions on GO sheets, Raman spectroscopy was performed using 532 nm laser excitation. In the spectrum of G-COOH (Figure 1a), two peaks around 1585 cm−1 and 1353 cm−1 were detected corresponding to G- and D- bonds. The G-band was related to the first-order scattering of the E2g mode and the D-band was attributed to the breaking of the translational symmetry of the C=C sp2 bond.57 Therefore, the

ratio between the intensities of the D- and G-bands is often used as an indication of the level of chemical modification in a graphitic carbon sample. During the chemical processing from G-COOH to the Ugi 4-CAP-modified graphene, graphene sheets were covalently functionalized through multicomponent assembly of amine, aldehyde and isocyanide with carboxylic acids on graphene surface. The Raman spectrum of Hydrophobic-MFGC (Figure 1d), Hydrophilic-MFGC (Figure 1g) and Amphiphilic-MFGC (Figure 1j) were characterized by a presence of D-band at 1347, 1353, 1350 cm-1 and G-band at 1589, 1587, 1585 cm-1 respectively, with an increased D/G ratio compared to the one in G-COOH (Figure 2B).58 This result indicates that the modification reaction does not damage the conjugated sp2 C=C network of graphene because the carboxylic acid groups attached to basal plane and edge of graphene sheet participated in Ugi multicomponent reaction. Moreover, this change suggests a decrease in the average size of graphitic domains.59 The functionalization of G-COOH was further characterized by UV–Vis spectroscopy, as shown in Figure 2. The GO suspension displayed a maximum absorption at 230 nm, which is corresponded to π-π* transitions of C=C bonds, and a shoulder at 293 nm corresponded to n-π* transitions of carbonyl groups (Figure 2a).34 Whereas, the G-COOH showed a bathochromic shift of the absorption peak to 258 nm upon activation process, along with an increase in the background absorbance (Figure 2b). This result could be attributed to the restoration of conjugation network within the graphene sheets, which might be related to the opening of the epoxide rings and some local changes in the microstructure of GO sheets.60 The characteristic peak of IL-NH2 (Figure 2d) appeared at 232 nm with a shoulder at 270 nm, while in the UV-Vis spectrum of Hydrophilic-MFGC (Figure 2e) and Amphiphilic-MFGC (Figure 2f), they showed a slight redshift to 235 and 278 nm. The HydrophobicMFGC displayed a maximum absorption at 260 nm, which is related to π-π* transitions of adjunct aromatic rings by Ugi 4-CAP functionalization (Figure 2c). As demonstrated in Figure 2A, GO and G-COOH could be well dispersed in water with long-term stability (vial a and b, respectively). Hydrophilic-MFGC also could be formed stable dispersion in water (vial e), whereas HydrophobicMFGC showed poor dispersion in water and aggregated (the lower phase is dichloromethane while water is the upper one). In other word, most Hydrophobic-MFGC could be transferred to the dichloromethane phase due to hydrophobic nature (vial c). Also, Amphiphilic-MFGC was dispersed in CH3OH with good stability (vial f).

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Figure 1. Raman and AFM experiments conducted at different stages of the oxidation/functionalization sequence. (a) Raman spectrum of a G-COOH exhibiting typical D- and G- bands. (b) AFM image from the G-COOH. (c) AFM profile along the blue line indicated in b. (d) Raman spectrum of hydrophobic-MFGC. (e) AFM image from hydrophobic-MFGC. (f) Height profile extracted along the blue line in e. (g) Raman spectrum of a hydrophilic-MFGC. (h) AFM image of a hydrophilic-MFGC. (i) Height profile extracted along the blue line in h. (j) Raman spectrum of an amphiphilic-MFGC. (k) AFM image from the amphiphilic-MFGC. (l) Height profile along the blue line in k.

FT-IR provides a powerful tool to determine the various functional groups in the GO and its derivatives. In the spectrum of GO, the most characteristic vibrations include the broad and intense O–H stretching vibrations around 30243390 cm−1, strong C=O stretching vibrations from carbonyl and carboxylic groups at 1730 cm−1, skeletal vibration of C=C from unoxidized sp2 C=C bonds at 1622 cm−1, C–OH stretching vibrations at 1361 cm−1, C–O–C peak at 1217 cm−1, C–O stretching vibrations at 1053 cm−1, and aromatic C-H peak at 860 cm−1 (Figure S1, see supporting information).61 The oxidation of GO by chloroacetic acid is expected to convert –OH and surface ether groups (C–O–C) into carboxyl ones (–COOH). By interaction of surface –OH groups with chloroacetic acid, C–O–CH2COOH would be replaced with C–OH. The C–O–C epoxide ring on the basal plane of GO would also be firstly opened under basic condition and converted to –OH and subsequently replaced with C–O–CH2COOH. As a result, the expectation was to observe a peak at 1217 cm−1 attributed to the disappeared C–O–C epoxide. The shift in the skeletal vibration of C=C from 1622 to 1575 cm−1 might be due to the change of microstructure in GO sheets. Moreover, two additional peaks were appeared at 2852 and 2925 cm−1 in the IR spectrum after chloroacetic acid reaction with GO, which might be correlated to

the symmetric and asymmetric stretching modes of –CH2– groups in chloroacetic acid.62 Furthermore, the peak at 1370– 1410 cm−1 was attributed to deformation vibration of –CH2– group. A small peak around 2310-2353 cm−1 could be associated with the O–H stretch from strongly hydrogen bonded –COOH groups. All these data confirmed covalent attachment of carboxylic acids groups to the GO sheets (Figure 3a).63 In comparison to the FT-IR spectrum of G-COOH, hybrid graphene exhibited characteristic peaks that clearly indicated the functionalization.

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Chemistry of Materials (Figure S2, see supporting information) that clearly indicated its functionalization. In figure 3c, the characteristic vibrations of the Hydrophilic-MFGC include the broad N–H stretching vibrations around 3230-3560 cm−1. The new bands at 2960, 2922, 2868, and 2839 cm−1 were corresponded to symmetric Vs (CH3), asymmetric Vas (CH2), asymmetric Vas (CH3) and symmetric Vs (CH2), respectively. The amide carbonyl-stretching mode (Amide I vibration stretch) appeared around 1631-1732 cm−1 and the peaks at 1573 cm−1 might be originated from the coupling of N–H bending and C–N stretching (Amide II vibration).67 In addition, the peaks at 1552 and 1521 cm−1 could be originated from imidazolium ring stretch. The peak at 1456 cm−1 was related to methylene scissoring, while the CH3(N) stretching, CH2(N) stretching, and ring in-plane asymmetric stretching arising from imidazolium ring (Imidazole H–C–C & H–C–N bending) were assigned at ca. 1164 cm−1 . Two peaks at 1377 and 1257 cm−1 might be originated from the C–N stretching vibration. Also, C–H bending in the imidazolium ring appeared at 804 and 621 cm−1.68 Similar results were obtained for Amphiphilic-MFGC (Figure 3d). Overall, these results confirmed the success of this strategy to produce hybrid materials by Ugi MCR.

Figure 2. (A) UV-Vis spectra of (a) GO and stable dispersion in water (0.8 mgmL-1), (b) G-COOH and dispersion in water (1 mgmL-1), (c) Hydrophobic-MFGC and stable dispersion in dichloromethane (0.5 mgmL-1) (the lower phase is dichloromethane while water is the upper one), (d) IL-NH2, (e) Hydrophilic-MFGC and dispersion in water (0.5 mgmL-1) (the lower phase is dichloromethane while water is the upper one), (f) Amphiphilic-MFGC and stable dispersion in methanol (0.5 mgmL1 ), (B) Raman spectrum with ID/IG for (a) G-COOH, (b) Hydrophobic-MFGC, (c) Hydrophilic-MFGC, (d) AmphiphilicMFGC.

As shown in Figure 3b, the Hydrophobic-MFGC sample exhibited the following characteristic feature of N–H stretching around 3200-3530 cm−1. The intense absorption peak at 2929, 2856 cm−1 was attributed to the asymmetric and symmetric methylene vibrations from cyclohexyl moieties. The new peak around 1651-1733 cm−1 could be assigned to an amide carbonyl-stretching mode (Amide I vibration stretch) and the band at 1577 cm−1 could originate from the coupling of N–H bending and C–N stretching (Amide II vibration stretch). Furthermore, the peak at 1452 cm−1 was related to methylene scissoring and the bands at 1373 and 1259 cm−1 might be originated from the C–N stretching vibration. The two peaks at 1161 and 931 cm−1 were correlated to the inplane and out-plane bending of C–H in benzene ring.64-66 The FT-IR spectrum of Hydrophilic-MFGC exhibited characteristic peaks in compared with G-COOH and IL-NH2

Figure 3. FT-IR spectra of (a) G-COOH, (b) HydrophobicMFGC, (c) Hydrophilic-MFGC and (d) Amphiphilic-MFGC. 1

H NMR is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, to identify the hydrogen framework of organic compounds. The 1H NMR spectrum of Hydrophobic-MFGC, Hydrophilic-MFGC and Am-

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phiphilic-MFGC in DMSO-d6 is shown in Figure 4. In spectra of Hydrophobic-MFGC, a multiplet at 0.84-1.75 ppm was assigned to the protons of cyclohexyl moieties and signals of NH appeared at 2.17 ppm. Furthermore, two peaks at 4.19 and 4.40 ppm might be originated from –OCH2CO– and –NCH2CO–, respectively. The peaks belonging to the aromatic protons were clearly visible at 7.21-7.43 ppm (Figure 4a).69 While using 1-(3-aminopropyl)-3methylimidazolium chloride (IL-NH2) as an amine component in Ugi 4-CAP, some new protons signals appeared, which was a clear indication of IL-NH2 participation in fourcomponent condensation. In 1H NMR spectrum of Hydrophilic-MFGC, signals of cyclohexyl and propyl chains were appeared at 0.84-1.75 and 2.08 ppm. A singlet at 2.15 ppm corresponding to NH protons and the characteristic chemical shifts of –OCH2CO– and –NCH2CO– near 4.21 and 4.43 ppm could be clearly distinguished. It is worth noting that imidazole ring protons resonance distinctly appeared at 7.667.80 ppm (Figure 4b).54 When an aromatic aldehyde was utilized instead of formaldehyde (4-chlorobenzaldehyde), the corresponding aromatic resonances were observed in Amphiphilic-MFGC at 7.25-7.39 ppm, together with imidazole ring protons (at 7.71-7.81 ppm) (Figure 4c). It should be noted that, due to low solubility of Ugi products and existence of impurities in solvent, some peaks at the range of 2.33.8 ppm were corresponded to products that overlapped with this region. Therefore, it was difficult to confirm the peaks representing –CH2– of benzyl group and CH of cyclohexyl moieties in Hydrophobic-MFGC, and aliphatic protons correspond to ionic liquid and –CH– of cyclohexyl species in Hydrophilic-MFGC and Amphiphilic-MFGC, because they were overlapped with solvent peak.70 Figure 4. 1H-NMR spectrum of Ugi adducts for (a) Hydrophobic-MFGC, (b) Hydrophilic-MFGC and (c) Amphiphilic-MFGC in DMSO-d6 as a solvents.

The elemental analysis, chemical characterization and morphological changes of hybrid materials were further analyzed by SEM and EDS. The EDS pattern verified the presence of C, N and O in the structure of Hydrophobic-MFGC (Figure 5a), and the Hydrophilic-MFGC sample represents Cl element in addition to C, N and O elements (Figure 5b).71 SEM imaging showed obvious morphological changes occurred during the multi-functionalization of graphene sheets. Images were obtained after evaporating a drop of the samples on silicon wafers. The SEM plot of GO sheets (Figure S3, see supporting information) revealed that the sheets were broken during carboxylation process and ultra-sonication. The G-COOH showed irregular particle grains with smooth surfaces (Figure 5c). The microstructure changes of Hydrophobic-MFGC (Figure 5d), Hydrophilic-MFGC (Figure 5e) and Amphiphilic-MFGC (Figure 5f) samples were caused by evolution of chemical processes and ultrasonic cracking. The above mentioned results indicated that Ugi multicomponent process is a very robust and valuable method for generating high levels of functionalization in one-pot operation.

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Figure 6. TGA curves of the G-COOH, Hydrophobic-MFGC, Hydrophilic-MFGC and Amphiphilic-MFGC.

Figure 5. EDS spectrum of (a) Hydrophobic-MFGC; (b) Hydrophilic-MFGC. SEM image of (c) G-COOH; (d) HydrophobicMFGC; (e) Hydrophilic-MFGC and (f) Amphiphilic-MFGC (The scale bar is 500 nm).

Unambiguous evidence of organic groups on the graphene sheets was further analyzed by TGA in inert atmosphere to 600 °C at a rate of 10 °C min-1. TGA plots under inert atmosphere of G-COOH, Hydrophobic-MFGC, Hydrophilic-MFGC and Amphiphilic-MFGC are shown in Figure 6. The G-COOH curve started to weight loss at room temperature and is followed by a dramatically weight loss between 130-270 °C, which is associated with the steam release, hydroxyl and carboxyl decomposition.72 A weight loss of 59.9%, occurring in the temperature range of 180– 350 °C, was observed for Hydrophobic-MFGC. This weight loss arises from the decomposition of the covalently grafted organic addends. Similarly, the Hydrophilic-MFGC and Amphiphilic-MFGC showed ~ 65% weight loss up to 450 °C, which was attributed to the decomposition of the imidazole moieties attached to graphene sheets.73 Further weight loss above 500 °C was related to the destruction of the graphene carbon network, which is good agreement with the behavior exhibited by functionalized carbon based materials.74,75

XPS is a powerful technique to achieve a deeper insight into the chemical nature and to characterize the elemental composition. In Figure 7A, the C 1s XPS spectrum of GCOOH clearly indicates a considerable degree of oxidation with four different components which can be attributed into sp2-hybridized C=C in aromatic ring (284.7 eV), C‒O (286.1 eV), C=O (287.3 eV), and C(O)OH (288.5 eV). According to the extracted data from XPS survey, in G-COOH sample the majority of oxygen groups, consisted of C(O)OH (13 atom %, carboxylic), which located at the edge and basal plane of graphene sheets, while C-O bonds (9.9 atom %, carbonyl) and C=O (2 atom %, carbonyl) that clearly exhibited decrease in the number of epoxide and hydroxyl groups by carboxylation process than to GO.76 In comparison to the C1s spectrum of G-COOH sheets, XPS data for the Hydrophobic-MFGC, and HydrophilicMFGC samples clearly exhibited a considerable degree of surface functionalization. In Figures 7B and 5C, the C1s peak of Hydrophobic-MFGC, and Hydrophilic-MFGC show new components at 285.7, 287.5 eV and 285.8, 287.4 eV respectively, corresponding to carbon bond to nitrogen (C‒N), which resulted from Ugi 4-CAP modification with N-atoms containing amine and isocyanide molecules and new amide bonds formation.76-79 Additionally, a detailed deconvolution of the C1s spectrums clearly exhibited a decrease in C(O)OH (5 atom %) components, and indicated the conversion of the COOH groups to amide groups through modification process. These findings were consistent with the FTIR results. Furthermore, the asymmetry of the N1s core-level peak of Hydrophobic-MFGC with binding energies located at 399.7 eV could be assigned to –CO–NH– (Figure 7B), while the asymmetric N1s XPS spectrum of Hydrophilic-MFGC was divided into two components, which indicated that nitrogen atoms attached to the G-COOH network located at two different binding states (Figure 5C).78 The peaks at 399.8 and 400.7 eV were attributed to amide,80 and imidazole,81 respectively. Beside, Hydrophilic-MFGC showed a new peak at 198.3 eV for Cl 2p specie, which comes from the Cl¯ counter ions of IL-NH2,82 indicating that GO has been successfully functionalized through Ugi multicomponent assembly. In summary, the XPS results were complementary to those obtained by FT-IR, 1H-NMR and Raman spectroscopy.

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Figure 7. (A) High-resolution C 1s spectra of G-COOH; (B) High-resolution C 1s and N 1s spectra of Hydrophobic-MFGC; (C) Highresolution C 1s, N 1s, and Cl 2p spectra of Hydrophilic-MFGC.

Enzyme immobilization by Ugi 4-CAP as contrast marker The recent advances in the understanding of bioconjugation chemistry, a chemical strategy to form covalently linked small molecules to proteins or oligonucleotides or to bond between two biomacromolecules, have opened up innumerable opportunities in the field of medicine and biochemistry.83 Enzyme-solid support conjugates are widely used for immobilization of enzyme to produce bio-catalysts, which have been particularly proven valuable, in order to be easily reused several times for the same reaction and to improve stability under a wide range of reaction conditions.84 It also decreases purification costs with preventing the contamination of the substrate and biocatalyst. It is highly desirable to have a procedure, which allows for the conjugation of protein or oligonucleotide e.g. an enzyme or a DNA fragment to nanoscaled materials.85-87 Previous studies applied nanoparticles for enzyme immobilization to increase enzyme activity. This property originates from high surface area that nanopar-

ticles create and other practical advantages they have.85-87 There are limited strategies to covalent immobilization of enzymes on nanoscaled supports with high catalytic activity and selectively controlling the covalent binding site onto the supports.88,89 Recently, a simple and rapid method with high loading capacity has been developed using multicomponent reaction to immobilized RML lipase on SBA-15 under mild condition.90 On the basis of the impressive ability of MCR for rapid assembly of small molecules libraries, covalent immobilization of Bacillus thermocatenulatus lipase (BTL) on G-COOH was performed via Ugi four-component reaction between BTL, formaldehyde and cyclohexylisocyanide in the presences of G-COOH. To the best of our knowledge, there is no report on immobilization of enzyme on carbon based materials via Ugi MCR. This coupling reaction was carried out in 0.1 M phosphate buffer at 25 °C for 30 min and then centrifuged. The immobilized BTL was filtered and washed thoroughly with the same buffer to remove nonspecific phys-

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ically adsorbed lipase. Lipase as a hydrolase enzyme, which hydrolysis short- and long-chain triacylglycerols, is broadly used in industry. To verify the efficiency of the proposed immobilization method, a broad range of lipase substrates from C4 to C18 versus the native lipase were used for analyzing the activity and substrate specificity of the grapheneimmobilized lipase in pH-STAT at 55 °C and pH 8.5.91 The graphene-immobilized lipase showed higher specific activities versus native lipase against all substrates. The control tests on the catalytic performance of G-COOH were also carried out under the same condition, but no catalytic activities were observed. Specific activity for grapheneimmobilized lipase was 12000 U/mg when C4 triacylglycerols were treated. The specific activity of grapheneimmobilized lipase for C6, C8 and C10 substrates was 8200, 9800 and 8350 U/mg, respectively, which was significantly greater than native enzyme. Despite positive trend in enzyme activity, this increase in specific activity continued to some extent and was not significant for C12, C14 and C16 triacylglycerols (Figure 8). In other word, the native lipase showed lower enzyme activity for all substrate compared to immobilized lipase and the maximum specific activity of native BTL enzyme was toward small triglyceride substrates such as tributyrin (C4). These observations are in accordance with a previous study.91

Native

Graphene-immobolized Enzyme

100 80 60 40 20 0 45

50 55 60 Temperature (°C)

65

Figure 9. Thermal stability of native and immobilized lipase. Specific activity was determined in a pH-STAT by using tricaprylin (C8) as substrate (55 °C in pH 8.5).

In addition, to investigate the time-stability of the enzyme, both free and immobilized lipase, were incubated at 60 °C for 60 min (Figure 10). The data showed that time-stability of the immobilized lipase was better than the native enzyme. The residual activity of graphene-immobilized lipase gradually decreased, while activity of native enzyme rapidly decreased during the incubation time. The activity of grapheneimmobilized lipase was 87 and 71% in compared with native lipase 71 and 57% after 10 and 20 min, respectively (p ≤0.05). These results clearly show that the high temperature leads to unfolding in protein structure and induce enzyme inactivation.89,91,92 Native

Graphene-immobolized Enzyme

100

Figure 8. Activities and substrate specificities of the native lipase and immobilized lipase in the presences of triacylglycerol with different acyl chain lengths as substrates. Activity was measured in a pH-stat at 55 °C and pH 8.5.

Immobilized lipase characterization The enzymatic activity of graphene-immobilized lipase and native enzyme in a range of temperature were assayed. The results showed that the optimized temperature for enzyme activity for both enzymes (graphene-immobilized lipase and native enzyme) was 60 °C, where the activity reached to 100%. When temperature reached to 65 °C the activity of both enzymes dramatically declined to 18%. But the main difference in activity of immobilized enzyme and native one was in 45, 50 and 55 °C, as the activity of graphene-immobilized lipase was 48, 65 and 87% (for 40, 45 and 50 °C respectively) versus native lipase was 32, 56 and 75% (for 40, 45 and 50 °C respectively) (Figure 9).89,91,92

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80 60 40 20 0 0

10

20 30 40 Time(min, 60 °C)

50

60

Figure 10. The residual activities of native and immobilized lipase under the time-dependent incubation at 60 °C. Specific activity was determined in a pH-STAT by using tricaprylin (C8) as substrate (55 °C in pH 8.5). The initial residual activity at 0 min was set as 100%, respectively.

The effect of pH on the activity of the free and immobilized enzyme was also examined (Figure 11). The results showed that lipase activity changes according to pH values. The best pH value for activity of lipase in both enzymes was ~ 8, where relative activity was 100%. The activity of graphene-immobilized lipase in acidic pH was lower than native enzyme, 25 and 34% activity versus 33 and 41% (in 5 and 6 pH values respectively), while at pH above 7, the activity of graphene-immobilized lipase was higher than native enzyme. These phenomena could be ascribed to various factors that influence on the enzyme activity such as inhibition of catalytically active site and conformational stability.89,91,92

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Graphene-immobolized Enzyme

Relative Activity (%)

100 80 60 40 20 0 5

6

7

8

9

10

pH

Figure 11. Effect of pH on the relative activities of free lipase and graphene-immobilized lipase. Specific activity was determined in a pH-STAT by using tricaprylin (C8) as substrate (55 °C in pH 8.5).

The reusability of immobilized lipase is a key factor in industrial application from an economic point of view. In this regard, graphene-immobilized lipase was reused to investigate its activity in this context. The immobilized lipase could be easily separated from the matrix with centrifuge at 7000 rpm for 10 min and washed thoroughly with phosphate buffer solution (pH 7.4). As shown in Figure 12, the activity of reused lipase gradually decreased to about 68% of its initial activity after 8 cycles. Therefore, the immobilized lipase onto graphene sheets through Ugi four-component condensation, shows excellent reusability for its bio-catalytic application. This could be attributed to exceptional chemistry of Ugi 4-CAP for covalent site-specific interaction.

We have presented conclusive evidence that Ugi 4-CAP is an efficient and powerful way to one-pot multifunctionalization of graphene materials, in which, starting materials according to a cascade of sequential and irreversible chemical reactions come together to anchor active functionality on graphene surface under extremely mild conditions (25 °C, water). By selecting the starting compounds, hybrid graphene with intrinsic hydrophobic-, hydrophilic-, or amphiphilic properties can be introduced. The graphenebased hybrid materials were characterized by using complementary spectroscopic and microscopic techniques. Importantly, we have proven that BTL enzyme was successfully immobilized on G-COOH surface with high bio-catalytic activity through proposed approach that serves as contrast marker. The Ugi 4-CAP is promising for providing an enormous level of diversity and complexity to revolutionize multi-functionalization and programmable manipulation of nanomaterials for a wide variety of next-generation technologies.

ASSOCIATED CONTENT Supporting Information. Experimental procedure of IL-NH2; FT-IR spectra of GO and IL-NH2; SEM image of GO. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author [email protected] (A. Rezaei) [email protected] (O. Akhavan)

ACKNOWLEDGMENT

100

The enzyme mixtures were a kind gift from Dr. Ali Asghar Karkhane at NIGEB, Tehran, Iran. The authors are grateful for the financial support by the “Iran National Science Foundation (INSF)”.

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40 30 20 10 0 1

2

3

4 5 Cycle number

6

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Figure 12. The reusability of immobilized lipase on graphene for 8 cycles. Specific activity was determined in a pH-STAT by using tricaprylin (C8) as substrate (55 °C in pH 8.5).

These results clearly show that not only lipase was successfully immobilized on graphene surface but also increases its activity as well. In addition, these interesting results confirmed results of previous studies, which revealed that immobilizing enzyme on nanoparticles surface leads to higher activity.85-89 Moreover, the results indicate that Ugi reaction is well suited for the assembly types of molecules on graphene materials to set complex scaffolds with high efficiency. CONCLUSION

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(72) Ciobotaru, C. C.; Damian, C. M.; Matei, E.; Iovu, H. Covalent Functionalization of Graphene Oxide with Cisplatin. Mat. Plast. 2014, 1, 1612-1618. (73) Peng, Y.; Wu, X.; Qiu, L.; Liu, C.; Wang, S.; Yan, F. Synthesis of carbon–PtAu nanoparticle hybrids originating from triethoxysilane-derivatized ionic liquids for methanol electrooxidation and the catalytic reduction of 4-nitrophenol. J. Mater. Chem. A 2013, 1, 9257–9263. (74) Titus, E.; Ali, N.; Cabral, G.; Gracio, J.; Ramesh Babu, P.; Jackson, M. Chemically Functionalized Carbon Nanotubes and Their Characterization Using Thermogravimetric Analysis, Fourier Transform Infrared, and Raman Spectroscopy. J. Mater. Eng. Perform. 2006, 15, 182–186. (75) Pagona, G.; Karousis, N.; Tagmatarchis, N. Aryl Dianium Functionalization of Carbon Nanohorns. Carbon 2008, 46, 604– 610. (76) Kim, T.; Lee, H.; Kim, J.; Suh, K. S. Synthesis of phase transferable graphene sheets using ionic liquid polymers. ACS Nano 2010, 4, 1612-1618. (77) Baker, S. E.; Cai, W.; Lasseter, T. L.; Weidkamp, K. P.; Hamers, R. J. Covalently bonded adducts of deoxyribonucleic acid (DNA) oligonucleotides with single-wall carbon nanotubes: synthesis and hybridization. Nano Lett. 2002, 2, 1413-1417. (78) Bhunia, P.; Hwang, E.; Yoon, Y.; Lee, E.; Seo, S.; Lee, H. Synthesis of Highly n‐Type Graphene by Using an Ionic Liquid. Chem. Eur. J. 2012, 18, 12207-12212. (79) Ramanathan, T.; Fisher, F.; Ruoff, R.; Brinson, L. Aminofunctionalized carbon nanotubes for binding to polymers and biological systems. Chem. Mater. 2005, 17, 1290-1295. (80) Wang, Z.; Zhang, Q.; Kuehner, D.; Xu, X.; Ivaska, A.; Niu, L. The synthesis of ionic-liquid-functionalized multiwalled carbon nanotubes decorated with highly dispersed Au nanoparticles and their use in oxygen reduction by electrocatalysis. Carbon 2008, 46, 1687-1692. (81) Zhang, H.; Cui, H. Synthesis and Characterization of Functionalized Ionic Liquid-Stabilized Metal (Gold and Platinum) Nanoparticles and Metal Nanoparticle/Carbon Nanotube Hybrids. Langmuir 2009, 25, 2604-2612. (82) Yang, Y.-K.; He, C.-E.; Peng, R.-G.; Baji, A.; Du, X.-S.; Huang, Y.-L.; Xie, X.-L.; Mai, Y.-W. Non-Covalently Modified Graphene Sheets by Imidazolium Ionic Liquids for Multifunctional Polymer Nanocomposites. J. Mater. Chem. 2012, 22, 5666-5675. (83) Gordon, M. R.; Canakci, M.; Li, L.; Zhuang, J.; Osborne, B.; Thayumanavan, S. Field Guide to Challenges and Opportunities in Antibody–Drug Conjugates for Chemists. Bioconjugate Chem. 2015, 26, 2198-2215. (84) Datta, S.; Christena, L. R.; Rajaram, Y. R. S. Enzyme Immobilization: An Overview on Techniques and Support Materials. 3 Biotech 2013, 3, 1-9. (85) Alwarappan, S.; Boyapalle, S.; Kumar, A.; Li, C.-Z.; Mohapatra, S. Comparative Study of Single-, Few-, and Multilayered Graphene Toward Enzyme Conjugation and Electrochemical Response. J. Phys. Chem. C 2012, 116, 6556-6559. (86) Kang, Y.; He, J.; Guo, X.; Guo, X.; Song, Z. Influence of Pore Diameters on The Immobilization of Lipase in SBA-15. Ind. Eng. Chem. Res. 2007, 46, 4474-4479. (87) Lee, Y.-M.; Kwon, O.-Y.; Yoon, Y.-J.; Ryu, K. Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and Its Electrochemical Properties. Biotechnol. Lett. 2006, 28, 3943. (88) Blank, K.; Morfill, J.; Gaub, H. E. Site‐Specific Immobilization of Genetically Engineered Variants of Candida Antarctica Lipase B. ChemBioChem 2006, 7, 1349-1351. (89) Zhang, G.; Ma, J.; Wang, J.; Li, Y.; Zhang, G.; Zhang, F.; Fan, X. Lipase Immobilized on Graphene Oxide as Reusable Biocatalyst. Ind. Eng. Chem. Res. 2014, 53, 19878-19883. (90) Mohammadi, M.; Ashjari, M.; Dezvarei, S.; Yousefi, M.; Babaki, M.; Mohammadi, J. Rapid and High-Density Covalent Immobilization of Rhizomucor Miehei Lipase Using a Multi Component Reaction: Application in Biodiesel Production. RSC Adv. 2015, 5, 32698-32705.

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Chemistry of Materials

(91) Karkhane, A. A.; Yakhchali, B.; Jazii, F. R.; Bambai, B. The Effect of Substitution of Phe 181 and Phe 182 With Ala on Sctivity, Substrate Specificity and Stabilization of Substrate at The Active Site of Bacillus Thermocatenulatus Lipase. J. Mol. Catal., B Enzym 2009, 61, 162-167.

(92) Hermanová, S.; Zarevúcká, M.; Bouša, D. Pumera, M.; Sofer, Z. Graphene oxide immobilized enzymes show high thermal and solvent stability. Nanoscale, 2015, 7, 5852–5858.

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