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INTRODUCTION
Graphene has attracted tremendous attention in recent years owing to its exceptional thermal,
mechanical, and electrical properties. Graphene, unlike other regular nanoparticles, provide
single carbon atoms thick graphite sheets with enormous surface area; perfect for uniform
attachment of Biomolecules.(Geim & Novoselov, 2007) But it is plagued with a problem when
considered with respect to the biological perspective. It has a very high surface area to volume
ratio and its surface is highly hydrophobic, therefore, making its suspension as monolayers a
very problematic issue in aqueous or polar aprotic solvents, where it is strongly dominated by
the tendency to agglomerate. This property makes the use of pristine graphene relatively less
usable in the biological aspect.(Kishore, Talat, Srivastava, & Kayastha, 2012)(Jiang, Zhang, Li, &
Niu, 2012)
One such alternative to this is the use of functionalized graphene oxide. Exemplary example to
that is the graphene oxide. It is graphene sheet functionalized with mixture of carboxyl,
hydroxyl, and epoxy functionalities.(Cao, Zhang, Feng, & Wu, 2011) Stability and high
functionality in conditions mimicking biological conditions make graphene viable for use in
cases such as polymer composites, biosensors, and drug delivery.(Dreyer, Park, Bielawski, &
Ruoff, 2010)
Nonetheless, even graphene oxide has its drawbacks like, it affects the enzyme activity
adversely in many cases, has relatively low loading as compared to graphene (and even some
other traditional immobilizers).(Y. Zhang et al., 2012)(J. Zhang et al., 2010)
Graphene shows extremely high loading of enzymes, even up to 60 times the traditional
immobilizers, but its conjugates are more expensive and cumbersome to make (due to
agglomeration in aqueous solutions). Perfectly reduced graphene also alters the structure of
the protein (even the secondary structure)(Y. Zhang et al., 2012)
Other methods of immobilization include binding an enzyme directly/ indirectly through
acovalent bond.(Kishore et al., 2012)(Zhou et al., 2012) Loading due to covalent binding will be
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much less compared to graphene. Apart from that the procedure would be more complex and
more time consuming than electrostatic binding (as in graphene oxide) and hydrophilic binding
(as in graphene).
So we are in need of a solution that gives us the benefits of both graphene and graphene oxide,
while minimizing the negative connotations.
OBJECTIVES
Synthesis of graphene/ graphene oxide non-fully-functionalized sheets Conjugation of graphene with enzymes such as -galactosidase, Lipase through a
combination of electrophilic and hydrophobic interactions
Testing of enzyme activity through enzyme activity assays Optimization of the conjugation process to obtain maximum output of the reaction
catalyzed by the respective enzymes
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by a mixture of carboxyl, hydroxyl, and epoxy functionalities(Cao et al., 2011) and covered with
strongly bound oxidative groups.(Rourke et al., 2011)They can be easily acquired from natural
graphite flakes by strong oxidation and subsequent exfoliation. In the past few years, graphene
oxide sheets and their derivatives have been extensively studied in the context of many
applications, such as polymer composites, biosensors, and drug delivery.(Dreyer et al., 2010)
Biocompatibility issues of graphene oxide on animal models have also been studied and
demonstrated non-toxic effects of the material under low dose administration(Wang et al.,
2011)
Recently, due to its inertia and low toxicity underphysiological conditions, the application of GO
has beenextending to biological systems. For example, polyethyleneglycol (PEG)-modified GO
has been used as a carrier forwater-insoluble cancer drugs(Zhuang Liu , Joshua T. Robinson,
2009) using covalent binding as a linking force.A GO sheet having a large specific surface area
and bondedfunctional groups is an ideal substrate forenzyme immobilization.(J. Zhang et al.,
2010)
However, electrostatic interaction as the driving force for enzyme binding to GO severelyaffected the activity of the enzyme. The enzyme loading on GO, though higher than that on
many classical materials, may still relatively low for practical applications. (Y. Zhang et al., 2012)
Graphene oxide sheets represent a unique type of building block with hydrophobic domains
on the planar region and hydrophilic carboxylic groups on the edges and also scattered
throughout the plane. Therefore, GOSs exhibit an amphiphilic character and can be used as
surfactants in numerous technological fields. For example, graphene oxide sheetshave beenused as dispersants to suspend otherwise insoluble carbon nanotubes in water.(Cao et al.,
2011)
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It is a promising material for barrier polymers because it wouldnt let even the smallest of
gasses diffuse through its plane. These can be coated in the form of a super thin coating or they
can be incorporated into the polymer matrix.(Min Yoo, Jin Shin, Wook Yoon, & Bum Park, 2013)
A matrix/ substrate for enzyme immobilization:Very large surface area and either a large amount of functional groups containing oxygen or a
large electron cloud ensures almost instantaneous enzyme immobilization on the surface of
graphene oxide or graphene respectively (J. Zhang et al., 2010)(Zhou et al., 2012)(Kishore et al.,
2012)(Y. Zhang et al., 2012)
Biosensors:The ability of graphene/ graphene oxide sheets to interact strongly with the aromatic groups of
ds-DNA make it a superb platform for sensing Biomolecules such as DNA orproteins.(Alwarappan, Liu, Kumar, & Li, 2010; Y. Zhang et al., 2012)
Drug delivery systems:The extremely high surface area is to volume ratio and favorable surface properties like
hydrophilicity/ hydrophobicity make it a vial alternative for drug delivery.(Zhuang Liu , Joshua T.
Robinson, 2009) Biocompatibility and non- toxicity are also important aspects which it fulfills.
(Cheng et al., 2013; Wang et al., 2011)
Enzyme immobilization:
Graphene, unlike other regular nano-particals, provide single carbon atoms thick graphite
sheets with enormous surface area; perfect for uniform attachment of biomolecules. In
addition, being made of carbon atoms, it does not alter native biochemical properties of
attached Biomolecules significantly. There are several ways to immobilize the enzymes on the
graphene/ graphene oxide surface
Some of them are:
Covalent attachment:
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For example amino groups can be attached covalently to carboxyl groups of the graphene oxide
sheet.(Zhou et al., 2012)
Fig: image showing schematic of modifying a functionalized group of graphene oxide and
subsequently attaching the enzyme concanavalin A.
Electrostatic interactions:The functional groups of graphene oxide which are negatively charged in the pH range of 4-11
interact with the hydrophilic surfaces on the enzyme.(Y. Zhang et al., 2013)(J. Zhang et al.,
2010)
Fig: image showing graphene oxide bound HRP (J. Zhang et al., 2010)
Hydrophobic interactions:If the protein surface is dominated by hydrophobic surfaces, then it would have a propensity
towards hydrophobic surfaces, such as the atomically flat, hydrophobic surface of graphene.(Y.
Zhang et al., 2012, 2013)
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Fig: image showing a schematic of enzyme HP35 on pristine graphene surface (Y. Zhang et al.,
2013)
Enzyme to be immobilized
-Galactosidase is enzyme of extreme industrial importance and has 2 main commercial
applications in food technology:
the reduction of lactose in dairy commodities for safe consumption by lactose intolerantpeoples
Production of galacto-oligosaccharides (GOS) for a balanced gastrointestinal florapreservation.
Lactose, an integral component of breast milk, causes a discomfort in children and adolescents
worldwide, causing abdominal pain, nausea, flatulence and bloating. The condition becomes
more severe with advancement of age due to dropping in the gastric b-galactosidase secretion.
Now a days, consumers are becoming more and more conscious regarding influence of diet on
health and demanding natural foods with beneficial health effects and luscious taste.
Therefore, commercially reduced lactose products are being manufactured for peoples acrossthe globe.(Boler & Jr, 2012)(Kishore et al., 2012)
Galacto-oligosaccharides (GOS) are non-digestible sugars containing two to five molecules of
galactose and one molecule of glucose or lactose connected through glycosidic bonds. In
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general, trans-galactosylation dominates early in the reaction, producing GOS with a high yield.
As lactose conversion increases, the enzymatic hydrolysis activity takes over trans-
galactosylation;resulting complete conversion of lactose into glucose and galactose
Units. GOS are classified as prebiotics or as bifidus growth factor as they specifically promote
the growth of bifidobacter(Boler & Jr, 2012)
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METHODOLOGY
Graphene from Graphene oxide and different degrees of reduced graphene oxide
Work would be starting from graphene oxide. Graphene oxide could be reduced to different
degrees using L-ascorbic acid as the reducing agent at the same conditions of temperature,
different reducing time intervals.
Formation of Graphene-Enzyme complexes
Formation of Graphene or graphene oxide could be loaded with the desired enzyme without
the use of any bridging agent by addition of enzyme directly to the suspended graphene
solution
Detection of Enzyme loadingTo detect the amount of enzyme loading on the graphene substrate, it could be centrifuged and
washed to remove all loosely attached enzyme. Solution could be used with fluorescence
spectrometry to determine the amount of enzyme loaded successfully. Adsorption isotherms
can be plotted to study the conjugation of enzymes onto the graphene sheets.
Detection of structural changes to the enzyme while loading onto the graphene
To detect the structural changes to the enzyme and the graphene sheet, CD spectroscopy could
be used for initial estimation and subsequently followed by FTIR
AFM (atomic force microscopy) could be used to visualize the graphene sheet alongside theattached enzyme which could not have been possible in traditional substrates, but for the
atomically flat graphene surface.
Detection of enzyme activity assays
Enzyme activity assays could be carried out to check the activity of the immobilized enzyme.
This can be done by measuring hydrolysis of o-nitrophenyl--Dgalactoside (ONPG).
Amount of o-nitrophenol formed can be measured by determining the absorbance at 420 nm
Enzyme loading of-Galactosidase to be carried over differently reduced Graphene Oxide
sheets.
Immobilization efficiency can be calculated as follows:
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References
Alwarappan, S., Liu, C., Kumar, A., & Li, C. (2010). Enzyme-Doped Graphene Nanosheets for
Enhanced Glucose Biosensing. Society, 114(30), 1292012924. Retrieved from
http://dx.doi.org/10.1021/jp103273z
Boler, B. M. V., & Jr, G. C. F. (2012). Direct-Fed Microbials and Prebiotics for Animals. (T. R.
Callaway & S. C. Ricke, Eds.), 1327. doi:10.1007/978-1-4614-1311-0
Cao, Y., Zhang, J., Feng, J., & Wu, P. (2011). Compatibilization of immiscible polymer blends
using graphene oxide sheets.ACS nano, 5(7), 59207. doi:10.1021/nn201717a
Cheng, C., Nie, S., Li, S., Peng, H., Yang, H., Ma, L., Zhao, C. (2013). Biopolymer functionalized
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coatings/anchors.Journal of Materials Chemistry B, 1(3), 265. doi:10.1039/c2tb00025c
Dreyer, D. R., Park, S., Bielawski, C. W., & Ruoff, R. S. (2010). The chemistry of graphene oxide.
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Geim, a K., & Novoselov, K. S. (2007). The rise of graphene. Nature materials, 6(3), 18391.
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Min Yoo, B., Jin Shin, H., Wook Yoon, H., & Bum Park, H. (2013). Graphene and graphene oxide
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Rourke, J. P., Pandey, P. a, Moore, J. J., Bates, M., Kinloch, I. a, Young, R. J., & Wilson, N. R.
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Zhang, Y., Zhang, J., Huang, X., Zhou, X., Wu, H., & Guo, S. (2012). Assembly of graphene oxide-
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Zhou, L., Jiang, Y., Gao, J., Zhao, X., Ma, L., & Zhou, Q. (2012). Oriented immobilization of
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