MTP midterm I.docx

7/27/2019 MTP midterm I.docx

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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

reduced graphene oxide with enhanced biocompatibility via mussel inspired

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.

Chemical Society reviews, 39(1), 22840. doi:10.1039/b917103g

Geim, a K., & Novoselov, K. S. (2007). The rise of graphene. Nature materials, 6(3), 18391.

doi:10.1038/nmat1849

Jiang, Y., Zhang, Q., Li, F., & Niu, L. (2012). Glucose oxidase and graphene bionanocomposite

bridged by ionic liquid unit for glucose biosensing application. Sensors and Actuators B:

Chemical, 161(1), 728733. doi:10.1016/j.snb.2011.11.023

Kishore, D., Talat, M., Srivastava, O. N., & Kayastha, A. M. (2012). Immobilization of -

galactosidase onto functionalized graphene nano-sheets using response surface

methodology and its analytical applications. PloS one, 7(7), e40708.

doi:10.1371/journal.pone.0040708


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Min Yoo, B., Jin Shin, H., Wook Yoon, H., & Bum Park, H. (2013). Graphene and graphene oxide

and their uses in barrier polymers.Journal of Applied Polymer Science, n/an/a.

doi:10.1002/app.39628

Rourke, J. P., Pandey, P. a, Moore, J. J., Bates, M., Kinloch, I. a, Young, R. J., & Wilson, N. R.

(2011). The real graphene oxide revealed: stripping the oxidative debris from the

graphene-like sheets.Angewandte Chemie (International ed. in English), 50(14), 31737.

doi:10.1002/anie.201007520

Wang, K., Ruan, J., Song, H., Zhang, J., Wo, Y., Guo, S., & Cui, D. (2011). Biocompatibility of

Graphene Oxide, 18.

Zhang, J., Zhang, F., Yang, H., Huang, X., Liu, H., Zhang, J., & Guo, S. (2010). Graphene oxide as a

matrix for enzyme immobilization. Langmuir : the ACS journal of surfaces and colloids ,

26(9), 60835. doi:10.1021/la904014z

Zhang, Y., Wu, C., Guo, S., & Zhang, J. (2013). Interactions of graphene and graphene oxide with

proteins and peptides. Nanotechnology Reviews, 2(1), 2745. doi:10.1515/ntrev-2012-

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Zhang, Y., Zhang, J., Huang, X., Zhou, X., Wu, H., & Guo, S. (2012). Assembly of graphene oxide-

enzyme conjugates through hydrophobic interaction. Small (Weinheim an der Bergstrasse,

Germany), 8(1), 1549. doi:10.1002/smll.201101695

Zhou, L., Jiang, Y., Gao, J., Zhao, X., Ma, L., & Zhou, Q. (2012). Oriented immobilization of

glucose oxidase on graphene oxide. Biochemical Engineering Journal, 69, 2831.

doi:10.1016/j.bej.2012.07.025

Zhuang Liu , Joshua T. Robinson, X. S. and H. D. (2009). NIH Public Access. journal of the

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