Reactions of Graphene Oxide and Buckminsterfullerene in the Aquatic Environment Yingcan Zhao Purdue University

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PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared

By Yingcan Zhao

Entitled REACTIONS OF GRAPHENE OXIDE AND BUCKMINSTERFULLERENE IN THE AQUATIC ENVIRONMENT

For the degree of Doctor of Philosophy

Is approved by the final examining committee:

Chad T. Jafvert Chair Timothy R. Filley

Inez Hua

Ronald F. Turco

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material.

Approved by Major Professor(s): Chad T. Jafvert

Approved by: Dulcy M. Abraham 6/21/2016 Head of the Departmental Graduate Program Date

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.

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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 3.4 Results and Discussion ...... 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 Materials and Methods ...... 63 4.3.1 Materials ...... 63 4.3.2 Irradiation of GO in Solar Spectrum Light ...... 63 4.3.3 Materials Analysis ...... 64 4.4 Results and Discussion ...... 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 Materials and Methods ...... 75 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 5.4 Results and Discussion ...... 80 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

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

1 1 3 singlet C60 ( C60), and then C60 is fast converted into triplet C60 ( C60) through intersystem crossing (ISC) (suggested mechanism shown in equation 1.1). Subsequently,

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

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

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

hϑ ISC O 1 3 2 1 C60 → C60→ C60 → C60+ O2 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.

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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.

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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).

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hϑ GO → 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.

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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.

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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

1 oxygen ( O2) 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)

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

1 ·− used at a time, as those for singlet oxygen ( O2 ) 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.

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

2+ ·− blue tetrazolium salt (referred to as NBT ) 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

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

1 containing furfuryl alcohol as a scavenging probe for O2, 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

a

b

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.

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

0.18 0.16 0.14 0.12 0.1 Spectrum of Lamp Light Irradiation 0.08 0.06 Spectrum of Solar (mW/cm2/nm) Irradiation 0.04

0.02 Spectrual Spectrual density ofIrradiance 0 250 300 350 400 450 500 550 600 650 700 Wavelength (nm)

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).

34

1 Measurement of O2

1 FFA was used to detect if O2 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

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

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

·− Detection of 퐎ퟐ 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

∙− 2+ ∙− superoxide anion (O2 ), as the reaction between NBT 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.

3.4 Results and Discussion

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 x (NADH + H+) + GO → (NAD+ + 2H+) + GOx− eq 3.1 2 2

46

b

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.

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

∙− 2+ O2 formation (i.e., NBT 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

2+ ∙− 1 primarily results from the reduced rate of the final reaction (i.e., NBT + 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

a

b

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.

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) ().

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·− 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

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·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.

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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.

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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 Materials and Methods

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).

4.4 Results and Discussion

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.

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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).

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a

b

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.

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Figure 4.2 continued

c

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.

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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

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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

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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.

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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

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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

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of time (i.e., weeks). The process involves the photoexcitation from ground-state C60 to

1 singlet C60 ( C60 where one of the 60 carbons in the molecule has one electron in the

1 3 excited state), followed by the fast conversion of C60 to triplet C60 ( C60) by intersystem

3 3 crossing. Finally, C60 is quenched by O2 through energy transfer from C60 to O2,

1 resulting in the formation of the reactive oxygen species (ROS) singlet oxygen, O2 [15].

More work has been done in our group to show that ROS species (including

1 ∙− O2 , 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

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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 Materials and Methods

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).

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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

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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.

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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

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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).

+ − − + 2− 퐻2퐶푂3 ⇔ 퐻 + 퐻퐶푂3 ; 퐻퐶푂3 ⇔ 퐻 + 퐶푂3 eq 5.1

Below pH 3, essentially all the inorganic carbon in the samples exists as CO2 either in the

퐶푂2,푎푞 aqueous phase (CO2,aq) or in the headspace (CO2,g). Henry’s constant (퐾퐻 = )can 퐶푂2,푔

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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].

5.4 Results and Discussion

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

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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.

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

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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).

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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.

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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

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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:

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3 CO (M) = ( × V ) ÷ V ÷ RT eq 5.2 2(g) 2 measured sampled

This procedure was confirmed with and applied to all CO2 analytical standards.

CO2 (M) Then, Henry’s constant ( k = (aq) = 0. 8317 ) was used to calculate the aqueous CO2(g) (M) 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 CO2 (measured) CO2 CO2 CO2 total

(days) (μL) (M, gas) (M, aqueous) (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

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Table 5.2. Summary of CO2 measurements for dark control samples

Time CO2 (measured) CO2 CO2 CO2 total

(days) (μL) (M, gas) (M, aqueous) (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.

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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.

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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

13 13 12 sunlight exposure, C labeled C60 should be used. The C/ C 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

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VITA

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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.