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Yingcan Zhao

REACTIONS OF GRAPHENE OXIDE AND BUCKMINSTERFULLERENE IN THE AQUATIC ENVIRONMENT

Doctor of Philosophy

Chad T. JafvertChair

Timothy R. Filley

Inez Hua

Ronald F. Turco

Chad T. Jafvert

Dulcy M. Abraham 6/21/2016

i

REACTIONS OF GRAPHENE OXIDE AND BUCKMINSTERFULLERENE IN THE AQUATIC

ENVIRONMENT

A Dissertation

Submitted to the Faculty

of

Purdue University

by

Yingcan Zhao

In Partial Fulfillment of the

Requirements for the Degree

of

Doctor of Philosophy

August 2016

Purdue University

West Lafayette, Indiana

ii

To my parents and Liang, for their love, support and encouragement.

iii

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my Ph.D. major advisor, Prof.

Jafvert, for his guidance, support and encouragement throughout my graduate research

studies during the past few years at Purdue. Even though sometimes he is extremely

busy, he would like to help me out with my hundreds of academic questions and

difficulties. I also enjoy the academic freedom he gave me to explore my own interest

in research. I have learned so much from him and if I have a chance to be faculty

member in environmental engineering area in the future, I hope to be someone like

you, Dr. Jafvert, patient, kind, generous and full of career passion.

I want to further thank one of my committee members, Prof. Inez Hua, who

provided me the opportunity to be a teaching assistant with her in Fall 2014 for the

course CE/EEE 350 Introduction to Environmental Engineering. I cherish the whole

semester working with her and five peer undergraduate TAs.

I want to thank all my committee member: Drs. Chad Jafvert, Timothy Filley, Inez

Hua and Ronald Turco. Without their guidance, this thesis would have been impossible

to complete. I also want to thank Nadya Zyaykina, the environmental engineering

laboratory manager. Thanks for her kindness and patience even though I bothered her

so many times. I also would like to thank Prof. Zhi Zhou who provided instruments for

iv

my gel electrophoresis experiments and give me helpful suggestions when I applied for

postdoc positions.

I want to express my thankfulness to my lab mates: Somi, Hsin-Se, Tengyi, Ming,

Xuda, Xuqing, Yining and Yichen; also Dr. Filley and his group: Tim Berry and Christy. I

will cherish all your help and the time we spent together. Thanks to all my friends here

at Purdue. You are like angels to make me feel we are just like a family.

Although being so far away from home, my parents Honglai Zhao and Wenyan

Zhang have never stopped offering me love and encouragement. Being their single

child, I know I owe them a lot. Thanks for their support and love, forever.

At the period of writing this thesis, I am excited to know that my husband and I

will have our first baby soon. She gives me so much courage that keeps me moving on. I

always wish to give her all the best things in life. Thank you for choosing us to be your

parents and welcome to our home, my little sweetie.

v

TABLE OF CONTENTS

Page

LIST OF TABLES ................................................................................................................... vii

LIST OF FIGURES .................................................................................................................. ix

ABSTRACT .......................................................................................................................... xiii

CHAPTER 1. INTRODUCTION ......................................................................................... 1

1.1 Scope and Significance ..................................................................................... 1

1.2 Fullerenes ......................................................................................................... 2

1.2.1 Physical and Chemical Properties of C60 ....................................................... 2

1.2.2 C60 Toxicity .................................................................................................... 3

1.2.3 C60 Photo-activity .......................................................................................... 4

1.3 Graphene Oxide ................................................................................................ 5

1.3.1 Graphene Oxide Applications ....................................................................... 6

1.3.2 Environmental Applications using Graphene Oxide-based Material ........... 7

1.3.3 Toxicity of Graphene Oxide........................................................................... 7

1.3.4 Environmental Transformation of Graphene Oxide ..................................... 8

1.3.5 High Electron Transfer Ability of Graphene Oxide........................................ 9

1.4 Chapter Outline .............................................................................................. 10

CHAPTER 2. ENVIRONMENTAL PHOtOCHEMISTRY OF SINGLE LAYERED GRAPHENE

OXIDE IN WATER ............................................................................................................... 12

2.1 Abstract........................................................................................................... 12

2.2 Introduction .................................................................................................... 13

2.3 Materials and Methods .................................................................................. 17

2.3.1 Materials ..................................................................................................... 17

2.3.2 Preparation of Aqueous Graphene Oxide ................................................... 17

vi

Page

2.3.3 Irradiation and GO Analysis ........................................................................ 18

2.3.4 ROS Measurement ...................................................................................... 19

2.4 Results and Discussion .................................................................................... 20

2.5 Conclusion ...................................................................................................... 30

2.6 Supporting Information .................................................................................. 32

CHAPTER 3. Light-IndepenDENT rEDOX REACTIONS OF GRAPHENE OXIDE IN WATER:

ELECTRON TRANSFER FROM NADH TO MOLECULAR OXYGEN PRODUCING REACTIVE

OXYGENM SPECIES (ROS) .................................................................................................. 37

3.1 Abstract........................................................................................................... 37

3.2 Introduction .................................................................................................... 38

3.3 Materials and Method .................................................................................... 40

3.3.1 Materials ..................................................................................................... 40

3.3.2 Oxidation of NADH ...................................................................................... 41

3.3.3 ROS Detection ............................................................................................. 41

3.3.4 Gel Electrophoresis ..................................................................................... 43


3.5 Conclusion ...................................................................................................... 58

CHAPTER 4. INDIRECT PHOTO-TRANSFORMATION OF GRAPHENE OXIDE IN WATER 59

4.1 Abstract........................................................................................................... 59

4.2 Introduction .................................................................................................... 60


4.3.1 Materials ..................................................................................................... 63

4.3.2 Irradiation of GO in Solar Spectrum Light ................................................... 63

4.3.3 Materials Analysis ....................................................................................... 64


4.5 Conclusion ...................................................................................................... 70

4.6 Environmental Significance ............................................................................ 71

vii

Page

CHAPTER 5. PHOTOREACTIONS OF AQU/NC60 CLUSTERS UNDER SIMULATED

SUNLIGHT: PHOTO MINERALIZATION AND PRODUCT CHARACTERIZATION .................... 72

5.1 Abstract........................................................................................................... 72

5.2 Introduction .................................................................................................... 72


5.3.1 Materials ..................................................................................................... 75

5.3.2 Aqu/nC60 Suspension Preparation .............................................................. 76

5.3.3 HPLC Measurement .................................................................................... 78

5.3.4 UV-Vis spectroscopy and pH measurement ............................................... 79

5.3.5 Headspace CO2 measurement .................................................................... 79


5.5 Conclusions ..................................................................................................... 88

CHAPTER 6. OVERALL SUMMARY AND RECOMMENDATION ..................................... 89

6.1 Summary ......................................................................................................... 89

6.2 Recommendations .......................................................................................... 91

REFERENCES ...................................................................................................................... 93

VITA ................................................................................................................................. 104

viii

LIST OF TABLES

Table Page

Table 5.1. Summary of CO2 measurements for samples exposed to light ....................... 87

Table 5.2. Summary of CO2 measurements for dark control samples ............................. 88

ix

LIST OF FIGURES

Figure .............................................................................................................................Page

1.1 C60 Buckminsterfullerene .............................................................................................. 3

1.2 The structure of graphene oxide (adapted from C.E. Hamilton, PhD Thesis, 2009, Rice

University) ........................................................................................................................... 6

2.1 (a) Photograph of 5 mg/L GO at pH 7, before (left vial) and after (right vial) irradiation

for 2 hours under lamp light irradiation; and (b) the change in the UV-visible light

absorption spectra of GO suspensions with increasing time of irradiation. .................... 22

2.2 The Raman spectra of GO before and after irradiating an aqueous GO suspension (100

mg/L) for 24 hours, where the spectra have been normalized to the intensity of the G

band. ................................................................................................................................. 24

2.3 (a) Evidence of superoxide anion formation by reaction with XTT at 470 nm, upon lamp

light irradiation of a 5 mg/L GO suspension at pH 7. (b) The change in the UV-visible light

absorption spectra for a suspension of GO (5 mg/L) and XTT (0.1 mM). ......................... 26

2.4 (a) Evidence for the increase in H2O2 concentration over time upon lamp light

irradiation of GO at pH 7. (b) The standard curve of H2O2 using DPD/HRP method. ..... 29

2.5 Proposed pathway for ROS production by photosensitization of GO in water. ......... 31

2.6 Images of GO (Provided by ACS Material LLC.). .......................................................... 32

x

Figure .............................................................................................................................Page

2.7 Spectral density of solar and employed lamp light irradiance as a function of time

(solar irradiance data was from ASTM G173-03 Reference Spectra). .............................. 33

2.8 Data points are the concentrations of FFA (initially at 0.2 mM) detected in suspensions

of 5 mg/L GO at pH 7. All samples were analyzed by HPLC after removing the GO with a

0.2-μm membrane filter. .................................................................................................. 34

2.9 Photograph of 5 mg/L GO in water with XTT (0.1 mM) at pH=7 at irradiation times of

0, 0.5, 1, 1.5, 2, 2.5, and 3 hr (left to right), before filtering out the GO particles. .......... 35

2.10 Data points are the concentrations of pCBA (initially at 5 μM) detected in suspensions

of 5 mg/L GO at pH 7after irradiation (), or incubation in the dark (). ....................... 36

3.1 Chemical structures of NADH and NAD+. ................................................................... 44

3.2 (a) The UV-visible absorption spectra of NADH (0.2 mM) and GO (5 mg/L) at pH 7 as a

function of time under dark conditions. (b) The loss of NADH (0.2 mM) with and without

GO (5 mg/L), calculated based on the change in light absorbance at 340 nm................ 46

3.3 (a) NBT2+ (0.2 mM) product formation indicating presence of superoxide anion at pH=7

(b) The effect of GO concentration in the production of superoxide anion within the

system containing NADH, NBT2+ and 0 mg/L GO (), 5 mg/L GO (), 10 mg/L GO (), 20

mg/L GO (). .................................................................................................................... 49

3.4 Evidence of superoxide anion using XTT as a scavenger. ........................................... 50

3.5 Evidence of H2O2 production via DPD/HRP product formation. ................................ 52

3.6 Scavenging for ·OH with pCBA in pH 7 phosphate buffered solutions. ...................... 53

xi

Figure .............................................................................................................................Page

3.7 (a) Gel image of pBR322 DNA after an 8 hr incubation at pH 7 in the different

experimental systems. (b) Quantitative analysis of DNA bands. ..................................... 56

3.8 (a) Gel images showing the time course of pBR322 DNA fragmentation, in GO

suspensions containing NADH at pH 7. The concentration of GO, NADH and pBR322 DNA

were 5 mg/L, 0.2 mM and 0.015 µg/µL, respectively. (b) Quantitative analysis of DNA

bands. ................................................................................................................................ 57

3.9 Proposed mechanism of GO acting as an electron shuttle to produce reactive oxygen

species(ROS) and subsequent DNA cleavage. .................................................................. 58

4.1 Characterization of GO. a) UV/Vis absorption spectra. b) Raman spectra. ................ 65

4.2 UV-visible attenuation spectra of samples (measured as absorbance); (a) 10 mg/L GO

with 100 mM H2O2 under irradiation with solar wavelength lamp light; (b) 10 mg/L GO

(with no H2O2) under irradiation with solar wavelength lamp light; and (c) 10 mg/L GO

with 100 mM H2O2 dark control. ....................................................................................... 67

4.3 Images of samples at specific experimental times: (a) GO before irradiation; (b) GO

after 48 hr of irradiation with solar-wavelength lamp light; and (c) GO and H2O2 after 48

hr of irradiation with solar-wavelength lamp light. All samples contain 10 mg/L GO, and

either zero or 100 mM H2O2. ............................................................................................ 69

4.4 Degradation of GO in the presence of H2O2 under irradiation by measuring TOC. ... 70

5.1 Merry-go-round photo-reactor ................................................................................... 78

5.2 HPLC analysis of Aqu/nC60 after zero, 5, and 15 days of light exposure. ................... 82

xii

Figure .............................................................................................................................Page

5.3 UV-Vis spectra of aqu/nC60 and irradiated C60 ........................................................... 84

5.4 pH changes of C60 irradiated and dark control samples. ............................................ 85

5.5 CO2 measurements as a function of time. .................................................................. 86

xiii

ABSTRACT

Zhao, Yingcan. Ph.D., Purdue University, August 2016. Reactions of Graphene Oxide and Bucminsterfullerene in the Aquatic Environment. Major Professor: Chad T. Jafvert. Due to unique physical and chemical properties, carbon-based nanomaterials,

including C60 and graphene oxide, now being used in an increasing number of

applications. Considering their widespread use, nanoparticles will inevitably find their

way to the natural environment. However, their environmental fate and transport have

not been intensively explored, resulting in a general lack of knowledge regarding their

risk assessment and life cycle exposure concentrations. To this end, this study has

investigated: (i) the photo mineralization of aqu/nC60 clusters under photo irradiation,

and (ii) environmental transformation of graphene oxide in the aquatic environment.

This study shows that CO2 was produced from aqu/nC60 when exposed to lamp light

within the solar spectrum (300-410 nm), suggesting mineralization was indeed occurring

to some extent. In addition, the ultraviolet-visible (UV-vis) spectrum and liquid

chromatographic separation of photo-irradiated samples indicated that decomposition

of C60 occurred. Aqueous graphene oxide suspensions produced reactive oxygen species

(ROS), including superoxide anion (O2·−) and hydrogen peroxide (H2O2) when exposed to

the same lamp light, under oxic conditions. The color of the GO suspension progressed

xiv

from pale to dark brown during the photoreaction process, consistent with changes in

the UV-vis spectrum. Raman spectra showed that the ratio of the ID/IG bands increased

as irradiation proceeded, suggesting an increased number of defects (e.g., functional

groups and vacancies) on the graphene oxide sheets. These defects may be the sites for

ROS production. In dark environments, GO was able to accept electrons from a well

know reducing agent and electron donor (NADH), oxidizing NADH to NAD+, and

transferring these electrons to molecular oxygen in water, producing the reactive

oxygen species (ROS) superoxide anion (O2·−) and hydrogen peroxide (H2O2). DNA

cleavage was observed in air-satured GO suspensions that contained NADH, suggesting

that ROS production could be a mechanism for DNA damage by GO within biological

cells. Indirect photochemical reactions involving GO were shown to occur in an

experiment in which hydroxyl radicals were generated by the photo-decay of hydrogen

peroxide. GO was oxidized by ·OH within a 48 hr irradiation period, as measured by

changes in the UV-Vis absorbance spectra over the wavelengths from 300 to 800 nm.

Spectral changes were consistent with visible color changes (i.e., fading) from 0 to 48

hours. Hence, both direct and indirect photochemical reactions might greatly affect the

lifetime and stability of GO in surface waters.

1

CHAPTER 1. INTRODUCTION

1.1 Scope and Significance

Due to the increasing use of carbon-based nano-materials (e.g. carbon nanotubes,

fullerenes such as C60, and graphene oxide (GO), it is anticipated that significant

quantities of these materials will find their way to the environment over the next few

decade, if used are not restricted to prevent their release. However, compared with the

vast number of studies to investigate new applications, studies on the environmental

fate and transport of many of these materials are limited. The overall objective of this

research was: (i) to investigate mineralization of C60 clusters in aqueous suspensions of

nC60 clusters under simulated solar irradiation; and (ii) to better understand the

photochemistry of GO in water and the mechanisms of light-independent reactions

involving GO in aquatic systems. The specific goals were fourfold: (1) To test whether

there is CO2 produced from Aqu/nC60 clusters under simulated sunlight; (2) to examine

photochemical transformation and reaction mechanisms of single-layered GO in

simulated sunlight; (3) to explore mechanisms of light-independent reaction of single-

layered GO in water by measuring ROS production in the presence of a common

biological reducing agent (NADH); and (4) to examine indirect photochemistry of GO.

Data on the physical and chemical changes to these two materials (C60 and GO),

2

including where appropriate, product (CO2) and byproduct (ROS) formation, could

provide useful information for overall environmental risk assessments for these

materials. First, by measuring the volume of CO2 in headspace of Aqu/nC60 cluster,

photo-mineralization was confirmed to occur. Second, reactive oxygen species (ROS)

were generated via GO under simulate solar irradiation. Third, a light-independent

reaction mechanism of GO was studied by measuring ROS generation in the presence of

NADH, a common biological reducing agent. Finally, how photochemically generated

hydroxyl radicals could affect aqueous dispersions of GO was examined.

1.2 Fullerenes

Buckminsterfullerene (C60) is an important nanomaterial that has drawn much

attention in recent years after it was first discovered by a research group at Rice

University in September, 1985 [1]. Due to its unique physical, chemical, and electrical

properties, C60 has been used in a wide range of applications, including biomedical,

cosmetic, and electronic applications [2-4]. Large scale production of carbon

nanomaterials, including fullerenes, is now done to meet industrial needs. For example,

Frontier Carbon Cooperation (FCC), produced nearly 1,500 tons of C60 in 2007, which is a

large expansion compared to the 300 ton produced in 2005 [2].

1.2.1 Physical and Chemical Properties of C60

Buckminsterfullerene (C60) is a cage-like molecule consisting of 60 carbon atoms

(C60) joining together with 12 pentagonal and 20 hexagonal aromatic rings. Because it is

3

not a nanoparticle, but rather a molecule, its solubility can be determined, and C60 has

an extremely low solubility in water [5, 6]. However, it is well recognized that aqueous

nano-clusters, or nC60, occur in water as stable aqueous colloidal suspensions. These

suspensions can be prepared by extended stirring, sonication in water, or through

solvent exchange using organic solvents [7-9].

Figure 1.1 C60 Buckminsterfullerene, image from ref [10]

1.2.2 C60 Toxicity

Several toxicity studies have shown that C60 can induce oxidative stress to the

brain of juvenile largemouth bass and may also be responsible for cytotoxicity, resulting

in DNA cleavage and cellular structure damage [11-13]. These results raise concerns

over these and other potential negative effects on human and animal health, thus

motivating additional research C60 toxicity and on its environmental fate and transport.

4

1.2.3 C60 Photo-activity

Many studies have reported on the reactivity and transport of C60 under natural

conditions to investigate its overall environmental fate. Photochemical transformation

is considered to be a potentially significant transformation path for C60 in environmental

systems. C60 is known to be strongly photoreactive in organic solvents. Hou and Jafvert

were the first to report that Aqu/nC60 underwent photo transformation under sunlight

conditions [14]. Results showed that nC60 undergoes structural changes and, through

reaction with dissolved molecular oxygen (O2) can generate reactive oxygen species

(ROS) [15, 16]. In nonpolar organic solvents (e.g. benzene), C60 is found to be an

efficient photosensitizer for 1O2 generation [17]. Ground-state C60 is firstly excited to

singlet C60 (1C60), and then 1C60 is fast converted into triplet C60 (3C60) through

intersystem crossing (ISC) (suggested mechanism shown in equation 1.1). Subsequently,

via energy transfer, 3C60 is quenched by O2, resulting in the formation of 1O2. Triplet C60

(3C60), as a form of C60 in the excited state, is able to convert ground state oxygen (3O2)

to singlet oxygen (1O2), followed by forming other ROS [14],

C60 hϑ→ 1C60

ISC→ 3C60

O2→ C60+ 1O2 eq 1.1

Lee et al. measured major photochemical transformation products of nC60 under UVC

(254 nm) light (not within the solar spectrum), which included mainly water-soluble

products with oxygen functional groups [18].

5

The rate of CO2 photo-production is an important indicator of photochemical

reactivity of organic materials under solar irradiation. Hou et al. [14] first suggested that

C60 may undergo some mineralization to CO2 by measuring the total organic carbon

(TOC) concentration under lamp light irradiation using lamps that emit light only within

the solar spectrum (8 UVA lamps that emit from 300 to 410 nm, centered at 350 nm).

The results showed a decrease in TOC from 65 mg/L to 11.3 mg/L after 65 days.

However, no previous study has shown mineralization to CO2, mainly because previous

experiments used concentration of Aqu/nC60 that were too low to properly detect

accumulation of CO2 above background, or conducted the experiments for only brief

periods of time [19, 20].

1.3 Graphene Oxide

Graphene oxide (GO) is a precursor material in graphene preparation and has a

large number of epoxy, hydroxyl, and carboxyl groups on its surface [21]. The structure

of graphene oxide is shown in Figure 1.2 (Figure 1.2 was adapted from C.E. Hamilton,

PhD Thesis, 2009, Rice University). The presence of these oxygenated functional groups

render GO extremely hydrophilic [22], making it an ideal material to use in aqueous

dispersions.

6

Figure 1.2 The structure of graphene oxide (adapted from C.E. Hamilton, PhD Thesis, 2009, Rice University)

1.3.1 Graphene Oxide Applications

Graphene and its functionalized derivatives, have been used in many fields

because of their unique physical, electronic, and optical properties [23-26]. As one

example, since 2012 the Defense Advanced Research Projects Agency (DARPA) has

focused on yielding low-cost infrared imaging devices based on graphene. As another

example, a number of research groups have found that GO or functionalized GO may

assist in cancer treatment. Sun et al. reported that nano-GO selectively killed cancer

cells in vitro and suggested that GO could be a promising new material for such medical

applications [26]. In addition, Yang et al. demonstrated that GO was an ideal photo-

thermal agent for photo-thermal therapy (PPT) treatment of cancer [27]. Therefore, GO

will for sure to be applied and produced in large amounts in next few decades.

7

1.3.2 Environmental Applications using Graphene Oxide-based Material

Graphene oxide has exceptional electron-transfer properties. Based on this fact,

GO has been considered as an ideal material to expand the light-response range of

other nanoparticles. For example, after reacting with BiOBr, GO changes the conduction

band and balance band of BiOBr to enhance its photocatalytic activity under visible light

irradiation [28]. Graphene oxide-TiO2 composites also have been intensively studied

and used for photo-degradation of pollutants, because GO can act as an electron shuttle

for TiO2 nanoparticles, improving the lifetime of the electron-hole pairs [29]. Moon et al.

found that TiO2-reduced GO has a high removal efficient for the photocatalytic oxidation

of As(III) [30].

1.3.3 Toxicity of Graphene Oxide

Akahaen et al. showed that hydrazine-reduced GO was more toxic to bacteria

than the parent GO, and suggested this is the result of the “sharper” nanowalls on the

reduced GO (RGO) [31]. For the cytotoxicity of GO to human cells, Liao et al. observed

that sonicated (smaller) GO exhibited higher hemolytic activity compared to larger GO

material. Viability assay experimental results revealed that both graphene and GO have

toxic effects to human skin fibroblasts [32]. Wang’s group reported that single-layer GO

had dose-dependent toxicity to human lung epithelial cells and fibroblasts and caused

obvious toxicity when doses were above 50 mg/L, suggesting the potential risk of GO to

human health [33]. Hu et al. found that GO could destroy cell membranes by direct

interactions between cell membranes and GO nanosheets [34]. In contrast, Chang’s

8

group used different sized GO and proposed that all the different sized materials

showed no toxicity to A549 cells, which are typical human lung cells [35]. The variability

among the results might be attributed to the various methods through which GO and

graphene can be made. However, there is no doubt that GO might cause some

toxicological effects to humans, motivating further study on the fate and effects of GO

in the natural environment.

1.3.4 Environmental Transformation of Graphene Oxide

Chemical transformation is a key factor affecting the characteristics of nano-

materials. Photo transformation is an important transformation process in the natural

environment. The π-bonds in GO absorb solar spectrum light, thus photochemical

transformation may be an important pathway in aqueous systems if light absorption is

followed by chemical reaction. Indeed, it is well known that photo-irradiation is a good

method for reducing GO [36, 37]. Matsumoto et al. used a 500 W Xenon lamp to study

the photoreaction of GO in water. Note Xenon lamps generally emit light below the

solar spectrum, down to about 280 nm. Matsumoto et al. observed that irradiation of

GO aqueous dispersions resulted in production of reduced GO (RGO), CO2 and H2 [38].

In addition, more defects were produced. These defects (i.e., non-aromatic carbon)

provided “sites” for either H2 or CO2 formation. Hou et al. recently reported that

intermediate photoproducts are of smaller size with fewer oxygen functionalities, which

obviously then would have different photo-reactivity compared to the unreacted GO

[39] (mechanism can be summarized as eq 1.2).

9

GO hϑ→ CO2 + rGO species + low MW PAH species (O2 dependent process) eq 1.2

Here, rGO: reduced graphene oxide; MW: molecular weight;

PAH: Polycyclic aromatic hydrocarbons)

Chowdhury et al. showed that direct photo-irradiation had an effect on GO aggregation

under both oxic and anoxic conditions; however, they noted negligible effects occurred

with respect to the overall surface of the material [40].

Chowdhury et al. also showed that pH, ion composition (including counter-ion valence),

and NOM concentration played significant roles in the overall stability of reduced

graphene oxide [41]. Under sunlight, nitrate and NOM in the natural aqueous

environment are known to generate hydroxyl radicals, which could further react with

graphene materials.

1.3.5 High Electron Transfer Ability of Graphene Oxide

Due to its high electron transfer ability, GO has been used in a variety of electrochemical

applications [42, 43]. Previous studies indicate that GO could enhance the

electrochemical reactivity of biomolecules, and has a remarkable electron transfer rate

compared to many other materials [44]. Based on its open surface and extensive length

of edges compared to other carbon-based nanomaterials (i.e., carbon nanotubes), GO

might be able to provide more active sites for electron transfer to biological chemical

10

species, promoting the reactivity of GO toward oxidation and or reduction of small

biomolecules such as NADH [45].

1.4 Chapter Outline

The goal of this dissertation is to investigate environmental reactions of two types

of carbon nano-materials, C60 and graphene oxide, under relevant environmental

conditions. With this goal in mind, studies were conducted to further elucidate whether

photo mineralization of aqu/nC60 clusters occurrs, to determine whether reactive

oxygen species are generated on the surface of GO under simulated sunlight, to explore

mechanisms of light-independent reactions of GO in water, and to determine how GO

behaves in the presence of hydroxyl radicals that are photochemically generated under

environmentally simulated light conditions.

In Chapter 2, the photochemical transformation of single-layered GO under lamps

that only emit light in the solar spectrum (λ =350 ± 50 nm) was investigated. Reduced

graphene oxide (RGO) was produced, evidenced by Raman Spectra measurements.

Color of GO suspension transitioned from pale to dark brown during the photoreaction

process, consistent with changes in measured light absorbance (i.e., attenuation)

spectra. Additionally, reactive oxygen species (ROS) including superoxide radical anion

(O2·−) and hydrogen peroxide (H2O2) were produced under oxic conditions.

Understanding the photochemical reactivity of GO in aqueous solutions will provide

information on evaluating the environmental impacts and cytotoxicity of this

nanomaterial.

11

In Chapter 3, mechanisms of light-independent reactions of graphene oxide with

β-nicotinamide adenine dinucleotide (NADH) in water is introduced. Reactive oxygen

species (ROS) were produced by electron transfer from the electron donor (NADH) to

GO, which shuttled these electrons to O2 in water. The addition of GO was found to

oxidize NADH to NAD+, evidenced by the decreased intensity of the 340 nm absorption

band of NADH and the increased intensity of the 260 nm absorption band of NAD+.

Tetrazolium salts (NBT2+ and XTT) were used as scavengers to indicate the production of

superoxide anion radicals. H2O2 was produced in the process. In the experimental

systems containing GO, NADH and O2, pBR322 DNA plasmid was cleaved, in spite of

finding no other evidence of ·OH formation. Similar processes involving GO in biological

cells may be the mechanism through which GO causes oxidative stress and cytotoxicity.

In Chapter 4, the indirect photochemical oxidation of GO was followed by

generating hydroxyl radical from hydrogen peroxide (H2O2) under solar wavelength

light. Light attenuation spectra in the UV and visible regions and visible color changes

suggested decomposition was occurring in the process. Total organic carbon (TOC) was

also found to decrease, which suggested mineralization of GO. This study indicates that

photochemical transformation is likely a significant fate pathway for

GO in the aquatic environment.

In Chapter 5, photo-mineralization of aqu/nC60 clusters is examined under

simulated sunlight. The volume of CO2 in the headspace increased for irradiation

samples, while the dark control samples showed no such increase. Photoproducts were

characterized also by using UV-Vis spectroscopy and HPLC.

12

CHAPTER 2. ENVIRONMENTAL PHOTOCHEMISTRY OF SINGLE LAYERED GRAPHENE OXIDE IN WATER

Reproduced from:

Yingcan Zhao, Chad T. Jafvert. Environmental Photochemistry of Single-Layered

Graphene Oxide in Water. Environmental Science: Nano 2 (2015) 136-142

with permission from Royal Society of Chemistry, Copyright 2015

2.1 Abstract

Graphene oxide (GO), as a precursor to produce graphene, is now in a variety of

potential applications; however, its environmental fate in natural systems remains

largely unknown. It can be easily suspended in water because of many hydrophilic

functional groups in the structure. Hence, photochemical transformation may be an

important pathway in aqueous systems, as it strongly absorbs sunlight within the solar

spectrum. In this study, the photochemical transformation of single-layered GO under

lamps that only emit light in the solar spectrum (λ =350 ± 50 nm) was investigated.

Holey reduced graphene oxide (RGO) was produced, evidenced by Raman Spectra

measurements. Color of GO suspension got from pale to dark brown during the

photoreaction process, which was concurrent with the results from UV-visible

13

absorption spectra. Additionally, reactive oxygen species (ROS) including superoxide

radical anion (O2·−) and hydrogen peroxide (H2O2) were produced under oxic conditions.

Understanding the photochemical reactivity of GO in aqueous solution will provide

information on evaluating environmental impact and cytotoxicity of this nanomaterial.

Keywords: graphene oxide, reactive oxygen species, photochemistry, ROS,

environmental fate.

2.2 Introduction

Many efforts are underway to discovery new ways to use graphene materials and

their derivative in electronic devices, for drug delivery, in biosensors, in solar energy

conversion, as catalysts, and in many other types of applications [23-26, 46-48]. Due to

the variety of industrial sectors in which manufactured graphene materials may find

application and due to expected future production rates, release and exposure to

graphene-based materials in natural and built environments may be anticipated, raising

concerns over their potential negative effects on human and ecosystem health if

precautions are not taken to control exposure. Unfortunately, knowledge regarding the

fate, transport, and toxicity of graphene and its derivatives, currently is limited,

especially with respect to aquatic environments. Graphene oxide (GO) is a precursor

material in the preparation of graphene, and despite its name, it has on its surface

several different types of functional groups, including epoxy, hydroxyl, and carboxyl

groups [21]. The presence of these functional groups on GO makes it hydrophilic and

easy to disperse in water [22].

14

Because it is easy to disperse in water, its toxicity to human cells has been

studied both as a potential aquatic pollutant, and as a potential material to selectively

kill cancer cells [26, 27]. For example, Liao et al. observed that sonicated (smaller) GO

exhibited greater hemolytic activity to human cells compared to larger GO materials.

Viability assay experiments revealed that both graphene and GO have toxic effects to

human skin fibroblasts [32]. Another research group reported that single-layer GO had

dose-dependent toxicity to human lung epithelial cells and fibroblasts and caused

obvious toxicity when doses were above 50 mg/L, indicating risk to human health [33].

Hu et al. found that GO could destroy cell membranes by direct interactions occurring

between cell membranes and GO nanosheets [34]. In contrast, another research group

used different sized GO and found that all the different sized materials showed no

toxicity to A549 cells, which are typical human lung cells [35]. The variability among the

results might be attributed to the various methods by which GO and graphene were

produced or suspended in water. However, there is little doubt that GO might cause

some toxicological effects to humans, motivating further study on the fate and effects of

GO in the natural environment. Environmental effects may include toxicity to micro-

organisms and other organisms up the food chain. Indeed, Akahaen et al. showed that

hydrazine-reduced GO was more toxic to bacteria than the parent GO, and suggested

this resulted from “sharper” nanowalls on the reduced GO (RGO) [31].

Because the disrupted π-bond structure in GO absorbs significant light within the

solar spectrum, environmental fate processes acting on GO are expected to include

photochemical processes. Indeed, it is well known that photo-irradiation is a good

15

method for “reducing” GO, at least with lamp light that occurs in UV regions that may or

may not occur solely within the solar spectrum. For example, in a study reporting on

photo-reduction of GO conducted by Matsumoto et al. [38], experiments were

performed used a Xenon lamp of unknown spectral output, although this type of lamp

generally emits light down to 280 nm, approximately 20 nm below the spectral limit of

solar light measured at earth’s surface. Matsumoto et al., however did measure CO2

and H2 generation from aqueous GO suspensions and noted a drastic decrease in H2

production if the Xenon lamp light was filtered through a 390 nm cutoff filter. While not

reported, it seems likely that a similar decrease in CO2 production would occur under a

390 nm cutoff filter. It is somewhat interesting that while the overall reaction can be

termed photo-reduction of GO, it is not clear if any of the remaining carbon on the GO

has actually been reduced, as simple loss of CO2 from carboxyl groups on GO is the

result of a rearrangement reaction, where the carbon-carboxylic acid bond is broken

and replaced with a carbon-hydrogen bond. Hence, although the average oxidation

state of the carbon in the GO is lower, it may result from loss of carbon that was already

highly oxidized, as previously reported to occur during photolysis of carboxylated multi-

walled carbon nanotubes [49], and not from any oxidation of specific carbon atoms in

the GO. Similar to information in the literature regarding photochemical carbonaceous

products, there is a general lack of information on the generation of reactive oxygen

species (ROS) by GO under solar light. Hou et al. irradiated an aqueous dispersion of

single layer GO with light not strictly within the solar spectrum (at energies in the range

3.94-4.43 eV; i.e., at λ = 280-315 nm), and similar to Matsumoto et al. [38], noted that

16

the GO became visibly darker over the irradiation period, but suggested through XPS

analysis that this occurred due to loss of hydroxyl groups through hemolytic removal of

·OH groups and formation of more conjugated π-bonds, rather than through

decarboxylation alone. Further, through electron paramagnetic resonance spectroscopy,

dose-dependent exponential growth in radical production on the GO surface was shown

to occur, presumably after loss of ·OH; however, it was reported that ·OH were not

observed, and a methodology of ·OH detection was not reported.

For other carbon-based nanomaterials, several reactive oxygen species have been

shown to be generated under sunlight conditions (λ > 300 nm). For example, singlet

oxygen (1O2) was generated in aqueous suspensions of C60 clusters (i.e., nano-

precipitates), oxidizing the C60 to more polar water soluble products [14-16]. Aqueous

dispersions of carboxylated and PEGylated single walled carbon nanotubes (SWCNTs)

produced significant ROS, including singlet oxygen (1O2), superoxide anion (O2·−), and

hydroxyl radial (·OH) [50, 51]. As a result, in this study the ability of aqueous dispersion

of single-layered GO to generate reactive oxygen species (ROS), including O2·− and H2O2

upon exposure to light within solar spectrum (λ =300-410 nm) was investigated. Based

on experimental results of this study and the previous work cited above, a mechanism

for ROS generation by photosensitized GO in water is proposed. Information of ROS

generation during solar irradiation is significant not only to evaluate ecological risks

associated with GO, but also to better understand the transformation pathways of

carbon within GO.

17

2.3 Materials and Methods

2.3.1 Materials

An aqueous dispersion of single layer graphene oxide (GO), synthesized by a

modified Hummer’s method, was purchased from Advanced Chemical Supplier (ACS)

Material, LLC (Medford, MA) and used as received. According to the manufacturer, the

material is approximately 80% single-layer GO (with the remainder being multi-layered)

and in the size range of 0.5 to 2.0 µm, with a thickness of 0.6 to 1.2 nm. More

information on the GO material is provided in the Supporting Information (SI). Furfuryl

alcohol (FFA), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

(XTT), p-chlorobenzoic acid (pCBA), N,N-diethyl-p-phenylenediamine hemioxalate (DPD),

horseradish peroxidase (HRP), and superoxide dismutase (SOD) were purchased from

Sigma-Aldrich (St. Louis, MO). All other chemicals were with the highest purity and used

as received. All aqueous samples were prepared with water purified with a Barnstead

Nanopure system (Dubuque, IA) after R/O pretreatment.

2.3.2 Preparation of Aqueous Graphene Oxide

The stock GO dispersion (10 mg/mL) was diluted with water (1:100), and

sonicated under low energy (8890R-MT ultrasonic bath from Cole-Parmer, Vernon Hills,

IL) for 2 hours to make a uniform dispersion of 100 mg/L GO. When not in use, the GO

suspensions were stored in the dark at room temperature. In most experiments,

samples were buffered to pH 7 with final phosphate buffer concentrations of 5 mM.

Buffers were prepared with phosphate salts (i.e., KH2PO4 and K2HPO4).

18

2.3.3 Irradiation and GO Analysis

For all irradiation experiments, samples were placed in a series of borosilicate

glass tubes sealed with PTFE-lined caps and exposed to sixteen 24 W black-light

phosphor lamps (RPR-3500Å lamps from Southern New England Ultraviolet, Branford,

CT) that emit light from 300 to 410 nm, with the maximum intensity occurring near 350

nm. A figure regarding to spectral density of irradiance as a function of wavelength was

provided (SI Figure 2.7), thus comparing the solar sunlight with our employed lamp light

source. All samples were irradiated in a Rayonet merry-go-round photochemical reactor

that rotated the samples at 5 rpm to ensure uniform light exposure of all samples over

the irradiation period. Dark control samples were prepared at the same time as

irradiated samples and were wrapped with aluminum foil and kept in a dark

environment over the same time period. All experiments were performed in duplicate

or triplicate. At specific times during the irradiation period, irradiated samples and dark

control samples were recovered for analysis. The UV-visible light absorbance spectra

were recorded with a Varian Cary 50 UV/Vis Spectrophotometer using quartz cuvettes

with 1 cm path lengths. The Raman spectra of samples were monitored by using an

excitation wavelength of 633 nm, and scanning from 1000 -3000 cm-1 to obtain

information on the characteristic D, G and 2D band intensities of GO. For all Raman

spectra measurements, irradiated and dark control samples were prepared and

analyzed in the absence of the phosphate buffers.

19

2.3.4 ROS Measurement

Reactive oxygen species (ROS) were detected by using specific chemical probes

that selectively react with each ROS at near diffusion-limited rates. Only one probe was

used at a time, as those for singlet oxygen (1O2 ) superoxide anion (O2·−), and hydroxyl

radical (·OH) require that they be added before the irradiation period, as the measured

response is due to accumulative ROS production; whereas analysis of hydrogen peroxide

(H2O2) was performed on samples by adding the necessary reagents after irradiation.

1O2 was monitored by the loss of FFA [52]. FFA was chosen to detect whether 1O2 was

produced. The FFA concentration at specific irradiation time was determined by

separation using HPLC (Varian 9012 gradient pump, 9050 UV/Vis detector and Prostar

autosampler) on a C18 column with the detection wavelength set at 219 nm. The mobile

phase was consisted of 50% methanol and 50% water at a flow rate of 1 mL/min. The

run time was 6 min and the injection volume was 100 μL. Previously, we used a nitro

blue tetrazolium salt (referred to as NBT2+) as a scavenger for O2·−, produced upon solar

light irradiation of functionalized carbon nanotubes (CNTs) [50]. However, in the

presence of NBT2+, GO was found to flocculate and settle; hence experiments using XTT

as a scavenging probe for O2·− were attempted with success. The reaction between XTT

and O2·− leads to a soluble product that was detected spectrophotometrically at 470 nm

after filtering samples through a 0.2 μm membrane filters to remove the GO which also

absorbances significant light at 470 nm. To confirm that transformation of XTT was

through reaction with O2·−, in some GO + XTT samples, superoxide dismutase (SOD) was

added, as it significantly decreases the steady-state concentration of O2·− by

20

enzymatically catalyzing its disproportionation to hydrogen peroxide (H2O2), confirming

any transformation of XTT was due to O2·−. p-Chlorobenzoic acid (pCBA) was added to

some samples to determine if ·OH was generated during irradiation.[50] pCBA reacts

rapidly ·OH resulting in loss of pCBA from the system. To detect the highly reactive

hydroxyl radical, pCBA was used as a scavenger. The concentration of pCBA was

determined by HPLC analysis using a C18 column and a UV detector set at 234 nm. The

mobile phase was 70% methanol and 30% water, with the water containing 20 mM

H3PO4, at a flow rate of 0.8 mL/min. The run time was 10 min and the injection volume

was 100 μL. All samples were filtered through 0.2-μm filter membranes before HPLC

analysis. H2O2 temporal concentrations were measured by horseradish peroxidase (HRP)

catalyzed oxidation of DPD, with the HRP and DPD added to samples after irradiation or

incubation in the dark.[53] Before analyzing samples for H2O2, irradiated and dark

control samples were filtered through 0.2-μm membrane filters to remove the GO,

which otherwise would interfere with DPD spectrophotometric detection. After

filtration, 1 mL of each sample was added to 1 mL phosphate buffer (pH=6), followed

sequentially followed by additions of 50 μL 30 mM DPD solution and 50 μL 30 U/mL

HRP. H2O2 concentrations were determined by comparing sample absorbances at 551

nm to that of know standards (i.e., from a standard curve).

2.4 Results and Discussion

Upon irradiation of an aqueous dispersion of single layer GO with light in the solar

spectrum, the GO dispersion become visibly darker as shown in Figure 2.1a. This is

consistent to previous observations noted in the introduction section that reported on

21

exposure of GO to shorter wavelength UV light [38, 54]. Similarly, light attenuation

(Figure 2.1b) of the GO suspension increased across the whole spectrum from 220 to

820 nm. Note that although the figure reports “absorbance”, the response is due to

both light absorbance and scatter because the samples are nanoparticle suspensions

that not only absorb light within this wavelength range, but also scatter the light, with

presumably much of the light attenuation at the higher wavelengths occurring due to

light scattering. The GO spectra before irradiation does exhibit two characteristic peaks:

A maximum at approximately 230 nm, which corresponds to π → π* transitions of the

aromatic C–C bonds, and a shoulder at 303 nm, which is due to n → π* transitions of C

O bonds [55]. Over the course of a 2 hr irradiation period, the absorption peak at

230 nm was gradually red-shifted to approximate 255 nm and the shoulder at 303 nm

disappeared, potentially indicating that some of the electronic conjugation within the

graphene sheets was restored as previously suggest upon reduction of GO with light at

shorter wavelengths [54, 56].

22

Figure 2.1 (a) Photograph of 5 mg/L GO at pH 7, before (left vial) and after (right vial) irradiation for 2 hours under lamp light irradiation; and (b) the change in the UV-visible

light absorption spectra of GO suspensions with increasing time of irradiation.

23

Raman spectroscopy is often used to probe differences in the electronic properties

of carbon nanomaterials [57]. Figure 2.2 shows the Raman spectral changes in GO as a

function of irradiation time. The spectra shows the D, G, and 2D bands, which are three

characteristic peaks of GO. The G band occurs at 1580 cm-1 and results from the

vibration of sp2-bonded carbon, and is an indication of the relative extent of aromaticity.

The D band at 1350 cm-1 is assigned to the vibration of sp3 carbon atoms (i.e.,

nonaromatic carbon). The relative intensities, or ratio of the D to G band intensities

(ID/IG) is often used as a qualitative measure for the degree of disorder caused by

nonaromatic sp3 carbon defects, that often occur at edges or as ripples or holes within

the GO structure [58]. Figure 2.2 shows that after 24 hrs of irradiation, the ID/IG ratio

increased from 0.451 (0 hr) to 0.678 (24 hr). This increase suggests an increase in the

number of defects (e.g., functionalized carbon) on the already functionalized graphene

oxide sheets. These defects may be sites for ROS production, which is discussed

subsequently.

24

Figure 2.2 The Raman spectra of GO before and after irradiating an aqueous GO suspension (100 mg/L) for 24 hours, where the spectra have been normalized to the

intensity of the G band.

By monitoring ROS production with the selective and highly reactive chemical

probes, formation of O2·−, but not 1O2 or ·OH, was detected. Irradiated samples

containing furfuryl alcohol as a scavenging probe for 1O2, showed no decay in furfuryl

alcohol after 24 hrs of irradiation (SI Figure 2.8). However, in GO suspensions

containing XTT, a significance increase in light absorbance at 470 nm occurred over time

upon irradiation and after filtering out the GO after irradiation. This increase in

absorbance occurs where the reaction product between XTT and O2·− as an absorbance

maximum [50] (Figure 2.3). Figure 2.3a also indicates that the addition of SOD almost

completely inhibited XTT reduction, further suggesting that XTT product formation was

25

caused directly by reaction of XTT with O2·− as SOD rapidly converts O2

·− to H2O2 through

a disproportionation reaction, reducing the amount of XTT product formed. In the

absence of XTT, irradiated and dark control GO samples showed little change in

absorbance at 470 nm over the same time period. Note that because the light

absorption spectral changes shown in Figure 3B (and reported at 470 nm in Figure 3A)

are on samples filtered through 0.2 m membrane filters (i.e, after removal of GO), the

increase in absorbance at 470 was due only to XTT product formation and not due to

the increase in light absorbance caused by the GO, as shown in Figure 2.1 on unfiltered

samples. An image showing the course of the reaction from 0 to 3 hours for a 5 mg/L

GO suspension containing 0.1 mM XTT, prior to filtration, is provided in the SI (Figure

2.9). Even with the increase in overall absorbance caused by the GO, the pink product

of reaction between O2·− and XTT is evident.

26

Figure 2.3 (a) Evidence of superoxide anion formation by reaction with XTT at 470 nm, upon lamp light irradiation of a 5 mg/L GO suspension at pH 7. (b) The change in the UV-visible light absorption spectra for a suspension of GO (5 mg/L) and XTT (0.1 mM).

Note: The symbols represent samples containing XTT (0.1 mM) alone (); GO (5 mg/L) alone (); GO (5 mg/L) and XTT (0.1 mM) (); GO (5 mg/L), XTT (0.1 mM) and SOD (40 U/mL) (); and the corresponding dark control samples of GO (5 mg/L) and XTT (0.1 mM) ().

27

With significant O2·− formation, there is a high probability that hydrogen peroxide

will form also, and potentially accumulate in solution. As noted above, H2O2 can be

formed by the disproportionation of O2·− with enzymes such as SOD accelerating the rate

of the reaction considerably. As the disproportionation reaction suggests, conversion

can occur also through transfer of another electron to the protonated form of

superoxide anion, HO2 · (hydroperoxyl radical), which upon the electron transfer,

extracts a proton from solution to form H2O2. Whether it occurs by disproportionation

or another electron transfer process, both electrons must originate from the GO in the

absence of an additional electron donor. Hence, accumulation of H2O2 was measured

by using the DPD-horseradish peroxidase (HRP) assay, with the DPD/HRP added to the

aqueous samples after irradiating the GO dispersions, and then after removing the GO

by filtration. Although H2O2 is somewhat reactive in this system, it has a much longer

half-live that the other ROS, even under solar light irradiation. Figure 2.4a clearly shows

that the H2O2 concentration within the GO dispersion increases over an irradiation

period of 4 hrs, whereas no increase occurred in dark control samples or in the absence

of GO. The H2O2 concentrations shown on Figure 2.4a were calculated from sample

absorbance valued measured at 551 nm after filtration, using the H2O2 standard curve

shown on Figure 2.4b. Hence, the initial values at time zero of 0.2-0.5 M are due to

absorbance reading at or below 0.005, and likely due to trace contamination resulting in

a small positive interference. Despite this, the results indicate that H2O2 was indeed

produced and accumulated during irradiation of 5 mg/L GO with light in the solar

28

spectrum, accumulating to over 3 M after an irradiation period of 4 hrs. Assuming a

carbon content of 80% in the original GO, the 5 mg/L GO concentration translates to a

molar concentration of 3 mM carbon. Hence, over the 4 hr irradiation period,

approximately 1 molecule of H2O2 was produced for every 1,000 carbon atoms in the

GO.

29

Figure 2.4 (a) Evidence for the increase in H2O2 concentration over time upon lamp light irradiation of GO at pH 7. (b) The standard curve of H2O2 using DPD/HRP method.

Note: Figure 2.4a represents evidence of H2O2 by reacting the accumulated H2O2 with DPD through the HRP catalyzed reaction, producing the red colored DPD Würster dye product that absorbs light at 551 nm. All samples contain DPD and HRP, added after irradiating samples which contained 5 mg/L GO (), or pure water (), or after incubating samples in the dark which contained 5 mg/L GO (); and with the DPD and HRP added after filtering out the GO through 0.2 μm membrane filters.

a

b

30

Although it is known that sunlight can cleave the oxygen-oxygen bond in H2O2 to form

·OH, the reaction is slow [51, 59]. Alternatively, ·OH may be formed more rapidly if

transfer of an addition electron from GO to H2O2 occurs, as was found to be the case for

carboxylated single walled carbon nanotubes under solar irradiation.[51] In order to

determine whether there is hydroxyl radical produced by GO, pCBA was added to some

samples, as it rapidly scavenges ·OH resulting in loss of pCBA. However, no pCBA decay

was observed for both irradiated and dark control samples over the 4 hour time period

of the experiments (SI Figure 10), suggesting that negligible ·OH was produced, or that

·OH scavenging by the GO was rapid and significant, reducing its pseudo-steady-state

concentration. Although scavenging of ·OH by GO is likely to occur, as the initial

electron transfer that results in its formation would occur at the GO surface such that

the site of its generation would be in close proximity to GO π bonds at which it could be

consumed, it is also likely that not much is produced, otherwise the concentration of its

precursor, H2O2 would not accumulate to such a degree, and the chromophore content

of the GO would not because enhanced as irradiation proceeds.

2.5 Conclusion

In summary, when exposed to light within the solar spectrum, aqueous

dispersions of single layered GO do become darker, indicating an increase in

chromophore content, or at least an increasing absorptivity by the existing

chromophores within GO, yet Raman spectroscopy indicates an increase in nonaromatic

defects. Clearly, upon exposure to light, electron transfer occurs from GO to O2,

forming O2·− and significant quantities of H2O2 as depicted in Figure 2.5, and because

31

these are both reduction reactions, this must result in an overall oxidation of GO carbon.

Although it is likely that some of the generated ROS reacts directly with the GO surface,

this clearly does not occur stoichiometrically, as evidenced by the buildup in the H2O2

concentration over a time period of several hours. These results suggest that future

studies should examine whether these electron transfer reactions are responsible for

some of the toxicological effects observed for GO. In a recent study[60] we report that

electrons from common biological reducing agents (i.e., NADH) can be shuttled through

single-walled carbon nanotubes to molecular oxygen in the dark, resulting in ROS

production and DNA cleavage. It is likely that a similar mechanism resulting in oxidative

stress may occur also in the case of GO, but this hypothesis is yet to be tested.

Figure 2.5 Proposed pathway for ROS production by photosensitization of GO in water.

32

2.6 Supporting Information

Figure 2.6 Images of GO (Provided by ACS Material LLC.).

33

Figure 2.7 Spectral density of solar and employed lamp light irradiance as a function of

time (solar irradiance data was from ASTM G173-03 Reference Spectra).

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

250 300 350 400 450 500 550 600 650 700

Sp

ectr

ual

den

sit

y o

f Ir

rad

ian

ce

(mW

/cm

2/n

m)

Wavelength (nm)

Spectrum of LampLight Irradiation

Spectrum of SolarIrradiation

34

Measurement of 1O2

FFA was used to detect if 1O2 was produced upon irradiation of GO. The FFA

concentration at each specific irradiation time was determined by HPLC analysis (Varian

9012 gradient pump, 9050 UV/Vis detector and Prostar autosampler) using a C18

column, and by detecting and quantifying FFA with the UV/Vis detector at a wavelength

of 219 nm. The mobile phase was 50% methanol and 50% water at a flow rate of 1

mL/min. The run time was 6 min and the injection volume was 100 μL.

Figure 2.8 Data points are the concentrations of FFA (initially at 0.2 mM) detected in suspensions of 5 mg/L GO at pH 7 after irradiation ( ) or incubation in the dark ( ). All samples were analyzed by HPLC after removing the GO with a 0.2-μm membrane filter.

35

No FFA loss was observed as irradiation time increased, indicating negligible

production of singlet oxygen. There are two possible reasons for this observation: Either

little 1O2 was generated, or after production it may have been rapidly quenched by the

GO. Both explanations lead to no net production of 1O2.

Detection of 𝐎𝟐·− by reduction of XTT

Figure 2.9 Photograph of 5 mg/L GO in water with XTT (0.1 mM) at pH=7 at irradiation times of 0, 0.5, 1, 1.5, 2, 2.5, and 3 hr (left to right), before filtering out the GO particles.

Note: Hence, the increase in color is due both to the increase in light absorbance of the GO and of the XTT product.

Measurement of ·OH

To detect the highly reactive hydroxyl radical, pCBA was used as a scavenger. The

concentration of pCBA was determined by HPLC analysis using a C18 column and a UV

detector set at 234 nm. The mobile phase was 70% methanol and 30% water containing

with the water containing 20 mM H3PO4, at a flow rate of 0.8 mL/min. The Run time was

10 min and the injection volume was 100 μL. All samples were filtered through 0.2-μm

filter membranes before HPLC analysis.

36

Figure 2.10. Data points are the concentrations of pCBA (initially at 5 μM) detected in suspensions of 5 mg/L GO at pH 7after irradiation (), or incubation in the dark ().

Note: All samples were analyzed by HPLC after removing the GO with a 0.2-μm membrane filter.

37

CHAPTER 3. LIGHT-INDEPENDENT REDOX REACTIONS OF GRAPHENE OXIDE IN WATER: ELECTRON TRANSFER FROM NADH TO MOLECULAR OXYGEN PRODUCING REACTIVE

OXYGENM SPECIES (ROS)

3.1 Abstract

There is growing interest in finding industrial and commercial uses for graphene oxide

(GO) because of this material’s unique properties. As with other micro-pollutants and

nanomaterials, increased production and use generally leads to increased

environmental exposure. However, very limited information exists on how GO is further

functionalized or mineralized in natural aquatic environments, or whether GO can

participate in reactions involving other natural or manmade chemicals. Because

generation of reactive oxygen species (ROS) by carbon-based nanomaterials previously

has been shown to occur, the hypothesis of this study was that ROS can be generated by

electron transfer from a common biological electron donor (NADH) to GO, which then

acts as an electron shuttle forming ROS from dissolved molecular oxygen (O2) in water.

Indeed, aqueous suspensions of 5 mg/L GO were found to oxidize 0.2 mM β-

nicotinamide adenine dinucleotide (NADH), evidenced by a decrease in absorbed light at

340 nm where NADH has an absorbance maxima, and by the increased absorbance at

260 nm where NAD+ has an absorbance maxima. Using tetrazolium salts (NBT2+ andXTT)

to scavenge superoxide anion radicals, production of O2∙− was shown to occur over a

38

period of about 30 hrs, and by using another colorimetric assay, H2O2 was shown to

accumulate. Hence, in the absence of light, GO can act as an electron shuttle, catalyzing

the oxidation of NADH (i.e., transfer of electrons to GO) and then pass these electrons

to molecular oxygen (O2), forming ROS in water. Although hydroxyl radicals (·OH) were

not detected, pBR322 DNA plasmid was cleaved in aqueous suspensions of GO

containing NADH and O2.

3.2 Introduction

Graphene has been intensely studied since it was first isolated in 2004 [61].

Graphene oxide (GO) is a graphene derivative, having a large number of oxygen atoms

covalently bound to its surface in the form of epoxy, hydroxyl, and carboxyl functional

groups. Because it is hydrophilic, GO is easy to disperse in water, and as a result of this

and other unique properties, GO has been studied for potential use in a number of

commercial applications, including as a drug delivery agent [62], in biosensor

development [23] and as a catalyst [63]. As a result, production rates and uses of GO

are expected to grow in the future. As with many other new materials and chemicals,

high production rates often lead to accidental or other unintended releases to the

environmental, yet little is known about the environmental fate and effects of GO in the

natural environment. To manage potential risks associated with this nanomaterial to

both humans and the environment, a much better understanding of the reactivity and

fate of GO in the environmental is needed.

39

The number of previous studies on environmental fate and health related effects of GO

are limited. In one study, GO was shown to exhibit dose-dependent toxicity by inducing

cell apoptosis and lung granuloma formation to human fibroblast cells and mice [64].

Dose-dependent hemolytic activity to GO also was shown by human red blood cells [32].

GO particle size has been inferred to be a major factor in hemolysis activity, as sonicated

(smaller) GO nanoparticles exhibited higher hemolytic activity than untreated (larger)

GO. Contrary to these studies, Chang et al. found that GO had no obvious toxicity to

A549 cells (a human lung carcinoma epithelial cell line), regardless of the size or dose of

GO [35]. Clearly, more information regarding the toxicity of GO is needed, with the

need for these new studies carefully documenting material characterization, including

the method of GO production, particle size, and level of oxidation.

Researchers generally agreed that production of reactive oxygen species (ROS)

upon exposure to carbon-based nanomaterials is a potential cause of toxicity. Indeed,

our recent studies revealed that carboxylated single-walled carbon nanotubes (C-CNT)

can shuttle electrons from common cellular reducing agents to molecular oxygen

produce ROS, including superoxide anion (O2∙−) and hydrogen peroxide (H2O2), and also

can result in DNA cleavage [60]. GO, similar to C-CNT, has a large proportion of defects

(i.e., partially oxidized carbon) which may somehow facilitate electron transfer. Zhang

et al. showed that electrochemically modified GO coated on a pre-anodized screen-

printed carbon electrode (SPCE) greatly enhanced electrocatalytic oxidation of NADH

compared to uncoated pre-anodized SPCEs. He proposed this as a way of quantifying

NADH in aqueous solution [65]. Under sunlight conditions, we have shown previously

40

that GO can act as an electron donor to form ROS from O2 [66], with these results

consistent to our other studies that have shown generation of ROS from functionalized

carbon nanotubes under solar light [50, 51]. However, whether ROS can be produced

through the reaction of molecular oxygen on the surface of GO in water without an

applied voltage or without photosensitization (i.e., in the dark, through chemical

reduction) has not been reported.

In this paper, we report on electron transfer from a well know biological

chemical reducing agent to GO, which in turn shuttles the electrons to molecular

oxygen, in the absence of light or an applied voltage. As in previous studies using C-

CNTs [60], β-nicotinamide adenine dinucleotide (NADH) was used as the reducing agent

because of its importance in living systems and because its oxidation to NAD+ is easy to

follow spectrophotometrically. A concentration of 0.2 mM NADH was employed in

experiments because it is similar in the concentration found in cells [51]. And finally,

because production of some reactive oxygen species was found to occur in these

systems, the ability of these ROS products to damage DNA was accessed in a simple

assay using pBR322, a double-stranded circular DNA plasmid containing 4,361 base

pairs.

3.3 Materials and Method

3.3.1 Materials

A water dispersion of graphene oxide, synthesized by a modified Hummer’s

method, was purchased from Advanced Chemical Supplier (ACS) Material, LLC (Medford,

41

MA) and used as received. According to the information provided by the manufacturer,

the single-layer ratio of GO was above 80%, the flake size was 0.5 to 2.0 µm, and the

thickness of the flakes was 0.6 to 1.2 nm. β-Nicotinamide adenine dinucleotide (NADH)

was obtained from EMD Chemical, Inc. (Gibbstown, NJ). Nitro blue tetrazolium salt

(NBT2+), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT),

N,N-diethyl-p-phenylenediamine hemioxalate (DPD), horseradish peroxidase (HRP),

superoxide dismutase (SOD), catalase, and p-chlorobenzoic acid (pCBA) were purchased

from Sigma-Aldrich (St. Louis, MO). A 0.5 µg/µL solution of pBR322 DNA plasmid (4361

base pairs) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA). UltraPureTM

agarose and SYBR Safe DNA gel stain were purchased from Life Technologies

Corporation (now part of Thermo-Fisher). Gel loading dye (6x) was obtained from New

England Biolabs, Inc.

3.3.2 Oxidation of NADH

The ability of GO to oxidize NADH to NAD+ was determined by monitoring over time the

UV-visible light absorbance spectra of a suspension of GO containing NADH. Data were

recorded with a Varian Cary-300 UV-Vis spectrophotometer with a 1 cm path length

quartz cuvette at room temperature.

3.3.3 ROS Detection

Nitro blue tetrazolium salt (NBT2+) was employed as a scavenger for detecting

superoxide anion (O2∙−), as the reaction between NBT2+ and O2

∙− produces a fairly stable

42

reduced product that absorbs light at 530 nm [67]. NBT2+, NADH, and GO were mixed in

a 5 mM phosphate buffered solution at pH 7, and the light absorbance at 530 nm was

monitored over time. Buffers were prepared with phosphate salts (i.e., KH2PO4 and

K2HPO4). To examine the effect of GO on superoxide generation, experiments were

conducted at different concentrations of GO while holding all other chemical

concentrations (i.e., NADH or NBT2+) constant. XTT also was used to detect superoxide

anion [68]. To ensure that reduction of NBT2+ and XTT occurred through electron

transfer from O2∙−, superoxide dismutase (SOD) was added, as it very effectively

scavenges O2∙− from solution (i.e., competes for O2

∙−), stabilizing the concentration of

NBT2+ or XTT (i.e., the less effective scavenger), if their decay only occurs through

reaction with O2∙−. In the presence of superoxide dismutase, O2

∙− is converted rapidly

into hydrogen peroxide (H2O2) with a reactant-to-product stoichiometric mole ratio of

2:1. In the absence of the enzyme, this same disproportionation reaction proceeds

slowly; however, H2O2 may also be produced if additional electron transfer occurs from

GO to the protonated form of O2∙− (the hydroperoxyl radical, HO2

∙ ). To measure the

concentration of H2O2, we used the same DPD/HRP method as previously reported [60].

And finally, because further electron transfer to H2O2 can produce hydroxyl radical

(∙OH), pCBA was added in some experiments as it is a well-known hydroxyl radical

scavenger, whose decay can be used to determine the steady-state concentration of

∙OH. The methods for detecting all reactive oxygen species were the same as reported

before [50, 66]. Unless noted otherwise, all experiments were conducted at pH 7 with a

43

final phosphate buffer concentration of 5 mM, and all reported datum points are the

average of duplicate or triplicate samples.

3.3.4 Gel Electrophoresis

Whether the type and amount of ROS production under our experimental

conditions was sufficient to damage DNA was accessed using pBR322 DNA. In

experiments, mixtures were prepared containing some of the following components:

PBR322 DNA (0.015 µg/µL), GO (5 mg/L), phosphate buffer (5 mM), SOD (40 U/mL),

NADH (0.2 mM), methanol (0.5 M), ethanol (0.5 M) catalase (400 U/mL). The total

volume of each sample was held at 40 µL and incubated in the dark for 8 hr at room

temperature. After incubation, 8 μL 6x gel loading dye was mixed with each sample

prior to loading 10 μL onto an agarose gel. Gel electrophoresis was performed at 80 V

for 30 min in 1×Tris-borate-EDTA (TBE) solutions with a PowerPacTM Basic Power Supply

(Bio-Rad Laboratories Inc., CA). GelDocTM XR and Gel Imaging System with Image LabTM

Software (Bio-Rad Laboratories Inc., CA) were used for capturing images. Quantitative

analyses of gel images were performed.


Oxidation of NADH. NADH has two specific light absorption bands between 240 and 400

nm, with absorption maxima located at 260 nm and 340 nm. The maximum absorbance

at 340 nm is due to the n-π* transition of the dihydronicotinamide moiety which is

coplanar with the carboxamide moiety, resulting in electron orbital conjugation

between these two parts [69, 70], with this maxima being stronger for NADH than NAD+.

44

The maximum absorbance at 260 nm is due to the π -π* transition of the adenine ring,

and this maxima is stronger for NAD+ than NADH. Figure 3.1 shows the structure of

NADH and the location on the molecule where oxidation occurs to form NAD+.

Figure 3.1 Chemical structures of NADH and NAD+.

Figure 3.2 shows the dark reaction spectral change over time of a 0.2 mM NADH

aqueous solution containing 5 mg/L GO. Figure 3.2 (a) shows that in the presence of

GO, the absorbance peak at 340 nm decreases as the absorbance peak at 260 nm

increases over a 45 hour time period. The decreasing absorbance at 340 nm is

attributed to the decreasing concentration of NADH, whereas the increasing absorbance

at 260 nm is attributed to NAD+ formation. These changes in the spectra indicate that

NADH was oxidized to NAD+ by reaction with GO under our experimental conditions.

Figure 3.2(b) shows that in the absence of GO, the loss of NADH in the system was

negligible, with only a few percent loss over a 48 hour period, whereas in the presence

of GO, nearly 30% loss occurred. Hence, based on the spectral data, we conclude that

45

GO was reduced chemically through an overall two-electron transfer reaction involving

each NADH molecule,

x

2(NADH + H+) + GO →

x

2(NAD+ + 2H+) + GOx− eq 3.1

46

Figure 3.2 (a) The UV-visible absorption spectra of NADH (0.2 mM) and GO (5 mg/L) at pH 7 as a function of time under dark conditions. (b) The loss of NADH (0.2 mM) with and without GO (5 mg/L), calculated based on the change in light absorbance at 340

nm.

b

47

ROS Production. Figure 3.3a provides evidence for O2∙− formation in solutions containing

NADH and GO, as this figure shows that reduction of the NBT2+ to its formazan product

occurs in solution. The purple reduced mono-formazan product of NBT2+ was

spectroscopically (at 530 nm) and visibly evident, suggesting production of superoxide

anion occurred over time. Addition of SOD to the reaction mixture completely inhibited

reduction of NBT2+, more strongly suggesting superoxide anion as the NBT2+-reducing

species. Control samples, containing NADH and GO, or NBT2+ and NADH, showed almost

no change absorbance at 530 nm indicating the primary reactants themselves do not

cause or effect the color change.

The change in absorbance over time was sigmoidal, with an initial lag period

followed by an apparent first-order increase stage, and a final plateau stage. Upon

examining Figures 1 and 2, this trend likely results due to the overall kinetics involved in

O2∙− formation (i.e., NBT2+ reduction). The likely mechanism is reduction of molecular

oxygen (O2), producing O2∙−, on the surface of electron-rich reduced GO. Before

substantial electron transfer from NADH to GO occurs, the GO surface has a relatively

high redox potential (i.e., low Fermi level). After this first step of the overall process has

occurred for some time (i.e., electron transfer to GO from NADH molecules), the GO

becomes sufficiently reduced making electron transfer to O2 more thermodynamically

feasible and kinetically facile, enhancing the rate of O2∙− production. After 45 hours of

reaction when approximately 30 % of the NADH has been consumed, the number of

electrons transferred to GO totals about 0.12 meq/L. These electrons transfer to O2 and

then to NBT2+ at stoichiometric ratios of 1:1 and 4:1 [67], respectively, indicating that as

48

much as 0.48 mM NBT2+ could be reduced assuming 100% transfer efficiency. However,

because the initial concentration of NBT2+ was only 0.2 mM, the plateau stage on Figure

1 primarily results from the reduced rate of the final reaction (i.e., NBT2+ + O2∙−) at lower

NBT2+ concentrations. Figure 3.3b shows that the rate of accumulation of the formazan

product is dependent on the GO concentration as expected [71].

49

Figure 3.3 (a) NBT2+ (0.2 mM) product formation indicating presence of superoxide anion at pH=7 (b) The effect of GO concentration in the production of superoxide anion within the system containing NADH, NBT2+ and 0 mg/L GO (), 5 mg/L GO (), 10 mg/L GO (), 20 mg/L GO (). Note: Figure 4.3 (a) If existing in reaction mixture, [GO] = 5mg /L, [NADH] = 0.2 mM, [NBT2+] = 0.2 mM, [SOD] = 40 U/mL). Symbols represent samples containing NBT2+, NADH and GO (),NBT2+, NADH, GO and SOD (40 U/mL) (), NADH and GO (), NBT2+ and NADH (). (b) Here y-axis stands for the absorbance at 530 nm after subtracting the intrinsic light attenuation caused by the respective GO concentration.

a

b

50

To further test for superoxide formation we used XTT as another probe, as the

product of XTT reduction absorbs light at 470 nm. Figure 3.4 shows the increase in light

absorption at 470 nm for the samples containing GO, NADH and XTT. Again, SOD was

added to confirm that the increase in absorbance was caused by superoxide radical.

Results concurred with those using NBT2+, as negligible increase in absorbance occurred

for samples containing SOD over the experiment. Control samples containing only

NADH and GO and those containing only XTT and NADH showed negligible change in

absorbance with reaction time.

Figure 3.4 Evidence of superoxide anion using XTT as a scavenger.

Note: Symbols represent samples containing XTT (0.1 mM), NADH (0.2 mM) and GO (5 mg/L) (), XTT (0.1 mM), NADH (0.2 mM), GO (5 mg/L) and SOD (40 U/mL) (), NADH (0.2 mM) and GO (5 mg/L) (), XTT (0.1mM) and NADH (0.2 mM) ().

51

With significant O2·− formation, there is a high probability that hydrogen peroxide

will form also, and potentially accumulate in solution. This may occur either through

transfer of another electron from the reduced GO to the protonated form of

superoxide, HO2· (hydroperoxyl radical), followed by proton abstraction from solution,

or through the overall disproportionation of O2·−, which involves reaction between HO2·

and O2·− . To test whether H2O2 was produced, the DPD/HRP method was used to

quantify H2O2, however, different from our previous study in which NADH was not used

[66], in this study any unreacted NADH was quenched by the addition of acid, as any

residual NADH displayed a positive interference. In this method, DPD is oxidized to a

red Würster Dye radical that absorbs light with a maxima at 551 nm. Figure 3.5 shows

that under light-independent conditions, there was an increase in light absorption at

551 nm due to DPD· formation in samples containing the DPD/HRP reagents, NADH (0.2

mM), and GO (5 mg/L). A visible change in color (to light magenta) was observed. From

the molar absorptivity of DPD· at 551 nm is equal to 21,000 ± 400 M-1 cm-1 [72], a

maximum concentration of H2O2 of 6.06 μM was found to accumulate after 6 hours of

reaction, followed by a decreasing concentration with time. Unlike the absorbance

changes occurring with NBT2+ or XTT, which are related to accumulated production of

O2·− and which are added at the initiation of the experiment, the DPD/HRP assay

measured the temporal concentration of H2O2 since the reagents are added at the time

of measurement. To further confirm that H2O2 was the reason for the color change, in

some experiments catalase was added before adding DPD and HRP. No change in

52

absorbance was detected since H2O2 is rapidly removed from solution by catalase.

Control samples containing GO or NADH alone also showed on change in visual color or

absorbance.

Figure 3.5 Evidence of H2O2 production via DPD/HRP product formation.

Note: Symbols represent samples containing NADH (0.2 mM) and GO (5 mg/L) (), XTT (0.1 mM), NADH (0.2 mM), GO (5 mg/L) and Catalase (400 U/mL) (), NADH (0.2 mM) alone (), GO (5 mg/L) alone (). Note that catalase was added just before adding DPD and HRP.

To test whether hydroxyl radical ·OH was produced in aqueous GO suspensions

containing NADH, pCBA was used as a ·OH scavenger. As shown in Figure 3.6, no

measureable ·OH was detected. This result is the same as what we observed for ROS

generation from GO under sunlight, where both O2·− and H2O2 were detected but not

53

·OH [66]. However, it is very possible that any ·OH formed on the surface of the GO is

rapidly consumed by the same surface, leading to a undetectable concentration in the

bulk aqueous phase [54].

Figure 3.6. Scavenging for ·OH with pCBA (5 µM) in pH 7 phosphate buffered solutions.

Note: Symbols stand for systems containing: pCBA (5 µM), NADH (0.2 mM), and GO (5 mg/L) (); pCBA (5 µM) and NADH (0.2 mM) (○), pCBA (5 µM), NADH (0.2 mM), and GO (5 mg/L) ().

Finally, as a way of assessing potential biological effects caused by ROS

production in the presence of GO and NADH, the ability of the ROS generated in these

experiments to cleave the pBR322 supercoiled DNA plasmid was assessed [60]. pBR322

DNA was used since it is a circular strand of double helix DNA which is often used for

evaluating chemicals thought to promote DNA damage. Figure 3.7 indicates that no

significant DNA cleavage was observed for samples containing DNA alone (0.015 µg/µL);

54

or DNA with NADH (0.2 mM); or DNA with GO (5 mg/L) (lanes A, B, and C, respectively).

This is consistent with those results described above in which the same controls

produced no measurable ROS. However, conversion of the supercoiled circular pBR322

(Form I) into the nicked form (Form II) was achieved in GO suspensions (5 mg/L)

containing the DNA and NADH (0.2 mM) after an 8 hr incubation period in a dark (lane

D). To test further whether DNA cleavage was due to ROS generation, different

scavengers for the specific ROS were added to some samples. By including the

superoxide dismutase (SOD) enzyme to the system (lane E), DNA cleavage was

prevented. Because O2∙− is a precursor to other ROS (i.e., H2O2, ·OH) this inhibition is

logical. By including the enzyme catalase to decompose any H2O2 that is produced (to

H2O and O2), DNA cleavage was prevented also (lane F). These results are consistent

with similar experiments conducted with carboxylated single walled carbon nanotubes

(C-SWCNTs) which found that quenching either O2∙− or H2O2 prevented pBR322 DNA

cleavage [60]. As in this other study [60] methanol and ethanol were added to some

samples, as they are well known ·OH scavengers (lanes G and H in Figure 5,

respectively). The addition of either ·OH scavenger prevented plasmid cleavage,

suggested that although ·OH could not be detected in the pCBA assay, it still may be the

responsible reactant responsible for DNA cleavage. As hypothesized for systems

containing C-SWCNT and NADH [60], it is well known that trace metals are always

associated with (i.e., form complexes with) DNA, and that many of these same trace

metals can be reduced by O2∙− and then through a Fenton’s type reaction, generate ·OH

from H2O2. Any ·OH formed at the surface of the DNA has a high probability to further

55

react with this surface, as it is generally known that ·OH can induce DNA cleavage by

hydrogen abstraction before diffusion into the bulk solution [73-75]. Consistent with

the experiments above in which ROS generation was observed to continue over time,

the amount of DNA damage was found to increase with incubation time (Figure 3.8).

56

a)

b)

Figure 3.7 (a) Gel image of pBR322 DNA after an 8 hr incubation at pH 7 in the different experimental systems. (b) Quantitative analysis of DNA bands.

57

a)

b)

Figure 3.8 (a) Gel images showing the time course of pBR322 DNA fragmentation, in GO suspensions containing NADH at pH 7. The concentration of GO, NADH and pBR322

DNA were 5 mg/L, 0.2 mM and 0.015 µg/µL, respectively. (b) Quantitative analysis of DNA bands.

58

3.5 Conclusion

This study shows that GO can act as a catalytic intermediate, shuttling electrons from

NADH to molecular oxygen forming O2∙− Additional electron transfer or

disproportionation of O2∙− produces H2O2. Although ·OH was not detected, DNA

cleavage was observed in air-saturated GO suspensions containing NADH. This result

raises concern regarding potential biological risk of GO. Based on The proposed scheme

is shown in Figure 3.9.

Figure 3.9 Proposed mechanism of GO acting as an electron shuttle to produce reactive oxygen species(ROS) and subsequent DNA cleavage.

59

CHAPTER 4. INDIRECT PHOTO-TRANSFORMATION OF GRAPHENE OXIDE IN WATER

4.1 Abstract

As a relatively new nanomaterial, graphene oxide (GO) has been found to have many

unique and potentially useful properties, leading to an increasing rate of commercial

production. This increased production has led to an increasing interest in GO’s

environmental fate, mechanisms of transport in the environment, and potential to

cause environmental or human toxicity. To further address the environmental fate of

GO, indirect photochemical transformation of GO under sunlight conditions was

investigated in this study. By photochemically generating low steady-state

concentrations of hydroxyl radicals from hydrogen peroxide under solar wavelength

light at concentrations similar to those found in some natural surface waters, we found

that reaction resulted in distinct changes to the GO. Light attenuation spectra in the

ultraviolet (UV) and visible (vis) spectral regions, and visible color changes occurred.

Total organic carbon (TOC) content was found to decrease, indicating some

mineralization of GO carbon occurred. Combined with previous results that show that

direct photochemical reactions of GO occur under solar light, presumably leading to

more reduced GO, the results suggest that environmental photochemical processes

involving GO are quite complex involving both oxidative and reductive processes,

60

however may be the dominate mechanisms by which this material is transformed in the

environment. This study also provides insight in ways of degrading or even mineralizing

GO by advanced UV/H2O2 oxidative processes in wastewater treatment.

4.2 Introduction

GO is a single layer of graphite that has been partially oxidized both on its

edges and its basal plane. Hence, it has a hybrid structure consisting of a mixture of sp2

and sp3 carbon atoms [76]. Its surface and edges contain a significant number of

hydroxyl and carboxyl functional groups, making its surface sufficiently hydrophilic to

disperse in water. These aqueous colloidal suspensions are quite stable over time. Due

to its unique chemical and electronic properties, GO has been used in, or suggested for

use in, a large number of applications in different areas [77-79]. With continued

increasing production and use in various application, GO is expected to be found in an

increasing number of industrial and consumer products. As a result, release of GO from

these products during their life cycles may lead to both human and environmental

exposure.

Currently, limited information is known about the environmental fate and

potential environmental effects of GO [64, 80, 81]. Because it is easy to suspend in

water, its further oxidation or reduction (i.e., environmental transformation) is likely to

occur at least partially through reaction with aqueous phase oxidizing or reducing

agents, including secretory plant enzymes. For example, Kotchey et al. showed that

horseradish peroxidase (HRP) could catalyze the oxidation of graphene oxide (in the

61

presence of H2O2) [82]. Interestingly, HRP failed to catalyze the oxidation of reduced

graphene oxide (RGO), which is a product of GO that has lost most of the oxygenated

groups on GO [83]. This result suggested that the existing oxygenated functional groups

on GO play a significant role its chemical, and presumably photochemical, reactivity.

Because the π-bonds in GO absorb solar spectrum light, photochemical

transformation also may be an important reaction pathway in aqueous environments, if

light absorption is followed by chemical reaction. Indeed, it is well known that GO can

be reduced under UV irradiation in the 280-450 nm wavelength range [36]. Matsumoto

et al. observed that irradiation of aqueous dispersions of GO under a 500 W Xenon

lamp, which generally emits light down to 280 nm approximately 20 nm below the

spectral limit of solar light measured at earth’s surface, resulted in production of

reduced GO (RGO), CO2, and H2 [38]. Irradiation of GO leads to more defects (i.e., non-

aromatic carbon) which provides new “sites” for either H2 or CO2 formation. Hou et al.

reported that irradiation under sunlight led to transformed GO particles of smaller size

and with fewer oxygen functionalities, with this “photo-weathered” material likely

having different photo-reactivity compared to the starting material [39]. Chowdhury et

al. showed that direct photo-transformation had great effects on GO aggregation rates

under both aerobic and anaerobic conditions; however, negligible effects occurred on

surface changes during photo-transformation in sunlight [40]. Our group has previously

reported that aqueous GO can generate several reactive oxygen species (ROS), including

O2∙− and H2O2, under solar wavelength light exposure [66]. No hydroxyl radicals were

not detected, as also reported by Hou et al. [54]. However, Zhou et al. found that GO

62

could react with Fenton’s reagent (iron species and hydrogen peroxide) under UV

irradiation, using a mercury lamp that emits primarily at 365 nm, converting the GO to

back to graphene sheets and then to graphene quantum dots (GQDs) stepwise [84].

These studies strongly suggest that indirect photochemical transformation in the

aquatic environment might be an important fate process for GO.

In addition to the presence of oxidizing and reducing agents in water, other

water quality parameters may affect the reactivity of GO in the environment. For

example, Walker et al. suggested that GO tends to be less colloidally stable in

groundwater than in surface waters due to the generally higher concentrations of

divalent cations (i.e., hardness) and lower concentrations of natural organic matter

(NOM) in groundwater [85]. Chowdhury et al. showed that the pH, ionic composition

(including valence of counter-ions) and amount of NOM play a significant and complex

role in the colloidal stability of reduced graphene oxide [41]. Because reduction or

further oxidation of GO in water will result in changes to its structural and chemical

properties the associated interactions with these other species in water also will be

modified.

Because hydroxyl radicals can be produced in natural aquatic environments from

reactions involving NOM, nitrate, and from photo-Fenton type reactions [86-88], in this

study, we examined changes to GO and possible product formation upon reaction of GO

with hydroxyl radicals (·OH), produced from photochemical cleavage of H2O2 of under

solar wavelength light irradiation. Light attenuation in the ultraviolet (UV) and visible

(vis) spectral regions, and visible color changes suggested that changes to the GO

63

surface were occurring. Total organic carbon (TOC) content was found to decrease,

indicating some mineralization of GO occurred.


4.3.1 Materials

A single layered graphene oxide (GO) dispersion (10 mg/mL) was purchased from

Advanced Chemical Supplier (ACS) Material, LLC (Medford, MA) and used as received.

According to the manufacturer, the material was synthesized by a modified Hummer’s

method and was approximately 80% single-layer GO (20% being multi-layered) with a

size range of 0.5 to 2.0 µm and a thickness of 0.6 to 1.2 nm. All other chemicals

including hydrogen peroxide solution with a concentration of 30 % (w/w) in H2O were of

the highest purity and used as received from Sigma Aldrich (St. Louis, MO). All aqueous

samples were prepared with water purified with a Barnstead Nanopure system

(Dubuque, IA) after R/O pretreatment. The original GO dispersion (10 mg/mL) was

diluted with water to make a 100 mg/L stock dispersion and was sonicated under low

energy (8890R-MT ultrasonic bath from Cole-Parmer, Vernon Hills, IL) for 2 hours to

make it as uniform as possible. When not in use, the GO stock dispersion was stored in

a dark environment at room temperature.

4.3.2 Irradiation of GO in Solar Spectrum Light

In all experiments, the graphene oxide initial concentration was 10 mg/L,

whereas the initial H2O2 concentration was 100 mM. Samples containing GO and H2O2

at these concentrations were placed in a Rayonet RPR-100 merry-go-round

photochemical reactor (Southern New England Ultraviolet Co.) with lamps that emit

64

only in the solar spectrum from 300 to 410 nm, with a maxima intensity at about 350

nm. More detailed descriptions of the photochemical reactor and the overall

experiment procedures can be found in a previous publication [66]. Samples were

removed (i.e., sacrificed) at specific times for analysis. Dark control samples containing

the same materials were wrapped with aluminum foil and stored in the dark over the

same time periods that the experimental samples were irradiated. All experiments

were performed in duplicate or triplicate.

4.3.3 Materials Analysis

A Varian Cary 50 UV/Vis Spectrophotometer along with a 1 cm path-length

quartz cuvette was used to record UV-vis absorption spectra. Samples were monitored

by a HORIBA LabRAM HR800 Raman spectrometer Raman spectroscopy with an

excitation wavelength of 633 nm. The samples for Raman spectrometric analysis were

prepared by dripping the suspension onto a silicon wafer, followed by evaporating the

water (i.e., drying) at room temperature. Total organic carbon (TOC) content of samples

was monitored by with a TOC-L CPH/CPN analyzer (Shimadzu, Japan).


Characterization of GO. The UV-vis spectrum of aqueous GO exhibits two

characteristic absorption bands (Figure 4.1a). One has a maximum at 230 nm, which

corresponds to the characteristic π to π* transitions of aromatic carbon-carbon bonds.

The other is a smaller peak at 303 nm due to n to π* transitions of the C=O bonds. In

the Raman spectrum, (Figure 4.1b), GO has a D band at 1355 cm-1, a G band at 1600 cm-

1, and a 2D band from 1000-3000 cm-1.

65

Figure 4.1 Characterization of GO. a) UV/Vis absorption spectra. b) Raman spectra.

Photo Degradation of GO with H2O2 in Water. Figure 4.2a showed the changes

in the light attenuation spectrum of a 10 mg/L suspension of GO containing 100 mM

H2O2 under lamp light irradiation. The spectrum is referred to as a light attenuation

spectrum, rather than an absorbance spectrum because the observed response

measured by the instrument is caused by both the amount of light that is absorbed by

the GO and by the amount of light that is scattered by the GO nanoparticles. The

response over the entire spectrum from 300 to 800 nm is underlain by a wide light

scattering band, as also evident on Figure 4.1(a). Over the course of the first 4 hours of

irradiation, there was a general increase in light attenuation across the UV-visible

spectrum, likely resulting from direct photochemical reduction of the GO, as occurs in

the absence of H2O2. In contrast, from 4 hours to 48 hours, a continual decrease in light

attenuation occurred, suggesting that the reaction of ·OH with GO was reducing the

66

number of aromatic functional groups within the GO nanoparticles. In contrast, for the

dispersion containing only GO (no H2O2), light attenuation across the spectrum

continued to increase from 0 to 18 hours, and remained relatively constant from 18 to

48 hours (Figure 4.2b). These results suggest that in the presence of 100 mM H2O2, the

resulting hydroxyl radical-mediated oxidation out-competes the direct photochemical

reduction process. The dark control samples containing GO and H2O2 showed negligible

difference in the UV-vis spectra over the total reaction period of zero to 48 hours

(Figure 4.2c).

67

Figure 4.2 UV-visible attenuation spectra of samples (measured as absorbance); (a) 10 mg/L GO with 100 mM H2O2 under irradiation with solar wavelength lamp light; (b) 10 mg/L GO (with no H2O2) under irradiation with solar wavelength lamp light; and (c) 10 mg/L GO with 100 mM H2O2 dark control.

a

b

68

Figure 4.2 continued

Figure 4.3 showed the visible difference between initial graphene oxide solution

and samples after solar-wavelength lamp light irradiation. The change in color of the 10

mg/L suspension of GO after 48 hours of irradiation in the absence of H2O2 can be seen

by comparing Figures 4.3a and 4.3b, indicating the much darker solution occurs after

irradiation. This visible change is in agreement with changes observed on the measured

UV-vis light attenuation spectra. In the presence of H2O2, irradiation resulted in the

initial color of the GO suspension to become less colored (and less turbid), as evidence

by comparing Figure 4.3a to Figure 4.3c.

c

69

Figure 4.3 Images of samples at specific experimental times: (a) GO before irradiation; (b) GO after 48 hr of irradiation with solar-wavelength lamp light; and (c) GO and H2O2 after 48 hr of irradiation with solar-wavelength lamp light. All samples contain 10 mg/L GO, and either zero or 100 mM H2O2.

Because no additional source of organic carbon, other than GO, was present in

the samples, the aqueous suspensions were analyzed for total organic carbon (TOC)

content at specific irradiation times. As shown in Figure 4.4, in the presence of H2O2,

GO showed a loss of around 80% total organic carbon content in 48 hrs. From 0 to 24

hrs, The TOC content significantly decreased by about 80%, but did not decrease by

much beyond this point during the next 24 hr period.

For samples that contained only GO (without H2O2), the TOC dropped to

around 60% after 24 hrs of irradiation. Additional loss of TOC did not occur during the

next 24 hours of irradiation as it appears that the residual organic carbon was stable

after this point in time. These results are consistent with the work of Hou et al. [39].

They observed a similar plateau in sample light absorbance and particle hydrodynamic

size, even after two months under natural sunlight. As expected, the dark control

70

samples containing only GO (without H2O2) or GO with H2O2 showed nearly no change in

the TOC measurement along the experimental time from zero to 48 hrs.

Figure 4.4 Degradation of GO in the presence of H2O2 under irradiation by measuring TOC.

4.5 Conclusion

In this study, the indirect (with H2O2 adding into the experimental system to

provide a nearly constant rate of hydroxyl radical production) photochemical

transformation of graphene oxide (GO) was examined under solar-wavelength light

exposure. Results indicate that GO reacts rapidly under these condition. Specifically,

transformation of GO in the presence of H2O2 showed a loss of 80% organic carbon

content in 48 hr, with visible color bleaching. In contrast, in the absence of H2O2,

around 40% of the organic carbon content (i.e., TOC) was lost and rather than color

71

bleaching, the GO suspension became darker in color. In the presence of H2O2, we

postulate that the dominate reaction involved hydroxyl radical attach on the GO carbon;

however this reaction was likely occurring simultaneously with some, and less rapid

photoreduction on the same particles.

4.6 Environmental Significance

This study indicates that as expected, GO is fairly reactive towards ·OH, which is

often photo-chemically produced in natural waters. The finding suggested that reaction

with ·OH might be an important transformation process of GO in the natural aquatic

environment. Also, this study provides insight into ways of degrading GO or even

mineralizing it during H2O2/UV processes in wastewater treatment systems.

72

CHAPTER 5. PHOTOREACTIONS OF AQU/NC60 CLUSTERS UNDER SIMULATED SUNLIGHT: PHOTO MINERALIZATION AND PRODUCT CHARACTERIZATION

5.1 Abstract

C60 is emerging in various applications, however, much remains to be learned regarding

its properties in aqueous systems in the natural environment. In this study,

mineralization of aqueous C60 clusters (aqu/nC60) was examined under simulated

sunlight in merry-go-round photo-reactor. CO2 was observed to be produced from

aqu/nC60 suspensions, indicating that some mineralization did occur. Additionally,

Ultraviolet-visible (UV- vis) absorbance spectra and HPLC analysis were performed to

follow the overall transformation process. This study shows that CO2 is one of the

products formed from aqu/nC60 clusters under solar irradiation, suggesting that major

changes occur to the overall structure of C60 upon exposure to sunlight.

5.2 Introduction

Many studies have evaluated the properties of C60 in order to discover how this

material can be used in more applications, including biomedical and electronic

applications [2, 4, 89]. One of the more interesting applications was to use it in a gel

formulation, where it was found to help control acne, and to provide other skin care

benefits by inhibition of neutrophil infiltration and sebum production [90]. Clearly, large

73

scale production of C60 and other fullerenes is likely to occur to meet industrial and

customers’ needs now and into the near future. Frontier Carbon Cooperation (FCC),

reported that nearly 1,500 tons/year carbon buckminsterfullerene were produced in

2007 [2]. Therefore, if this production rate continues to increase over the next few

decades, fullerenes will appear in the natural environment through live cycle release

and also possibly through accidental release without doubt. Many toxicity studies have

shown that C60 can induce oxidative stress to the brain of juvenile largemouth bass and

may also be responsible for cytotoxicity, resulting in DNA cleavage and other cellular

structure damage [11-13]. Hence, clearly there are concerns over negative effects of C60

on human and animal health motivating much research directed at providing data for a

more thorough risk assessment analysis of C60 and other fullerenes.

C60 is very hydrophobic [5, 6]. However, it is well known that clusters (i.e.,

nano-sized precipitates) of C60, termed nC60, form stable colloidal suspensions in water.

These colloidal suspensions can be easily formed by adding solid C60 to water, and then

stirring the mixture for an extended period of time, or by sonicating the mixture in

water for a shorter period of time [7-9]. If released to the aqueous environment, C60 will

no doubt influence the water quality of the receiving stream, and it found in water at

high concentrations, it will exist as these clusters. Photo-chemical transformation has

been proposed to be a possible transformation pathway of nC60 clusters. Our group first

reported that C60 underwent photochemical transformation in sunlight and under

simulated solar lamp lights [14]. The color of the nC60 suspensions transitioned from

yellow brown to light yellow, to nearly clear upon exposure to light for extended periods

74

of time (i.e., weeks). The process involves the photoexcitation from ground-state C60 to

singlet C60 (1C60 where one of the 60 carbons in the molecule has one electron in the

excited state), followed by the fast conversion of 1C60 to triplet C60 (3C60) by intersystem

crossing. Finally, 3C60 is quenched by O2 through energy transfer from 3C60 to O2,

resulting in the formation of the reactive oxygen species (ROS) singlet oxygen, 1O2 [15].

More work has been done in our group to show that ROS species (including

1O2 , O2∙− and ·OH) are formed upon irradiation of nC60 [14-16]. Lee et al. also

characterized major photochemical transformation products of nC60 under UVC (254

nm), mainly including water-soluble products with oxygen functional groups [18]. The

studies in which solar wavelength light has been used collectively suggest that photo-

transformation in sunlight is a potentially important process controlling the

environmental fate of C60, and that the photoproducts have properties distinct from the

original material.

One of the measurements not previously made on C60 photochemical

experiments under solar light conditions has been the determination of CO2 evolution to

indicate the degree of mineralization that may occur from direct photolysis of C60, alone.

Hou et al. [14]first suggested that C60 may undergo significant mineralization to CO2 by

monitoring the total organic carbon (TOC) concentration in 65 mg/L nC60 samples upon

exposure to lamp light (8 UVA lamps, that emit from 300 to 410 nm), with the TOC

decreasing from 65 mg/L to 11.3 mg/L after 65 days of continuous exposure. Other

studies have not been able to assess mineralization, mainly because the time period of

irradiation was relatively short, or the concentration of nC60 was too low to detect an

75

increase in CO2 above background [19, 20]. Su was the first to perform photo-

mineralization studies of nC60 clusters over long time periods, indicating some

mineralization did occur in sunlight [91]; however, these results were somewhat

ambiguous, as dark control samples also showed an increase in CO2 as a function of

time.

In this effort, the photochemical reaction of aqueous C60 clusters (Aqu/nC60),

was studied, focusing on mineralization to CO2, pH changes, and photoproduct

characterization under simulated sunlight irradiation conditions. This effort should help

to better understand the overall photochemical transformation process of nC60 in the

natural aqueous environments.


5.3.1 Materials

Sublimed C60 fullerene (99%) was purchased from the MER Corporation (Tucson,

AZ, USA). The 85% H3PO4 solution for acidification was obtained from Mallinchrodt

Chemicals. Toluene and methanol that were used for liquid chromatography were

HPLC grade with over 99.99% purity (Advantor Performance Materials Inc.,

Philipsburg, NJ). Other chemicals were of the highest purity available and used as

received. All aqueous samples were prepared using water produced by a Barnstead

Nanopure system (Dubuque, IA).

76

5.3.2 Aqu/nC60 Suspension Preparation

Aqu/nC60 clusters were prepared following a reported method [92]. Purchased C60

powder was pulverized using an agate mortar and pestle for around 10 min, producing a

fine black powder. The C60 pulverized power was added to water at a given mass to

volume ratio (i.e., C60 600 mg/L). The solution was sonicated in a bath sonicator

(Branson Ultrasonics Corporation, Danbury, CT) for approximately 5 hours, and then

mixed continuously for 10 days on an orbital shaker (New Brunswick Scientific Co., Inc.,

Edison, NJ) to obtain a relatively homogeneous Aqu/nC60 cluster suspension. After the

extensive mixing, the suspension was allowed to sit for 4 days to settle large particle

aggregates, and the supernatant (around 90% of the solution by volume) was removed

and stored in the dark when not in use. Filtration was not used, however the

concentration of this stock suspension was determined by HPLC to be 12.8 mg/L. The

stock suspension was buffered to pH 7 with a final concentration of 5 mM phosphate

buffer (the measured pH was 7.08).

For lamp light irradiation studies, 10 mL Aqu/nC60 stock solution subsamples

were placed in 16 mL borosilicate glass vials. These vials were tested and were shown

not to absorb solar-wavelength light. The headspace to liquid ratio was 3 to 5, to

ensure sufficient oxygen was available for complete oxidation of the C60 to CO2 to

theoretically occur. Each vial was sealed with a natural rubber sleeve stopper (13 × 20

mm I.D. × O.D., VWR Inc.) and two tight-ties (5.6”, VWR Inc.) to prevent gas leakage.

Samples were irradiated with sixteen 24 Watt black-light phosphor lamps (RPR-3500Å

lamps from Southern New England Ultraviolet, Branford, CT) that emit light from 300 to

77

410 nm, with the maximum intensity occurring near 350 nm. Samples were irradiated

with the lamps placed within a Rayonet merry-go-round photochemical reactor placed

in a fume hood (Figure 5.1). The temperature during lamp irradiation was at “room

temperature” by using two cooling fans, built inside the reactor. Samples were placed

in a circular pattern near the center of the merry-go-round reactor, and rotated at the

speed of 5 rpm to ensure uniform light exposure. Dark control samples were prepared

using the same method as the irradiated samples; however, they were wrapped with

aluminum foil and kept in a dark environment while the experimental samples were

being irradiated. All experiments were performed in duplicate or triplicate. At specific

times during the irradiation period, irradiated samples and dark control samples were

sacrificed for analysis.

78

Figure 5.1 Merry-go-round photo-reactor

5.3.3 HPLC Measurement

The C60 content of each sample was measured by HPLC after toluene-salt

extraction. The extraction method was a slightly modified method reported previously

[14], with the only change being to extend the mixing time. Specifically, for each sample,

a 3 mL aqu/nC60 subsample, 3 mL toluene, and 1.2 mL 0.1 M Mg(ClO4)2 (5:5:2) were

combined together in a 15 mL glass centrifuge tube and placed on a wrist shaker and

stirred overnight. The addition of Mg(ClO4)2 was found to increase transfer efficiency of

C60 to the toluene phase, presumably by making the aqueous phase more ionic (more

79

hydrophilic). The C60 concentrations in the toluene extracts were determined by HPLC

analysis using a Cosmosil Buckprep column and photodiode array (PDA) detector,

monitoring absorbance at 336 nm. Pure toluene (HPLC grade) was used as the mobile

phase at a flow rate of 1 mL/min. The run time was 12 min and the injection volume was

100 μL.

5.3.4 UV-Vis spectroscopy and pH measurement

The UV-visible light absorbance spectra were recorded with a Varian Cary 50

UV/Vis

Spectrophotometer using quartz cuvettes with 1 cm path lengths. The pH value of all

samples were measured with an Accumet 925 pH/ion Meter (Fisher Scientific Inc.).

5.3.5 Headspace CO2 measurement

After irradiation or incubation in the dark, all samples were acidified before

analyzing the CO2 content in the headspace. The purpose of acidification was to

decrease the pH so that the total inorganic carbon (TIC) was essentially all CO2 under

equilibrium conditions at low pH (eq 5.1).

𝐻2𝐶𝑂3⇔𝐻+ + 𝐻𝐶𝑂3−; 𝐻𝐶𝑂3

−⇔𝐻+ + 𝐶𝑂32− eq 5.1

Below pH 3, essentially all the inorganic carbon in the samples exists as CO2 either in the

aqueous phase (CO2,aq) or in the headspace (CO2,g). Henry’s constant (𝐾𝐻 =𝐶𝑂2,𝑎𝑞

𝐶𝑂2,𝑔 )can

80

be applied to calculated the distribution of CO2,aq and CO2,g. Once the gas phase CO2

concentration has been measured, the total inorganic carbon (TIC) in the sample can be

calculated by adding the amount of CO2 measured in the gas phase to the amount

calculated in the aqueous phase (calculated by Henry’s constant).

For acidification, concentrated H3PO4 (60 μL/10mL) was injected into each vial.

Then samples were placed on a vortex mixer for 5 minutes. To ensure complete

equilibrium was achieved, the acidified samples were stored in the dark overnight

before performing headspace analysis. An overpressure method (OPM) was applied

when removing headspace gas for analysis, by first adding 3 mL of pure nitrogen gas to

the headspace, followed by immediately removing 3 mL from the headspace, to ensure

pressure equalization of the syringe needle with the surrounding air upon removal of

the needle from the tube septa. The added volume of gas was accounted for in mass

calculations. The CO2 in the headspace was measured using a PDZ-Europa trace gas

analyzer in conjunction with a 20/20 PDZ-Europa isotope ratio mass spectrometer.

More details regarding the methods can be found in Su’s master’s thesis [91].


C60 concentrations were measured by HPLC in samples at specific times after

initiating irradiation (Figure 5.2). HPLC analysis of samples revealed an obvious

response at a retention time of approximately 8.5 min, measuring absorbance at 336

nm [93]. As reported previously, this peak corresponds to molecular C60. In the

irradiated samples, the C60 concentration decreased rapidly, with greater than 70% of

81

original C60 response lost after 15 days of irradiation. This result is consistent with

studies by Hou et al. [14] which also showed a significant loss over time. One study has

suggested that much of the loss may be due to a decreasing extraction efficiency as

intermediate products are generated over time, decreasing the extraction efficiency of

any remaining C60 remaining in the clusters [20]. However, Hou has clearly shown this is

not the case for the nC60 clusters he used (prepared with tetrahydrofuran (THF), and

subsequently concluded at the addition of Mg(ClO4)2 to Aqu/nC60 also resulted in nearly

complete extraction of the molecular C60 to the toluene phase. Additional peaks

appeared on the chromatograms at retention times of 2 to 3 min after 15-days of

irradiation, indicating potential organic intermediate products of C60 were accumulating.

The dark control samples were almost the same over the entire incubation period,

indicating the stability of C60 in the dark over this time period.

82

Figure 5.2 HPLC analysis of Aqu/nC60 after zero, 5, and 15 days of light exposure.

83

Figure 5.3 shows the changes in the UV/vis absorbance (i.e., attenuation)

spectrum of aqu/nC60 over a 10 day irradiation period. There are two obvious peaks at

270 and 350 nm on the spectra at zero, 5 and 10 days, and a broad band between 410

and 555 nm, indicating C60 is present in nanoparticle (cluster) form, as this broad band

results primarily due to light scattering rather than light absorption [8]. At all

wavelengths from 300 to 800 nm, the absorbance (or more precisely, the light

attenuation) decreased with time. Despite the response being the result of both

molecular light absorption and light scattering by particles, and also the result of

partially oxidized C60 intermediate products, the measured response at 350 nm has been

used by some researchers to “quantify” the concentration of nC60 in aqueous

suspensions, [20], despite the ability of HPLC methods to separate and quantify the

molecular form of C60. However, the absorbance spectra do indicate an overall decrease

in response, which is a good indication of the general loss of aromatic carbon during the

10-day irradiation period. This result is consistent with previous studies and with the

HPLC analysis. For all dark control samples, no change in the spectra occurred over time

(data not shown).

84

Figure 5.3 UV-Vis spectra of aqu/nC60 and irradiated C60

Since the Aqu/nC60 suspensions in the experiment were buffered, the change in

pH was small with a slight decrease in pH occurred for the irradiated samples (from 7.07

to 6.81 after 20 days of irradiation), likely due to photo-oxidation, resulting in the

formation of photoproducts containing acidic functional groups such as carboxyl groups

and polyhydroxyl groups on the C60 cage structures (Figure 5.4). An additional reason

for the pH drop may be attributed to the production of CO2, which between pH 4 to 7

will exist as bicarbonate (HCO3-) and protons (H+) in solution.

85

Figure 5.4 pH changes of C60 irradiated and dark control samples.

After calculating all CO2 present in each sample tube from the headspace

analysis data, Figure 5.5 shows an appreciable increase in CO2 concentration over the 20

day irradiation period for the irradiated samples. CO2 increased by four times above the

initial background concentration in the irradiated samples. This results clearly shows

that some mineralization did occur upon exposure of Aqu/nC60 clusters to light within

the solar spectrum.

Production of CO2 from aqu/nC60 is quite a complex process since different

functionalized forms of C60 and potential C60-fragments may produce CO2 at different

rates, making it difficult to kinetically analyze the data, however, the data can provide a

86

good estimate on the temporal mass balance conversion of parent C60 to the final

product, CO2. Because each sample initially contained 12.8 mg/L C60, the initial total

organic carbon mass in each tube as 10.7 moles. Hence, with a net increase in CO2 of

0.08 moles, approximately 7% of the C60 carbon had been mineralized after 20 days of

constant light exposure.

Figure 5.5 CO2 measurements as a function of time.

Tables 5.1 and 5.2 show the details regarding the calculated mass recoveries of

inorganic carbon in the headspace and aqueous phase. Because the CO2 concentration

is diluted during the over-pressurization step, the final concentration is adjusted by

multiplying by 3/2 using the following equation:

87

CO2(g) (M) = (3

2× Vmeasured) ÷ Vsampled ÷ RT eq 5.2

This procedure was confirmed with and applied to all CO2 analytical standards.

Then, Henry’s constant ( k =CO2(aq)(M)

CO2(g) (M) = 0. 8317 ) was used to calculate the aqueous

phase CO2(aq)(M) concentration. The total CO2 mass produced in the system was

calculated by adding the mass of CO2 in the aqueous and gas phases:

CO2total mass = [CO2]g × Vg + [CO2]aq × Vaq eq 5.3

Table 5.1. Summary of CO2 measurements for samples exposed to light

Time

(days)

CO2 (measured)

(μL)

CO2

(M, gas)

CO2

(M, aqueous)

CO2 total

(μmol)

0 15.79 0.0011832 0.00098407 0.016939838

10 51.16 0.00383359 0.0031884 0.054885506

20 76.4 0.00572491 0.00476141 0.081963499

88

Table 5.2. Summary of CO2 measurements for dark control samples

Time

(days)

CO2 (measured)

(μL)

CO2

(M, gas)

CO2

(M, aqueous)

CO2 total

(μmol)

0 15.79 0.0011832 0.00098407 0.016939838

10 14.97 0.00112175 0.00093296 0.016060125

20 19.23 0.00144097 0.00119845 0.020630342

5.5 Conclusions

This study unequivocally shows that aqueous colloidal dispersions of nC60 exposed

to light within the solar spectrum undergo photo-transformation, with some of the

reactions resulting in CO2 evolution. Within only 20 days of continuous light exposure,

approximately 0.7% or the C60 carbon was converted to CO2 in phosphate buffered

water. This further indicates that photo-transformation of C60 in sunlight can result in

C60-cage opening reactions, reducing the number of carbon atoms on the oxidized

organic material to less than 60. A slight decrease in the pH of irradiated samples is

consistent with CO2 formation.

89

CHAPTER 6. OVERALL SUMMARY AND RECOMMENDATION

6.1 Summary

This study focused on photochemistry and light-independent reactions of carbon-

based nanomaterials (i.e. C60 and graphene oxide), as environmental processes could

significantly affect the environmental fate and transport of these materials in the

aquatic environment. Similar research reports and papers focusing on the

environmental fate, transport, and toxicity of these carbon nanomaterials is few

compared to studies on the potential applications of them. The data presented in this

thesis shows for the first time: (i) Photo-mineralization of aqueous nC60 occurs. This

work on CO2 generation recently was included in a paper authored by Berry et al [94]

that included the author of this dissertation as a coauthor for this contribution. (ii)

Under sunlight, GO dispersed in water results in significant ROS formation, with

chemical changes occurring on the GO surface. This work on the photochemistry of GO

was recently published with the author of this dissertation as the first author [66]. (iii)

Chemically mediated reduction reactions of aqueous suspensions of single-layered GO

lead to the production of ROS in oxygenated water. The data presented on ROS

generation by GO under sunlight conditions, and under dark reducing conditions, should

inform regulators and policymakers regarding potential environmental impacts of GO.

90

First, by measuring reactive oxygen species (ROS), the photochemical

transformation of single-layered GO under lamps that only emit light in the solar

spectrum (λ =350 ± 50 nm) was investigated. ROS including superoxide radical anion

(O2·−) and hydrogen peroxide (H2O2) were found to be produced under oxic conditions.

Raman Spectra measurements, indicated in increase in the ratio of the ID/IG absorption

bands, suggesting the GO was becoming more reduced. UV-visible absorption spectra

measurements also indicated that GO was undergoing reduction, and the changes in the

spectra obviously were consistent with visible color changes to the GO suspension (i.e.,

from pale to dark brown).

Second, the light-independent reaction of graphene oxide with β-nicotinamide

adenine dinucleotide (NADH) in water was examined. In GO suspensions, oxidation of

NADH to NAD+, evidenced by the decreased intensity of the 340 nm absorption band of

NADH and the increased intensity of the 260 nm absorption band of NAD+. In the

presence of NADH, GO acted as an electron shuttle in water to produce reactive oxygen

species (ROS) by accepting electrons from NADH, transferring these electrons to

molecular oxygen. The production of superoxide anion radical was confirmed by using

tetrazolium salts (NBT2+ and XTT) as scavengers. A DPD/HRP assay was used to detect

H2O2, and was shown to accumulate over time. In GO suspensions containing NADH and

O2, cleavage of the pBR322 DNA plasmid occurred, in spite of finding no detectable ·OH.

Third, the reaction of GO with hydroxyl radicals, produced from the photolytic

cleavage of H2O2 under simulated sunlight was studied. In the presence of 100 mM

H2O2 and light, GO decomposition was evident by following the change in the light

91

attenuation spectra with the UV and visible regions, and also by simple visual changes in

the suspension color. It may be deduced that significant mineralization of GO occurred,

as evidenced by the decrease in the total organic carbon (TOC) content. This research

study indicates that photochemical transformation is likely a significant reaction

pathway for GO in surface waters. This study also provided insight into potential ways of

degrading GO, or even completely mineralizing it, through the use of UV/H2O2 advanced

oxidation methods in wastewater treatment systems.

Finally, mineralization of C60 carbon in aqueous dispersion of aqu/nC60 clusters

was found to occur by simple exposure to light within the solar spectrum. The volume of

CO2 in the headspace increased for light irradiated samples, while the CO2 content in the

dark control samples stayed almost the same. After 20 days under continuous light

exposure approximately 0.7% of the initial C60 carbon was converted to CO2.

6.2 Recommendations

To better understand the mineralization process of aqu/nC60 clusters under

sunlight exposure, 13C labeled C60 should be used. The 13C/12C ratio would further

confirm that the measured CO2 was derived from C60.

Graphene oxide (GO) is usually prepared by a modified Hummer’s method.

However, even a small change in the procedure can make a large difference in the

properties of the GO product with respect to the content of the specific number and

types of functional groups. In the future studies, different types of GO (with different

functional group content) could be studied to more completely understand the fate and

92

reactivity of GO in the environment. Using this approach, the environmental chemistry

of GO, and potentially other functionalized nanomaterials, will be better understood,

including how the weathering of GO proceeds in the environment. Considering that

graphene oxide currently is used for industry processes, it is expected that it, and

materials that contain it, will be eventually disposed in landfills. In landfills, the

environment is considerably different that those environmental conditions studied here.

For example, reduced conditions over a wide range of pH conditions may occur. Thus, is

critical that reactions involving GO under many other environmental conditions (i.e.,

landfills, sediments, soils, etc.) be explored to identify environmental compartments

were GO may be more problematic. In Chapter 3, GO was found to induce DNA damage

by producing reactive oxygen species (ROS) in the absence of light, but in the presence

of a well-known biological reducing agent (NADH). Addition work such as this on the

mechanisms of potential adverse biological effects at the molecular level within cells is

warranted.

8

REFERENCES

93

REFERENCES

1. Diederich F, Whetten RL: Beyond C60: the higher fullerenes. Accounts of chemical research 1992, 25(3):119-126.

2. Murayama H, Tomonoh S, Alford JM, Karpuk ME: Fullerene production in tons

and more: from science to industry. Fullerenes, Nanotubes and Carbon Nanostructures 2005, 12(1-2):1-9.

3. Mauter MS, Elimelech M: Environmental applications of carbon-based

nanomaterials. Environmental Science & Technology 2008, 42(16):5843-5859. 4. Osawa E: Perspectives of fullerene nanotechnology: Springer Science & Business

Media; 2002. 5. Heymann D: Solubility of fullerenes C60 and C70 in seven normal alcohols and

their deduced solubility in water. Fullerene Science & Technology 1996, 4(3):509-515.

6. Jafvert CT, Kulkarni PP: Buckminsterfullerene’s (C60) octanol− water partition

coefficient (K ow) and aqueous solubility. Environmental science & technology 2008, 42(16):5945-5950.

7. Deguchi S, Alargova RG, Tsujii K: Stable dispersions of fullerenes, C60 and C70,

in water. Preparation and characterization. Langmuir 2001, 17(19):6013-6017. 8. Fortner J, Lyon D, Sayes C, Boyd A, Falkner J, Hotze E, Alemany L, Tao Y, Guo W,

Ausman K: C60 in water: nanocrystal formation and microbial response. Environmental Science & Technology 2005, 39(11):4307-4316.

94

9. Andrievsky GV, Kosevich MV, Vovk M, Shelkovsky VS, Vashchenko LA: On the production of an aqueous colloidal solution of fullerenes. J Chem Soc, Chem Commun 1995(12):1281-1282.

10. Kroto HW, Heath JR, O'Brien SC, Curl RF, Smalley RE: C 60: buckminsterfullerene.

Nature 1985, 318(6042):162-163. 11. Dhawan A, Taurozzi JS, Pandey AK, Shan W, Miller SM, Hashsham SA, Tarabara

VV: Stable colloidal dispersions of C60 fullerenes in water: evidence for genotoxicity. Environmental science & technology 2006, 40(23):7394-7401.

12. Oberdörster E: Manufactured nanomaterials (fullerenes, C60) induce oxidative

stress in the brain of juvenile largemouth bass. Environmental health perspectives 2004, 112(10):1058.

13. Sayes CM, Fortner JD, Guo W, Lyon D, Boyd AM, Ausman KD, Tao YJ, Sitharaman

B, Wilson LJ, Hughes JB: The differential cytotoxicity of water-soluble fullerenes. Nano letters 2004, 4(10):1881-1887.

14. Hou W-C, Jafvert CT: Photochemical transformation of aqueous C60 clusters in

sunlight. Environmental science & technology 2009, 43(2):362-367. 15. Hou W-C, Jafvert CT: Photochemistry of aqueous C60 clusters: evidence of 1O2

formation and its role in mediating C60 phototransformation. Environmental science & technology 2009, 43(14):5257-5262.

16. Hou W-C, Kong L, Wepasnick KA, Zepp RG, Fairbrother DH, Jafvert CT:

Photochemistry of aqueous C60 clusters: wavelength dependency and product characterization. Environmental science & technology 2010, 44(21):8121-8127.

17. Arbogast JW, Darmanyan AP, Foote CS, Diederich FN, Whetten R, Rubin Y,

Alvarez MM, Anz SJ: Photophysical properties of sixty atom carbon molecule (C60). The Journal of Physical Chemistry 1991, 95(1):11-12.

95

18. Lee J, Cho M, Fortner JD, Hughes JB, Kim J-H: Transformation of aggregated C60 in the aqueous phase by UV irradiation. Environmental science & technology 2009, 43(13):4878-4883. 19. Hartmann NB, Buendia IM, Bak J, Baun A: Degradability of aged aquatic

suspensions of C< sub> 60</sub> nanoparticles. Environmental Pollution 2011, 159(10):3134-3137.

20. Hwang YS, Li Q: Characterizing photochemical transformation of aqueous nC60

under environmentally relevant conditions. Environmental science & technology 2010, 44(8):3008-3013.

21. Dreyer DR, Park S, Bielawski CW, Ruoff RS: The chemistry of graphene oxide.

Chemical Society Reviews 2010, 39(1):228-240. 22. Barroso-Bujans F, Cerveny S, Verdejo R, del Val J, Alberdi J, Alegría A, Colmenero

J: Permanent adsorption of organic solvents in graphite oxide and its effect on the thermal exfoliation. Carbon 2010, 48(4):1079-1087.

23. Jung JH, Cheon DS, Liu F, Lee KB, Seo TS: A Graphene Oxide Based Immuno‐

biosensor for Pathogen Detection. Angewandte Chemie International Edition 2010, 49(33):5708-5711.

24. Lightcap IV, Kosel TH, Kamat PV: Anchoring semiconductor and metal

nanoparticles on a two-dimensional catalyst mat. storing and shuttling electrons with reduced graphene oxide. Nano letters 2010, 10(2):577-583.

25. Song Y, Qu K, Zhao C, Ren J, Qu X: Graphene oxide: intrinsic peroxidase catalytic

activity and its application to glucose detection. Advanced Materials 2010, 22(19):2206-2210.

26. Sun X, Liu Z, Welsher K, Robinson JT, Goodwin A, Zaric S, Dai H: Nano-graphene

oxide for cellular imaging and drug delivery. Nano research 2008, 1(3):203-212.

96

27. Yang K, Wan J, Zhang S, Tian B, Zhang Y, Liu Z: The influence of surface chemistry and size of nanoscale graphene oxide on photothermal therapy of cancer using ultra-low laser power. Biomaterials 2012, 33(7):2206-2214.

28. Liu J-Y, Bai Y, Luo P-Y, Wang P-Q: One-pot synthesis of graphene–BiOBr

nanosheets composite for enhanced photocatalytic generation of reactive oxygen species. Catalysis Communications 2013, 42:58-61.

29. Morales-Torres S, Pastrana-Martínez LM, Figueiredo JL, Faria JL, Silva AM: Design

of graphene-based TiO2 photocatalysts—a review. Environmental Science and Pollution Research 2012, 19(9):3676-3687.

30. Moon G-h, Kim D-h, Kim H-i, Bokare AD, Choi W: Platinum-like behavior of

reduced graphene oxide as a cocatalyst on TiO2 for the efficient photocatalytic oxidation of arsenite. Environmental Science & Technology Letters 2014, 1(2):185-190.

31. Akhavan O, Ghaderi E: Toxicity of graphene and graphene oxide nanowalls

against bacteria. Acs Nano 2010, 4(10):5731-5736. 32. Liao K-H, Lin Y-S, Macosko CW, Haynes CL: Cytotoxicity of graphene oxide and

graphene in human erythrocytes and skin fibroblasts. ACS applied materials & interfaces 2011, 3(7):2607-2615.

33. Wang K, Ruan J, Song H, Zhang J, Wo Y, Guo S, Cui D: Biocompatibility of

graphene oxide. Nanoscale Res Lett 2011, 6(8). 34. Hu W, Peng C, Lv M, Li X, Zhang Y, Chen N, Fan C, Huang Q: Protein corona-

mediated mitigation of cytotoxicity of graphene oxide. Acs Nano 2011, 5(5):3693-3700.

35. Chang Y, Yang S-T, Liu J-H, Dong E, Wang Y, Cao A, Liu Y, Wang H: In vitro toxicity

evaluation of graphene oxide on A549 cells. Toxicology letters 2011, 200(3):201-210.

97

36. Guardia L, Villar-Rodil S, Paredes J, Rozada R, Martínez-Alonso A, Tascón J: UV light exposure of aqueous graphene oxide suspensions to promote their direct reduction, formation of graphene–metal nanoparticle hybrids and dye degradation. Carbon 2012, 50(3):1014-1024.

37. Kim SR, Parvez MK, Chhowalla M: UV-reduction of graphene oxide and its

application as an interfacial layer to reduce the back-transport reactions in dye-sensitized solar cells. Chemical Physics Letters 2009, 483(1):124-127.

38. Matsumoto Y, Koinuma M, Ida S, Hayami S, Taniguchi T, Hatakeyama K, Tateishi

H, Watanabe Y, Amano S: Photoreaction of graphene oxide nanosheets in water. The Journal of Physical Chemistry C 2011, 115(39):19280-19286.

39. Hou W-C, Chowdhury I, Goodwin Jr DG, Henderson WM, Fairbrother DH,

Bouchard D, Zepp RG: Photochemical transformation of graphene oxide in sunlight. Environmental science & technology 2015, 49(6):3435-3443.

40. Chowdhury I, Hou W-C, Goodwin D, Henderson M, Zepp RG, Bouchard D:

Sunlight affects aggregation and deposition of graphene oxide in the aquatic environment. Water research 2015, 78:37-46.

41. Chowdhury I, Mansukhani ND, Guiney LM, Hersam MC, Bouchard D: Aggregation

and Stability of Reduced Graphene Oxide: Complex Roles of Divalent Cations, pH, and Natural Organic Matter. Environmental science & technology 2015, 49(18):10886-10893.

42. McCreery RL: Advanced carbon electrode materials for molecular

electrochemistry. Chem Rev 2008, 108(7):2646-2687. 43. Dumitrescu I, Unwin PR, Macpherson JV: Electrochemistry at carbon nanotubes:

perspective and issues. Chemical Communications 2009(45):6886-6901. 44. Tang L, Wang Y, Li Y, Feng H, Lu J, Li J: Preparation, structure, and

electrochemical properties of reduced graphene sheet films. Advanced Functional Materials 2009, 19(17):2782-2789.

98

45. Shao Y, Wang J, Wu H, Liu J, Aksay IA, Lin Y: Graphene based electrochemical sensors and biosensors: a review. Electroanalysis 2010, 22(10):1027-1036. 46. Geim AK, Novoselov KS: The rise of graphene. Nature materials 2007, 6(3):183-

191. 47. Geim AK: Graphene: status and prospects. science 2009, 324(5934):1530-1534. 48. Wang X, Zhi L, Müllen K: Transparent, conductive graphene electrodes for dye-

sensitized solar cells. Nano letters 2008, 8(1):323-327. 49. Bitter JL, Yang J, BeigzadehMilani S, Jafvert CT, Fairbrother DH: Transformations

of oxidized multiwalled carbon nanotubes exposed to UVC (254 nm) irradiation. Environ Sci Nano 2014, 1:324-337.

50. Chen C-Y, Jafvert CT: Photoreactivity of Carboxylated Single-Walled Carbon

Nanotubes in Sunlight: Reactive Oxygen Species Production in Water. Environmental Science & Technology 2010, 44(17):6674-6679.

51. Chen C-Y, Jafvert CT: The role of surface functionalization in the solar light-

induced production of reactive oxygen species by single-walled carbon nanotubes in water. Carbon 2011, 49(15):5099-5106.

52. Haag WR, Hoigne J: Singlet oxygen in surface waters. 3. Photochemical

formation and steady-state concentrations in various types of waters. Environmental science & technology 1986, 20(4):341-348.

53. Bader H, Sturzenegger V, Hoigne J: Photometric method for the determination

of low concentrations of hydrogen peroxide by the peroxidase catalyzed oxidation of N, N-diethyl-< i> p</i>-phenylenediamine (DPD). Water Research 1988, 22(9):1109-1115.

99

54. Hou X-L, Li J-L, Drew SC, Tang B, Sun L, Wang X-G: Tuning radical species in graphene oxide in aqueous solution by photoirradiation. The Journal of Physical Chemistry C 2013, 117(13):6788-6793.

55. Paredes J, Villar-Rodil S, Martinez-Alonso A, Tascon J: Graphene oxide

dispersions in organic solvents. Langmuir 2008, 24(19):10560-10564. 56. Radich JG, Kamat PV: Making graphene holey. Gold-nanoparticle-mediated

hydroxyl radical attack on reduced graphene oxide. ACS nano 2013, 7(6):5546-5557.

57. Dresselhaus MS, Jorio A, Hofmann M, Dresselhaus G, Saito R: Perspectives on

carbon nanotubes and graphene Raman spectroscopy. Nano letters 2010, 10(3):751-758.

58. Eda G, Chhowalla M: Chemically Derived Graphene Oxide: Towards Large‐Area

Thin‐Film Electronics and Optoelectronics. Advanced Materials 2010, 22(22):2392-2415.

59. Hou W-C, BeigzadehMilani S, Jafvert CT, Zepp RG: Photoreactivity of

Unfunctionalized Single-Walled Carbon Nanotubes Involving Hydroxyl Radical: Chiral Dependency and Surface Coating Effect. Environ Sci Technol 2014, 48:3875-3882.

60. Hsieh H-S, Wu R, Jafvert CT: Light-independent reactive oxygen species (ROS)

formation through electron transfer from carboxylated single-walled carbon nanotubes in water. Environmental science & technology 2014, 48(19):11330-11336.

61. Novoselov KS, Geim AK, Morozov S, Jiang D, Zhang Y, Dubonos S, Grigorieva I,

Firsov A: Electric field effect in atomically thin carbon films. science 2004, 306(5696):666-669.

100

62. Liu Z, Robinson JT, Sun X, Dai H: PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. Journal of the American Chemical Society 2008, 130(33):10876-10877.

63. Pyun J: Graphene oxide as catalyst: application of carbon materials beyond

nanotechnology. Angewandte Chemie International Edition 2011, 50(1):46-48. 64. Wang K, Ruan J, Song H, Zhang J, Wo Y, Guo S, Cui D: Biocompatibility of

graphene oxide. Nanoscale Res Lett 2011, 6(8):1. 65. Zhang L, Li Y, Zhang L, Li D-W, Karpuzov D, Long Y-T: Electrocatalytic oxidation of

NADH on graphene oxide and reduced graphene oxide modified screen-printed electrode. Int J Electrochem Sci 2011, 6(3):819-829.

66. Zhao Y, Jafvert CT: Environmental photochemistry of single layered graphene

oxide in water. Environmental Science: Nano 2015, 2(2):136-142. 67. Bartosz G: Use of spectroscopic probes for detection of reactive oxygen species.

Clinica Chimica Acta 2006, 368(1):53-76. 68. Sutherland MW, Learmonth BA: The tetrazolium dyes MTS and XTT provide new

quantitative assays for superoxide and superoxide dismutase. Free radical research 1997, 27(3):283-289.

69. Baumgarten B, Hones J: Spectroscopic investigation of dihydronicotinamides‐II.

dihydronicotinamide adenine dinucleotide complexes with dehydrogenases. Photochemistry and photobiology 1988, 47(2):201-205.

70. Fisher P, Fleckenstein J, Hönes J: Spectroscopic investigation of

dihydronicotinamides‐I: conformation, absorption, and fluorescence. Photochemistry and photobiology 1988, 47(2):193-199.

101

71. Fu H, Zhu D: Graphene oxide-facilitated reduction of nitrobenzene in sulfide-containing aqueous solutions. Environmental science & technology 2013, 47(9):4204-4210.

72. Zuo Y, Hoigne J: Formation of hydrogen peroxide and depletion of oxalic acid in

atmospheric water by photolysis of iron (III)-oxalato complexes. Environmental Science & Technology 1992, 26(5):1014-1022.

73. Pogozelski WK, Tullius TD: Oxidative strand scission of nucleic acids: routes

initiated by hydrogen abstraction from the sugar moiety. Chemical reviews 1998, 98(3):1089-1108.

74. Chevion M: A site-specific mechanism for free radical induced biological

damage: the essential role of redox-active transition metals. Free Radical Biology and Medicine 1988, 5(1):27-37.

75. Cooke MS, Evans MD, Dizdaroglu M, Lunec J: Oxidative DNA damage:

mechanisms, mutation, and disease. The FASEB Journal 2003, 17(10):1195-1214. 76. Dikin DA, Stankovich S, Zimney EJ, Piner RD, Dommett GH, Evmenenko G,

Nguyen ST, Ruoff RS: Preparation and characterization of graphene oxide paper. Nature 2007, 448(7152):457-460.

77. Williams G, Kamat PV: Graphene− semiconductor nanocomposites: excited-

state interactions between ZnO nanoparticles and graphene oxide†. Langmuir 2009, 25(24):13869-13873.

78. Krishnamurthy S, Lightcap IV, Kamat PV: Electron transfer between methyl

viologen radicals and graphene oxide: Reduction, electron storage and discharge. Journal of Photochemistry and Photobiology A: Chemistry 2011, 221(2):214-219.

79. Fan W, Lai Q, Zhang Q, Wang Y: Nanocomposites of TiO2 and reduced graphene

oxide as efficient photocatalysts for hydrogen evolution. The Journal of Physical Chemistry C 2011, 115(21):10694-10701.

102

80. Zhang W, Wang C, Li Z, Lu Z, Li Y, Yin JJ, Zhou YT, Gao X, Fang Y, Nie G: Unraveling Stress‐Induced Toxicity Properties of Graphene Oxide and the Underlying Mechanism. Advanced Materials 2012, 24(39):5391-5397.

81. Duch M, Budinger G, Urich D, Soberanes S, Chiarella S, Gonzalez A, Radigan K,

Chandel N, Hersam M, Mutlu G: Graphene oxide but not graphene causes severe acute lung injury and pulmonary fibrosis in mice. Am J Respir Crit Care Med 2011, 183:A2283.

82. Kotchey GP, Allen BL, Vedala H, Yanamala N, Kapralov AA, Tyurina YY, Klein-

Seetharaman J, Kagan VE, Star A: The enzymatic oxidation of graphene oxide. ACS nano 2011, 5(3):2098-2108.

83. Gómez-Navarro C, Weitz RT, Bittner AM, Scolari M, Mews A, Burghard M, Kern K:

Electronic transport properties of individual chemically reduced graphene oxide sheets. Nano letters 2007, 7(11):3499-3503.

84. Zhou X, Zhang Y, Wang C, Wu X, Yang Y, Zheng B, Wu H, Guo S, Zhang J: Photo-

Fenton reaction of graphene oxide: a new strategy to prepare graphene quantum dots for DNA cleavage. Acs Nano 2012, 6(8):6592-6599.

85. Lanphere JD, Rogers B, Luth C, Bolster CH, Walker SL: Stability and transport of

graphene oxide nanoparticles in groundwater and surface water. Environmental engineering science 2014, 31(7):350-359.

86. Page SE, Arnold WA, McNeill K: Assessing the contribution of free hydroxyl

radical in organic matter-sensitized photohydroxylation reactions. Environmental science & technology 2011, 45(7):2818-2825.

87. Zepp RG, Hoigne J, Bader H: Nitrate-induced photooxidation of trace organic

chemicals in water. Environmental science & technology 1987, 21(5):443-450. 88. Zepp RG, Faust BC, Hoigne J: Hydroxyl radical formation in aqueous reactions

(pH 3-8) of iron (II) with hydrogen peroxide: the photo-Fenton reaction. Environmental Science & Technology 1992, 26(2):313-319.

103

89. Colvin VL: The potential environmental impact of engineered nanomaterials. Nature biotechnology 2003, 21(10):1166-1170.

90. Inui S, Aoshima H, Nishiyama A, Itami S: Improvement of acne vulgaris by

topical fullerene application: unique impact on skin care. Nanomedicine: Nanotechnology, Biology and Medicine 2011, 7(2):238-241.

91. Su D: Product characterization and headspace analysis of solar irradiated

aqueous C60 clusters. 2013. 92. Duncan LK, Jinschek JR, Vikesland PJ: C60 colloid formation in aqueous systems:

effects of preparation method on size, structure, and surface charge. Environmental science & technology 2007, 42(1):173-178.

93. Bachilo SM, Benedetto AF, Weisman RB: Triplet state dissociation of C120, the

dimer of C60. The Journal of Physical Chemistry A 2001, 105(43):9845-9850. 94. Berry TD, Clavijo AP, Zhao Y, Jafvert CT, Turco RF, Filley TR: Soil microbial

response to photo-degraded C 60 fullerenes. Environmental Pollution 2016, 211:338-345.

13

VITA

104

VITA

Yingcan Zhao, a single child of Honglai Zhao and Wenyan Zhang, was born in

Heilongjiang Province, China in 1990. She received her B.S. in environmental science

from Shandong University in China in June 2012. After coming to Purdue for Ph.D. study

in Fall 2012, she developed a special interest in environmental nanotechnology

including photo mineralization of Aqu/nC60 clusters and environmental reactions of

graphene oxide. She has already published two papers in scientific journals on the topic.

Her paper “Environmental Photochemistry of Graphene Oxide in Water” was selected as

the cover of the journal “Environmental Science: Nano”. She was also a recipient of

Joseph P. Chu Fellowship, Ron Wukasch Environmental Engineering Scholarship, Pai Tao

Yeh Fellowship (twice) while at Purdue.

Personally, she likes traveling, swimming and playing instruments. She hopes to

travel all over the world.

reactions of graphene oxide and buckminsterfullerene in

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