CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

Carbon Quantum Dots (CQDs) as Probes of Biological Systems

A thesis submitted in partial fulfillment of the requirements

For the degree of Master of Science in Chemistry

By

Osma Gomez

May 2021
 Signature Page
This thesis of Osma Gomez is approved

Dr. Jussi Eloranta Date

Dr. Brenton A.G. Hammer Date

Dr. Daniel Tamae Date

Dr. Joseph A. Teprovich, Chair Date

California State University, Northridge

ii
 Dedication

I would like to dedicate this thesis to my aunt, Silvia Vilma Guerra. She has had an
overwhelming influence on my life and has been a key to my research. I hope we get closer to
finding a cure to breast cancer.

May you rest in peace.

You are truly missed and have been in my thoughts ever since you passed away.

iii
 Acknowledgement

First and foremost, I have to thank Dr. Joseph A. Teprovich. I have spent four years in his

laboratory and been very impressed by all his knowledge, confidence and support throughout this

program. I entered the master’s program with minimum physical chemistry background.

Regardless of my academic background, Dr. Teprovich always had sufficient amounts of patience

and kindness to tolerate me and hold me accountable to different tasks. I am going to miss being

in his lab!

I would also like to show gratitude to my committee members, Dr. Jussi Eloranta, Dr.

Brenton Hammer and Dr. Daniel Tamae. Dr. Eloranta was my first advisor through my chemistry

undergraduate program. His teaching style and enthusiasm in physical chemistry made a strong

influence to pursue a chemistry master’s degree. Dr. Hammer for reinforcing my organic

chemistry skills, mentorship and encouragement. Dr. Tamae for his biochemistry collaboration in

my thesis project. Even though they all had other priorities, I feel very honored and moved to have

a great thesis committee for they have given me unfailing support, mentorship and encouragement

throughout my master’s program

I would like to thank my collaborator, Dr. Paula Fischhaber for all the bioimaging help,

guidance and support in my research as well as life experience outside of research. I would also

like to thank Dr. Mike Kaiser for troubleshooting instrumentation and guidance. Michael and Dee

Dee for their help and patience and prompt response.

I also like to thank all grad students in the chemistry graduate program who had made this

journey doable. They have given me great support, laughter and encouragement. I hope it won’t

be the last and we continue our friendship beyond the master’s degree.

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 I would like to finally thank my sister, friends and family for their unfailing and genuine

support. They helped me relieve stress and pushed me to greater scholastic achievement beyond

this program. Their motivation and attentiveness were very helpful. I wouldn’t be here if it wasn’t

for them.

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 Table of Contents

Signature Page ii

Dedication iii

Acknowledgement iv

List of Schemes x

List of Tables xi

Abstract xii

Introduction 1

1.1 Properties of light 1

1.2 Absorption of Light 3
1.3 Luminescence 4
1.4 Jablonski Diagram 6
1.5 Lifetime 9
1.6 Quantum Yield 10
1.7 Stokes Shift 11
1.8 Frank-Condon Principle 12

Quantum Dots 15

2.1 Background 16
2.2 Fullerene based carbon dots 18
2.3 LiBH4-C60 CQDs 19
2.4 Gadolinium Doped CQDs 20
2.5 Chiral Doped CQDs 20
2.6 Lawsone CQDs 21

Biological Systems 23

3.1 Cancer 23
3.2 MRI Contrasting Agents 24

Methodology 25

vi
 4.1 Fourier Transform Infrared Spectroscopy 25
4.2 Ultraviolet-visible Spectroscopy 28
4.3 Fluorescence Spectroscopy 33
4.4 Nuclear Magnetic Resonance 34
4.5 MTT assay 37
4.6 Fluorescence Microscopy 38
4.7 X-Ray Diffraction (XRD) 42
4.8 Circular Dichroism 43

Results 47

5.1 Synthesis of CQDs 47

5.1.1 Synthesis of 70:30 LiBH4-C60 CQDs 47
5.1.2 Synthesis of 70:30 LiBH4-C60-GdCl3 CQDs 49
5.1.3 Synthesis of 70:30 LiBH4-C60-EDC/NHS-Trp CQDs 52
5.1.4 Synthesis of Lawsone CQDs 54
5.2 Cell Toxicity 57
5.2.1 MTT Assay of 70:30 LiBH4-C60 CQDs 58
5.2.2 MTT Assay of 70:30 LiBH4-C60-GdCl3 CQDs 59
5.2.3 MTT Assay of GdCl3 60
5.3 Fluorescent Microscopy 60
5.3.1 LiBH4-C60 CQDs 61
5.4 Circular Dichroism 63
5.4.1 LiBH4-C60-EDC/NHS-Trp CQDs 63
5.5 Nuclear Magnetic Resonance T1 Relaxation 64
5.5.1 LiBH4-C60-CQDs 65
5.5.2 LiBH4-C60-GdCl3 CQDs 66

Bioimaging Implications and Conclusion 67

References 69

vii
 List of Figures

Figure 1 - Electromagnetic Wave 2

Figure 2 - Single Beam Spectrophotometer Schematic Diagram 3
Figure 3 - Luminescence Energy Diagram 4
Figure 4 - Jablonski diagram showing the absorption, emission and phosphorescence 8
processes and time scales
Figure 5 - Stoke Shift with minimal self-absorption 12
Figure 6 - Frank Condon Principle transition representation of two electronic states 13
and an absorption transition
Figure 7 - FTIR Schematic 27
Figure 8 - Dual Bean UV-Vis Spectrometer Schematic 29
Figure 9- Electronic excitations diagram 31
Figure 10- Energetic electron transitions 32
Figure 11- Fluorescence Schematic 34
Figure 12- General NMR Schematic 35
Figure 13- NMR T1 Relaxation 37
Figure 14- Fluorescence Microscopy Schematic 39
Figure 15- Filter Cube 41
Figure 16- XRD Basic Schematic 42
Figure 17- Scattering & Diffraction: The Bragg’s Law 43
Figure 18- Unpolarized light with Circular & Linear Polarized Light 44
Figure 19- Circular Dichroism Schematic 45
Figure 20- FTIR of CQDs 47
Figure 21- CQDs Emission Map 48
Figure 22- CQDs Excitation & Emission Spectrum 48
Figure 23- CQDs Photostability spectrum 49
Figure 24- Emission Map of Gd-CQDs 50
Figure 25- FTIR of Gd-doped CQDs 51
Figure 26- XRD on Gd doped CQDs 52
Figure 27- 0 Hour Emission Spectra 55
Figure 28- 1 Hour Emission Spectra 55

viii
Figure 29- 12 Hours Emission Spectra 56
Figure 30- 24 Hours Emission Spectra 56
Figure 31- 575 nm excitation spectra 56
Figure 32- Qualify effects inhibit on cell metabolism spectrum 57
Figure 33- Dialyzed CQDs comparison to control 58
Figure 34- Dialyzed CQDs comparison to the lowest concentration 50ug/mL 58
Figure 35- Undialyzed CQDs comparison to control 59
Figure 36- Undialyzed CQDs comparison to the lowest concentration 50ug/mL 59
Figure 37- Gd-CQDs comparison to control 59
Figure 38- Gd-CQDs comparison to the lowest concentration 50ug/mL 59
Figure 39- Pure Gd MTT assay comparison to control 60
Figure 40- Pure Gd MTT assay comparison to the lowest concentration 50ug/mL 60
Figure 41- Chiroptical properties of tryptophan enantiomers and their respective 63
chiral CQDs
Figure 42- FTIR of Enantiomeric CQDs 64
Figure 43- NMR Initial CQD Results 65
Figure 44- Initial Gd NMR Results 66

ix
 List of Schemes

Scheme 1- Tautomeric forms of 2-hydroxy-1, 4-naphthoquinone 22

Scheme 2- NADH Oxidoreductase Reaction 37
Scheme 3- Synthesis overall pathway of Gd-CQDs 50
Scheme 4- Synthesis overall pathway of chiral functionalization of CQDs 53
Scheme 5- Cross-coupling reaction of EDC/NHS to CQD mechanism 53
Scheme 6- Tryptophan added to CQD mechanism 54
Scheme 7- Synthesis overall pathway of generating lawsone CQDs 54

x
 List of Tables

Table 1- Initial Optimization Experiments for Fluorescence Microscopy 61

Table 2- New Fluorescence Microscope Images 61
Table 3- MDA-MB-231 Cells with three different focal planes 62
Table 4- Initial CQD NMR Results 64
Table 5- Gadoderitol NMR Results 64
Table 6- MRI CQDs T1 & T2 Results 65
Table 7- Initial GD NMR Results 65
Table 8- MRI Gd-CQDs T1 & T2 Results 66

xi
 Abstract

Carbon Quantum Dots (CQDs) as Probes of Biological Systems

By

Osma Gomez

Master of Science in Chemistry

In order to better understand and treat disease, there is an essential need to develop a

multimodal probe that can obtain chemical information about processes that occur at both the

microscale (cellular level) and the macroscale (tissues, organs). The current focus of this project

is to determine if CQDs can be used for fluorescent microscopy imaging at the cellular level and

MRI contrast agents for tissues and organs on the macroscale. The cell line utilized for

fluorescence imaging and cytotoxicity tests are the MDA-MB-231 breast cancer cells. The

cytotoxicity of the CQDs and gadolinium doped CQDs has been evaluated through MTT assays

indicates that they do not affect cell viability up to concentration of 500𝜇g/mL. Fluorescence

microscopy studies show that the CQDs are useful as dyes and that they localize in different parts

of the cell. NMR and MRI experiments demonstrates that the CQDs shorten the T1 and T2

relaxations of water protons.

xii
 Additional work has demonstrated that it is possible to functionalize the surface of the

CQDs with chiral amino acids. Lawsone (henna plant) has been demonstrated as an interesting

candidate for the production of CQDs which is a natural sustainable carbon source for their

production.

xiii
 Introduction

Light plays a vital role in our everyday lives and has become an essential tool in meeting

the demands of our 21st century world. Light-based technologies protect health and safety,

provide sustainable energy, enable space explorations, advance lighting options in rural areas,

enable communication via the Internet, and hold the promise of limitless possibilities to improve

the human condition and protect the earth. However, in physics, light is defined as all portions of

the electromagnetic spectrum.1 Visible light is a small portion of the electromagnetic radiation

spectrum (400-700 nm) and will be primary focus of this thesis. In particular, carbon quantum

dots (CQDs), consist of fluorescent carbon nanoparticles that exhibit luminescence in distinctive

forms, that has been studied for the past decade2 because of their potential applications electronics,

fluorescence imaging, sensing, etc.3 It has been demonstrated that these novel nanomaterials have

good photostability and tunable luminescence.4 The purpose of this work is to develop a new

fluorescent CQD platform that can be used as a multi-modal probes of biological systems with a

focus on fluorescent imaging and MRI contrasting agents. A quantum dot is a quantum

mechanically confined system, which means that its optical properties are a function of its size.

For example, as the size of the quantum dot is increased, the photoluminescence shifts from the

blue portion of the visible spectrum toward the red portion of the visible spectrum. However, one

of many drawbacks of the first-generation QDs is that they are typically composed of cadmium

and other transition metals, which is toxic to biological systems. Due to this, alternative efforts

were initiated to replace cadmium and other transition metals with carbon.

1.1 Properties of light

Photons are electromagnetic radiation, which have both particle and wave character. For

all electromagnetic radiation, the oscillating components of the electric and magnetic fields are

1
perpendicular to each other along the direction of propagation. For simplicity, we can look at a

plane-polarized electromagnetic radiation wave in Figure 1.

Figure 1: Electromagnetic Wave

Electromagnetic waves can be described by their wavelength, energy, and frequency shown in

equation 1.1.1.

ℎ𝑐
𝐸= = ℎ𝜐 (1.1.1)
𝜆

%
where ℎ is Planck’s constant (6.626 × 10!"# 𝐽 ∙ 𝑠), 𝑐 is the speed of light 32.998 × 10$ & 6, 𝜆 is

the wavelength (m) and 𝜐 is the frequency (𝑠 !' ).

The Poynting Theorem states that the work done electromagnetic force is equal to the

decrease in energy stored in the field when accounting for the energy which flowed out through

the surface.5 The Poynting Vector (S) is a vector defined by Equation 1.1.2.

1
𝑆⃗ = 9𝐸:⃗ × 𝐻
:⃗< (1.1.2)
𝜇(

𝜇) is known as the permeability of the vacuum, E is the electric field and H is the magnetic field

vectors.6 It represents the energy flux density, 𝑆 ∙ 𝑑𝑎, as the energy per unit time transported by

the field across a surface da. According to the Poynting Theorem, the irradiance (intensity) that is

related to an electromagnetic wave of light is proportional to the square of the amplitude of the

2
electric field.6 When light interacts with a substance, this substance will be changed, mainly

through its velocity and its intensity. Additionally, some materials are able to alter light differently

in each spatial direction.

1.2 Absorption of Light

When a molecule absorbs a photon, electrons get promoted from a lower energy level

(ground state) to a higher energy level (excited state). In band theory, the ground state is described

as the conduction band and the excited state as the valence band.

When light is absorbed by a sample, the intensity of the beam of light is decreased after

passing through a sample.9 In a UV-Vis spectrophotometer, a light source generates a wide

spectrum of electromagnetic radiation. This light passes through a monochromator that selects a

particular wavelength of light to send to the sample. The intensity of this selected wavelength of

light (Io) is reduced as it passes through the sample. The intensity of the light that passes through

the sample (I), is then sent to a detector where it is measured. A block diagram in Figure 2 shown

below describes how this process occurs in a single beam spectrophotometer.

Figure 2: Single Beam Spectrophotometer Schematic Diagram

UV-vis spectrometers measure the transmittance (T) of a sample, which is the ratio of the

intensity of the light passing through the sample to the intensity of the incident light before entering

the sample shown in equation 1.2.1.

*
𝑇=* (1.2.1)
!

3
T can range anywhere from 0 to 1 or 0 to 100% if measured in percent transmittance. Absorbance

on the other hand is defined by equation 1.2.2.

*
𝐴 = log') 3* 6 = − log') 𝑇 (1.2.2)
!

If no light is absorbed, 𝐴 = 0 and 𝐼 = 𝐼( .7 If 80% of the light is absorbed, 20% is transmitted and
*
𝐼 = +)! , therefore 𝐴 = 1.30.

1.3 Luminescence

Luminescence is the emission of light due to the transition of electrons from a higher

energy state to lower energy state. Each energy level can be further divided into vibrational states,

in which the electrons can occupy in an orbital. Luminescence is categorized based on the excited

state and the source of energy that permitted the excited state. The cause of excitation, which is

the instance when electrons are promoted to an excited state, is typically achieved through the

absorption of ultraviolet radiation or visible light. If the luminescence results from a singlet excited

state to a singlet ground state, it will be referred to fluorescence as shown in Figure 3.

Figure 3: Luminescence Energy Diagram

The term singlet state indicates that an electron pair has opposite spins. In fluorescence, there is

an emission of a photon with a lower energy and a longer wavelength in comparison to the

4
absorbed photon. This is due to vibration relaxation of the excited state and translations motion of

the atoms in the molecules or the solvent. The vibrational energy can be transferred to another

molecule by collision, to rotational motion in the same molecule or to a different vibrational mode

within the same molecule. The process of fluorescence occurs very quickly, within 10-9 s to 10-6 s.

However, if luminescence is due to the transition of an electron from a triplet excited state

to a singlet state, luminescence will be categorized as phosphorescence as shown in Figure 3. In

this process, the electron pair will have their spins aligned in the same direction with one in the

ground state and the other in the excited state. The triplet state occurs when the spin quantum

number changes through collision. During this event, there is intersystem crossing, in which the

molecule goes from an excited singlet state to a triplet state as seen in Figure 3. The triplet state

has higher vibrational energy and a lower electronic energy in comparison to a singlet state due to

a reduced interelectronic repulsion in the triplet state. In phosphorescence, the molecule will lose

vibrational energy and it will go to the lowest excited triplet state. The process of phosphorescence

is relatively slow, 10-4 to 102 s. A molecule can undergo both phosphorescence and fluorescence

which can be difficult to distinguish each process. However, fluorescence has a higher probability

of occurrence than phosphorescence for most molecules because phosphorescence involves a spin

forbidden transition.

A fluorescence emission spectrum is obtained when the excitation wavelength is fixed, and

the wavelength of emission is scanned. On the contrary, an excitation spectrum is obtained when

the emission wavelength is fixed, and the wavelengths are scanned to determine which give rise

to the emission at a particular wavelength. The spectrofluorometer is a more sensitive technique

in terms of limits of detection compared to a UV-vis. These two spectroscopic techniques can be

used to determine the effect that different chemical and environmental conditions have on a system

5
and help to understand its electronic structure. The maximum emission or excitation wavelength

and/or intensity may change with variables such as temperature, concentration, quenchers, energy

transfer, or any other process that involves energy transfer.

1.4 Jablonski Diagram

The Jablonski diagram is an energy diagram and describes the absorption and emission

process of light from a system. It is named after Professor Alexander Jablonski, who is known as

the father of fluorescence spectroscopy. These diagrams can demonstrate the numerous molecular

processes that can happen in the excited state. An example of a Jablonski diagram is shown in

Figure 4.

The diagram’s vertical axis describes energy, meanwhile the rest of the diagram has

columns that characterizes a specific spin multiplicity. In some diagrams, the same spin

multiplicity has divided energy levels placed in different columns, and each column has horizontal

lines that symbolize eigenstates. The bold horizontal lines are limits of electronic energy states

that contain a series of vibrational states.

These diagrams use curved and straight lines to show transitions between eigenstates. The

curved lines show when the molecule gives off heat to its surrounding, vibrational relaxation. The

straight lines show an energy conversion in the system. If the arrow points upward, it is gaining

energy. If the arrow points downward, it is losing energy.

Absorption, the upward red arrow in Figure 4, occurs when the molecule absorbs energy

in the form of electromagnetic radiation. The electron is excited from a ground electronic state to

a vibrational level in the excited state. For example, in Figure 4, the arrow shows that the electron

is excited to the 6th vibrational state of an excited electronic state, however, the absorption process

may result in excitation to different vibrational state depending on the excitation energy. In other

6
words, the molecule may transition to a higher energy than the bottom energy level of the singlet

excited state. In the Jablonski diagram, the arrow points to a particular energy level. The likelihood

of ending at any particular state of energy and the likelihood of a certain photon absorption follows

a rule depends on the Frank-Condon principle, which will be discussed in a later section. In the

excited singlet state, the net spin is zero, and the spin doesn’t change when the molecule undergoes

an absorption event. The electron transitions to a different eigenstate according to the amount of

energy gained. Only certain wavelengths of a photon are possible for absorption which correspond

to the wavelength of the energy difference between the two different eigenstates. After absorption,

there are several ways in which the molecule can return to ground state: non-radiative and radiative

processes. The non-radiative process are vibrational relaxation, internal conversion and

intersystem crossing. Radiative processes are fluorescence and phosphorescence.

One way of dissipating energy is through vibrational relaxation, which is a non-radiative

process. This is demonstrated as a curvy, downward arrow between vibrational levels, and the

electrons will not change from one electronic level to another. This can be seen in Figure 4, as a

gray arrow. During vibrational relaxation, the molecule gives some of its energy as heat to its

surrounding and is transferred through collision between the excited molecule and the surrounding

molecules or solvent. Other times, the kinetic energy can stay within the same molecule. The

electron will relax down to a lower energy level of the excited state and remains in the same

electronic state. This process occurs very quickly on the order of 10-14 to 10-12 seconds.

7
 Figure 4: Jablonski diagram showing the absorption, emission and phosphorescence processes
and time scales
Under certain circumstances, vibrational energy can overlap the electronic energy levels in

which the electron transitions from a vibrational level of higher electronic state to another

vibrational level in a lower electronic state. This process is known as internal conversion. In

internal conversion, the molecules dissipate energy, and can occur simultaneous with vibrational

relaxation. It is also demonstrated as a curved arrow pointing downward from one electronic

energy state to another on the Jablonski diagram. The process is a non-radiative loss of energy

between electronic levels of the same spin state.

Another way for the dissipation of energy is through the emission of a photon, which is

termed as fluorescence. It is illustrated in the Jablonski diagram as a blue straight line, pointing

downward from the first electronic state to the ground electronic state. At higher electronic

energies than the first electronic state, it is more probable that the energy dissipates in the form of

vibrational relaxation and internal conversion, which is the reason most fluorescence is observed

between the first excited state and ground state. Therefore, the energy of fluorescence is always

less that the absorption of energy because a portion of the electron’s energy is dissipated through

8
vibrational relaxation and internal conversion. The emitted energy of fluorescence is the same

energy as the difference between the eigenstates. Florescence is a relatively slow process compared

to absorption and may compete with other non-radiative that can occur on the same time scale.

Another relaxation process described in the Jablonski diagram is intersystem crossing. This

transition is from an excited singlet state to an excited triplet state and the electron changes spin

multiplicity. It will have a net spin that is not zero. It is illustrated on the Jablonski diagram as a

straight, horizontal arrow from one column to another column. It isn’t changing energy, and it can

be in the excited vibrational state in the triplet state. It is just jumping from the singlet state to a

triplet state. The process is slower than fluorescence, and it is known as the forbidden transition,

which is less likely to occur. These transitions are forbidden by the electric dipole selection rules.

After the intersystem crossing to a triplet state (T1), the electron can then undergo vibrational

relaxation within that electronic state. If the electron then transitions back to the ground state

through a radiative process, then it described as phosphorescence. However, the electron can also

undergo another intersystem crossing to the ground state (S0) and then undergo vibrational

relaxation. This is a non-radiative process.

In general, emitted light is lower energy than the absorbed light. Phosphorescence has a

lower energy than fluorescence. Phosphorescence is slower than fluorescence, and vibrational

relaxation warms up the surroundings. The diagram also demonstrates which types of transitions

take place in a specific molecule, and it is dependent on the time scale of the electronic transitions.

The transitions that occur fastest have a higher probability of occurrence, based on the selection

rules.

1.5 Lifetime
Lifetime is an important parameter in fluorescence. The lifetime is the average time the

molecule remains in the excited state following excitation. Emission occurs is a random event and

9
it will emit at a photon of light after a given period of time, which will result in a decay of the

excited state. There are different decay laws, such as non-exponential decays and multi-

exponential decays.

The lifetime of the excited states becomes the average lifetime of the entire fluorescent

molecule population. When the excited state population is diminished by some process (i.e.,

vibrational relaxation solvent collision, fluorescence, phosphorescence, etc.), the fluorescence

average lifetime will decrease. The average lifetime (𝜏̅) measurements can provide information

about energy transfer rates, including the average time the molecule spends in the excited state

before returning to the ground state. The average lifetime is defined by equation 1.5.1 with a two-

exponential decay:
0
𝛼' 𝜏'+ + 𝛼+ 𝜏++ + ⋯ ∑0/1' 𝛼/ 𝜏/+
𝜏,-. = (𝜏̅) = = = 𝑓' 𝜏' + 𝑓+ 𝜏+ + ⋯ = N 𝑓/ 𝜏/ (1.5.1)
𝛼' 𝜏' + 𝛼+ 𝜏+ + ⋯ ∑0/1' 𝛼/ 𝜏/
/1'

In the expression above, 𝜏/ are the decay times, and 𝛼/ represent the amplitudes of each component

at 𝑡 = 0, and 𝑛 is the number of decay times. The values of 𝛼/ can depend on many factors, such

as concentration, absorption, quantum yields and intensities of each fluorescence molecule at the

observed wavelength.

1.6 Quantum Yield

All the processes shown in the Jablonski diagram have a certain probability of occurrence.

Absorption is the process that generates an excited state, and all the processes occur from or

between different excited states. For each process, there is a quantum yield, which is a measure of

the amount of the excited states decaying by each method, such as florescence, internal conversion,

etc.

However, for this thesis, the topic of concern is quantum yield of fluorescence. In this case,

every photon that is absorbed will not cause a florescence event. The quantum yield for

10
fluorescence is the ratio of the number of photons emitted relative to the number of photons

absorbed. It is an essential way of measuring the efficiency of a process. The quantum yield can

range between zero and one. Zero indicates that no absorption events resulted in a fluorescence

event, while a quantum yield of one indicates that every absorbed photon resulted in a fluorescence

event. A larger quantum yield will exhibit visibly brighter emission. In terms of the kinetic model,

it is a ratio of rate constants. It is the ratio of the fluorescence rate constant divided by the sum of

all the rate constant that causes deactivation of the exited state shown in equation 1.6.1.

234567897:; =>5;5:8 B"

Φ2 = ?@856@7A =>5;5:8
=B (1.6.1)
" C B#$% CB#%

k 2 represent the rate constant of fluorescence, k DEF is the rate constant of intersystem crossing

and k DF is the rate constant of internal conversion.

1.7 Stokes Shift

The stoke shift is an important topic in understanding fluorescence. Referring to the

Jablonski diagram, the energy of the emission of a photon is less than the energy of the absorption,

which arises due to the loss of energy through vibrational relaxation. Sir George Gabriel Stokes

noticed that fluorescence occurs at longer wavelength and lower energies than the absorption, and

the Stokes shift was named after him. The Stokes shift is the difference in the maximum

wavelength of the absorption and emission for a molecule arising from the same electronic

transition. The larger the absorption and emission peaks are, and minimal overlapping is shown in

Figure 5 is ideal. This eliminates any self-absorption interaction or interferences caused by the

sample.

11
 Figure 5: Stokes Shift with minimal self-absorption

A larger stoke shift prevents spectral overlap between emission and absorption which helps

reduce interference and allows detection of fluorescence. This removes the quenching of

fluorescence and provides a better signal for certain applications such as fluorescence microscopy.

1.8 Frank-Condon Principle

As described in the Jablonski Diagram in Figure 4, when light is absorbed, the atom or

molecule transitions to an excited state. The Frank-Condon principle allows for calculation of the

intensities of transitions between electronic energy levels and vibrational states. The atom or

molecule can only exist in discrete electronic states and absorbs energy quanta proportional to the

separation between the allowed energy states. The energy of the quanta of absorption or emission

is relative to the length of the arrow. The absorption of energy permits the rearrangement of inertia-

free electrons, and it is an instantaneous process. As the molecule or atom absorbs energy, the

nuclei remain still while the electrons move. The nuclei and solvent medium do not have time to

readjust since they are heavier, and the relative momentum does not get affected by electronic

transitions. This can be illustrated by a diagram of potential energy versus the distance between

the nuclei, as seen in Figure 6. The arrow will point to the upper curve from the lower curve. The

12
curves show the two electronic states, ground and excited states. The vertical arrow shows the

transition of the electrons from the lowest vibrational state of the ground state to a specific

vibrational state of the excited state of a molecule. For these diagrams, there aren’t any horizontal

transitions because the nuclei don’t have time to move, and only the electrons are moving. The

arrow shown represents a possible transition between the two electronic states. Each of these

arrows corresponds to different energy, which determine the peak wavelength for each transition.

In addition, each arrow will provide a relative intensity of the transitions. These are determined by

two components: the solvent environment and the molecular overlap between the vibronic

eigenfunctions.

Figure 6: Frank Condon Principle transition representation of two electronic states and an

absorption transition

The ground vibrational state will have a wavefunction for vibrational motion, which is

solved from the Schrodinger equation. The squaring of this wave function provides the probability,

and the probability is highest at the center. This illustrates that the projection of the center to the

horizontal axis is where the atom will be in position. In addition, the transition starts from the

13
center of the vibration wave function in the ground state, and it will end at the overlap of the

vibrational wave function in the excited state. The probability of the transition between the ground

and the excited state is proportional to the value of Ψ2. There needs to be a good overlap between

these states. As the vibrational states increase in each electronic state, there will be more nodes.

All vibrational excited states have large lobes at the end of the potential curve and those are

classical turning points. There are several horizontal lines, within each electronic state, which

represent specific vibrational and rotational states. There is zero-point for vibronic motion

corresponding to a nonvibrating state in liquids and solids. However, some energy diagrams omit

the rotational sub-states for each electronic state. An electronic state will remain the same for all

vibrational or rotational states that lie within the same electronic state.

After absorption, which is considered an instantaneous process, the excited state atom or

molecule will not interact with the surrounding solvent atoms or molecules. After, the atom or

molecule dissipates the energy in the form of heat due to the change in vibrational energy. The

excited atom or molecule adopts a minimum free energy, and it will transition to a lower excited

state energy, which is called the equilibrium excited state. In addition, the electron relaxes to the

lowest vibrational level of the excited state. The orientations and position of the excited state atoms

or molecules and the solvent molecules adjust to a different arrangement of solvent molecules. The

lower energy state will have a lower energy organization of the solvent and solute molecules.

After, the equilibrium configuration of the excited state molecule and solvation shell is

achieved, the electron can then undergo fluorescence then transition back to the ground state. Once

fluorescence occurs, the molecules and solvation shell will return to its equilibrium state through

a vibrational relaxation process.

14
 Quantum Dots

Quantum dots (QDs) are small semiconducting particles with typical sizes that range from

one to ten nanometers. QDs are artificial clusters of semiconductive atoms that have the ability to

confine the electron’s motion due to their small size. One of the most important properties of QDs

is the ability to fine tune their bandgap which allows one to control the frequency of absorbance

and emission. In this way, it is possible for their optical and electrical properties to be adjusted

according to their applications. QDs absorb photons of light and then emit longer wavelength

photons. The frequency of absorbed and emitted from the QD can actually be controlled without

significant cost and without the use of high-end technology. There are different types of quantum

confined semiconductors which include quantum wires, quantum wells, and quantum dots.

Quantum wires are confine electrons or holes in two special dimensions and allow free propagation

in the third dimension. Quantum wells confine electrons or holes in one dimension and allow free

propagation in two dimensions. Electrons exist in discrete energy levels in the bulk semiconductor.

In these materials, there is a forbidden range of energy levels known as bandgap. The lower energy

level is called the valence band (VB) and the higher energy level above the bandgap is called a

conduction band (CB). By absorbing light, an electron can rise from the valence band to the

conduction band. This action leaves behind a hole in the valence band and the hole, and the

electron together are called an exciton. The average distance between the electron and the hole

and an exciton is called the excited Bohr radius. When the size of the semiconductor falls below

the Bohr radius, the semiconductor is called a quantum dot and its properties can differ

significantly from the bulk material. The exciton, electron hole pair, has a limited lifetime and

will eventually recombine. The recombination process is usually irradiated process which includes

a photon release such as fluorescence.

15
 Generally, the smaller the size of the crystal the larger the bandgap, the greater the

difference in the energy between the valence band and conduction band becomes. Therefore, more

energy is needed to excite the dot and simultaneously more energy is released when it returns to

its resting state. This equates to higher frequencies of light emitted after excitation of the dot as

the crystal size grows smaller, resulting in a color shift from red to blue in the light emitted. In

addition to such tuning, a main advantage with the quantum dot is its high level of the control

particularly to their size.

2.1 Background

Semiconducting quantum dots (QDs) were discovered in 1981 are the first generation and

most common form of fluorescent nanomaterials studies.8 They are very small semiconductor

crystals on the order of nanometers. These novel materials exhibit remarkable novel magnetic,

mechanical, physicochemical and optoelectronic properties.9 However, the first generation of QDs

typically contain heavy metal elements such as cadmium, whose potential health and

environmental hazards have been documented.10

Quantum dots are made largely from the elements in the second and sixth group of the

periodic table. Cadmium chalcogenides (CdS, CdSe, CdTe), zinc (ZnSe, ZnS, ZnTe), and the third

and fifth groups, phosphides and indium arsenide.11 Quantum dots can be engineered to

fluorescence in different wavelengths based on their physical dimensions. Even though QDs have

received immense interests for their applications in medicine and biology. Studies have shown

with CdSe-core quantum dots are toxic to both cell cultures and live animals.12 Cadmium has been

reported to transport in cardiac cells and liver cells by a number of pathways.13, 14 Cadmium has

shown to transport primarily through diffusion in the renal cortical epithelial cells.15 Other

evidence suggest that cadmium can be transported via calcium channels.16, 17

Additionally,

16
cadmium competes with calcium (Ca), zinc (Zn) and copper (Cu) for uptake in hepatic and renal

cells.15, 18, 19 Since these metals (Ca, Zn, Cu) are vital to cellular metabolic, homeostatic, and repair

mechanisms, the inhibition of such processes by cadmium can lead to cell death.13

There is a need to develop a more sustained and environmentally friend form of quantum

dots to replace the first generation of quantum dots based on transition metals. Carbon dots is a

general class of materials typically referring to small carbon nanoparticles with various levels of

surface passivation. They have emerged as a new class of quantum dot-like fluorescent

nanomaterials.20 Carbon dots (CQDs and CNDs) were discovered much later in 2004 followed by

graphene quantum dots (GQDs) in 2006.21 These nanomaterials are also quantum confined

systems in which their bandgap can be changed based on their size. The valence (LUMO) and a

conduction (HOMO) band are separated by some finite energy gap known as a bandgap. When

an electron from the valence band attains ample energy to overcome the bandgap, due to an

absorption of a photon, a hole is left behind in the HOMO

Numerous approaches with a large array of techniques and starting materials have been

utilized for the synthesis of fluorescent carbon dots. The synthesis methods can be divided into

two processes: top-down and bottom-up. Some examples of top-down approaches are laser

ablation, chemical oxidation in strong acid and electrochemical synthesis. Compared with the top-

down approach, some bottom-up strategy examples are hydrothermal and solvothermal. For the

purposes of this thesis, the method used for the synthesis of the CQDs was a bottom-up approach.

Early synthesis used top-down procedures used “brute-force” experimental schemes, using

high energy impact upon a carbon source thereby generating the fluorescent carbon

nanoparticles.22 As such, laser ablation was most often used to produce inorganic nanoparticles

nanotubes from solid substrates.2 Electrochemical methodologies have been employed to

17
synthesized quantum dots. For example, graphite rods were used for the electrochemical cell to

function as both anode and cathode. The electrolyte solution was sodium hydroxide and ethanol.

As the current passed through the chemical circuit, the graphite rods dissolved producing carbon

dots.23

New methods such as hydrothermal and solvothermal processes are now widely employed

to produce CQDs. Carbon quantum dots were generated by glucose and its derivatives as the

carbon source and heated in water in microwave-assisted hydrothermal method.24 Tang and his

research group took measures of 1 to 9 minutes. The only main different between hydrothermal

and solvothermal is the solvent used for the reaction. If water is used as the solvent, it is referred

as hydrothermal method while if any organic liquid is used as the solvent, it is referred as

solvothermal method.25

Alternative methods have been employed to incorporate heteroatoms into the CQD

structure. Heteroatom doping has been shown to have a significant impact of the photophysical

properties of CQDs. For example, nitrogen doped carbon dots were developed by combining citric

acid and urea through a hydrothermal process to synthesize nitrogen doped carbon dots.26 Some

specific precursors for hydrothermal conditions was heating up to 180℃ for 60 minutes followed

by cooling to room temperature. As a result, there exhibits a shift towards the red emission of the

spectrum upon the addition of nitrogen heteroatoms.

2.2 Fullerene based carbon dots

Fullerene and its derivatives were the first class of carbon-based nanostructures to be

discovered (1985) and their chemistry and properties have remained the focus of extensive

investigations.27 Fullerene are mainly produced through the vaporization of graphite by resistive

heating under carefully defined conditions or the combustion of hydrocarbons in fuel-rich flames.28

18
Generally, they are closed hollow cages made of sp2-hybridized carbon atoms arranged into 12

pentagons and twenty hexagons carbon atoms.29 In one interesting study of fullerene-based carbon

dots, fullerene was conjugated with tetraethylene glycol (TEG), to generate TEG-fullerene

conjugates, which eventually aggregated into larger nanoparticles at higher concentrations.30

Fullerenes have indicated extraordinary physicochemical properties, and functionalization

capabilities that can be employed for the next-generation biomedical applications, particularly in

MRI contrasting agents and photodynamic therapy (PDT).31

Another prominent feature of fullerenols (hydroxyl functionalized C60) is its ability to

scavenge free radicals, which is attributed to their strong conjugated 𝜋-system to capture

electrons.32 Low toxicity and high water solubility was discovered which open new prospects for

further clinical studies. Also, fullerene in organic solvents exhibits five stages of reversible

oxidation/reduction processes, and therefore can work either as an electrophile or nucleophile.33

These characteristics were taken to account for selecting an appropriate carbon precursor to form

the next generation CQD.

2.3 LiBH4-C60 CQDs

The carbon quantum dot of interest in this thesis was made by reacting a carbon precursor,

a fullerene, and a complex metal hydride, lithium borohydride.34 The resulting CQDs possess

good photostability and the ability to tune its photophysical properties. The solvent used to react

LiBH4 with C60 is tetrahydrofuran. Upon removal of solvent under vacuum, annealing at 300℃

under an inert atmosphere for one hour the CQD precursor is obtained. Once the black CQD

precursor is obtained, it is then exposed to air, and causes the rapid oxidation of the material. This

rapid oxidation results in a color change to red-orange and the resulting material becomes

fluorescent and extremely water soluble.

19
2.4 Gadolinium Doped CQDs

Gadolinium (III)-based contrast analyses have occurred extensively used in clinical

magnetic resonance imaging (MRI). So far, there are at least nine formulations of Gd-containing

contrasting agents approved for human and they are assisting more than 10 million MRI scans per

year.35 However, MRI active Gd-chelates can be toxic to humans when Gd escapes the chelating

ligand.

The first Gd-doped C-dot that combine fluorescence with a strong MRI contrast ability was

in 2012 and were synthesized by adding gadopentetic acid into tris(hydroxymethyl)aminomethane

(Tris base) and betaine hydrochloride followed by pyrolysis in air at 250℃ which led to a Gd

doped carbon dot.36 The Gd-doped C-dot showed promising results for cell cytotoxicity studies

and the dots are water soluble with strong T1 weighted MRI contrast comparable to commercial

Gadovist. It served as a dual probe for both fluorescent and MRI studies purposes. In light of this

evidence, we decided to incorporate Gd into our CQDs by addition of Gd using a different

approach into our CQDs.

Since we already know that fullerene has unique chemical properties, researchers began to

look at biomedical applications. In particular, water-soluble derivatives of fullerenes possess

distinct potential for medicinal applications, since the fullerene cage guards the encapsulated metal

ion from external chemical attack and metal ion release in the body.37

2.5 Chiral Doped CQDs

One future work to consider is functionalizing CQDs with chiral groups. Chirality exists

extensively in nature and plays vitals roles in life and material sciences. Chirality has a distinct

geometry of atoms, molecules, particles, etc., defined by describing the fact that the mirror image

of an object is not superimposable with the original.38 Chiral functionalization of CQDs would

20
broaden applications to chiral recognition, chiral separation, asymmetric catalysis, chiral sensing

as well as other bio-applications.39 To this day, chirality at the nanometer scale is still being

investigated. Chiral quantum dots are a class of nanomaterials with chiral and fluorescence

properties and have shown great interest due to easy functionalization, low cost, very low toxicity

and favorable biocompatibility.40

The research on chiral CQDs indicated that levorotatory CQDs (L-CQDs) and

dextrorotatory CQDs (D-CQDs) present different biological effects.40 For instance, L-CQDs

indicated chirality dependent enhancement in cellular glycolysis, while D-CQDs are selective in

energy metabolism of cells.41 D-CQDs are able to boost photosynthesis better and collect more

carbohydrate in mung bean plants, which can be possibly used as fertilizer for agricultural

application42

One approach that has been investigated was using sodium citrate and ammonium

bicarbonate to synthesize CQDs by a facile green hydrothermal process.43 Dialysis was conducted,

and finally transferred to a round bottom where 1-(3-dimethylaminopropyl)-3-ethylcarbosiimide

hydrochloride (EDC-HCL) and L-tryptophan was added to the mixture to generate L-CQDs.

Based on this and similar work, we decided to try a similar scheme using our CQD to prepare

chiral analogues.

2.6 Lawsone CQDs

Another future work to consider is using inexpensive plant-based precursors to generate

CQDs. Lawsone (2-hydroxy-1, 4-naphthoquinone), is a natural naphthoquinone present in the

henna leaf extract with several cytotoxic activities and used as precursor for synthesis of various

pharmaceutical compounds and is in quinones.44 Quinones contain a benzyl ring with two

21
carbonyl groups in the ortho- or para- position as seen in Scheme 1 depending on the tautomeric

form.

Scheme 1: Tautomeric Forms of 2-hydroxy-1, 4-naphthoquinone

In Scheme 1, there are 3 possible tautomeric forms. However, the 1,4-naphthoquinone structure

is the most stable form followed by 1,2-naphthoquinone and finally 1,2,4-naphthotrione.45 This

is due to the intramolecular hydrogen bonds stability in the 1,4-naphthoquinone and the dipole

moment cancellation of the carbonyl groups. Their chemical structures enable these molecules to

interfere in several biochemical processes regarding redox homeostasis and inhibition of electron

transport.46

The benzyl group as well as the carbonyl bonds contain conjugated pi systems that can

undergo pericyclic reactions to form larger polymer macromolecules. Due to these conjugated pi

systems, lawsone exhibits very strong light absorption. The benzyl groups in lawsone are

ubiquitous precursors for producing carbon dots (CDs) with red/near-IR or multicolor emissions,

primarily those that are substituted with functional groups such as amino (-NH2), hydroxyl (-OH)

or sulfhydryl (-SH).47, 48
It has been demonstrated that certain structure presented in these

precursors would lower the energy gaps of CDs by generating large sp2 domains through

carbonization and dehydration reaction.49, 50

22
 Biological Systems

3.1 Cancer
Tumor metastasis to distant sites is a major cause of cancer morbidity and mortality. There

is a dire need for novel imaging modalities and contrast agents in order to improve patient

outcomes.

Cancer cells are abnormal cells that divide uncontrollably, forming tumors. A tumor

consists of many cancer cells that destroy the healthy, normal cells that surrounds the tumor and

eventually damage the healthy tissue and organs. Cancer cells emerge through a series of

epigenetic and genetic changes. Some of the changes may be caused through inheritance or

carcinogens in the environment. These tumors can be benign, malignant or metastatic. Benign

tumors grow slowly and do not spread. Malignant tumors have the capability to invade nearby

tissue and organ and spread throughout the body. Metastasis is the terminal stage of the cancer.

There are various types of cancer cells, but the four main types of cancer are carcinomas, sarcomas,

leukemia and lymphomas. For the focuses of this thesis, the main focus will be on a triple negative

breast adenocarcinoma cell line, MDA-MB-231. This cell line is an epithelial, human breast cancer

cell line that was founded from a pleural effusion of a 51year old Caucasian female with a

metastatic mammary adenocarcinoma.51

Cancer cells differ from normal cells in several ways. Cancer cells proliferate in an

uncontrolled way. In contrast, normal cells grow as part of development or to repair injury. Normal

cells have a limited lifespan and can self-destruct when it becomes old or damaged. In contrast,

cancer cells developed a loss of tumor suppressor genes and activation of oncogenes. A way for a

cell to resist death is to restore their telomeres as it continues to divide. In addition, cancer cells

can extend to other parts of the body by not responding to any signal. On the contrary, normal cells

are specific and remain within its boundary.

23
 The process of a normal cells transforming into a cancer cell goes through several stages

to become more abnormal. These stages maybe hyperplasia, dysplasia and finally cancer. The

development of cancer initiates when a cell proliferates abnormally.

In many cases, the immune system recognizes and tries to eliminate the cancer cells.

However, during treatment, the cancer cells do not remain the same and continually mutate.

Different cancers have different mutation routes. For this reason, many times, the cancer cells are

resistant to chemotherapy and antibiotics.

3.2 MRI Contrasting Agents

Many biological investigations in biotechnology and molecular biology necessitates

florescent dyes labeling for specific proteins, antibodies or cell detection. Such dyes are used to

detect by attaching the target molecule or molecules and aid to the problem. As fluorescent

nanomaterials with bright fluorescence, exhibit high quantum yield, resistance to photobleaching,

biocompatibility and low biotoxicity, carbon dots have shown great potential for use in fluorescent

bioimaging.52

Magnetic resonance imaging (MRI) has also been extensively performed as a noninvasive

imaging technology with high spatial resolution in clinical diagnoses.53 In particular, Gadolinium

(Gd) exhibits exceptional contrasting efficiency because of its unique magnetic properties.

Gadolinium (III) is a fair large paramagnetic species with seven unpaired electrons and allows

water to enter the inner solvation sphere. In the inner solvation sphere, its able to affect the

relaxation time of water to highlight features in an MRI image. Currently there are many

commercially available gadoliniums chelates. In these systems, gadolinium is chelated by a

multidentate ligand such as an EDTA or porphyrin derivative. However, one of the issues with

these contrasting agents is that gadolinium can actually escape and spread throughout other parts

of the body causing lesions as well as other potentially complications.54

24
 Methodology
The work presented in this thesis came to fruition through several collaborations between

laboratories at The University of Texas at Austin, Savannah River National Laboratory (SRNL),

and California State University, Northridge, in California, USA. Thus, a description of each

instrumentation will be presented, which was utilized for either qualitative or quantitative or both

for analysis.

4.1 Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy, known as FTIR, is widely used in food analysis,

petrochemical engineering, pharmaceutical industry, polymer science, organic synthesis,

manufacturing, research and product development. It is used to identify and/or characterize new

materials, as well as verify known specimens. FTIR spectrometers have the following benefits: it

has high resolution of about 0.1 – 0.005 cm!' , it has a rapid scan time of all frequencies, the signal-

to-noise ratio is higher than the prior generations, the scan range is between 1000 and 10 cm!' ,

and the wavenumber has high accuracy, therefore, the error is within ±0.01cm!' .

Infrared spectroscopy examines the interaction between matter and infrared (IR) radiation.

Infrared is a region in the electromagnetic spectrum that has a longer wavelength than visible light.

It has a smaller wave number and carries less energy. The range of infrared region can be divided

into three regions: far-infrared region, mid-infrared region and near-infrared region. The IR region

is characterized by molecular vibration. There are various types of vibrations, but the main

categories are anti-symmetric, symmetric, deformation vibration, wagging, rocking and twisting

vibrations. When the sample molecules absorb infrared radiation of specific wavelengths, the

molecules have a change of dipole moment, and the vibrational energy levels transfer to the excited

state from the ground state. The vibrational energy gap governs the frequency of the absorption

peak. The intensity of the absorption peak is associated with the change of the dipole moment, and

25
the number of vibration peaks correlates to the number of vibrational degrees of freedom in the

molecule. Therefore, the infrared spectrum can provide information of the molecular structure.

The improvements of the IR spectrometers led to the invention of the Fourier Transform

Infrared (FTIR) Spectrometer. Most FTIR spectrometer comprise of a radiation source, Michelson

interferometer, sample compartment, detector, A/D converter, amplifier and a computer. Within

the interferometer, there is a beam-splitter, a fixed mirror and a movable mirror. As the source

generates the IR radiation, the beam travels from the source and enters the interferometer. Then, it

enters the beam-splitter. It has a material that reflects IR radiation and permits IR travel through

the transparent part. From the beam splitter, the beam is split and directed at equal amounts to the

moveable and fixed mirror. In other words, fifty percent of the radiation travels to the moveable

mirror and the remaining goes to the fixed mirror. Then, the beams reflect the mirrors, and

recombines, which causes interference. Finally, the light is directed at the sample. The sample

absorbs some of the light, and specific amounts of light will travel through the sample material.

The remaining light that is transmitted through the sample carries the information into the detector

to produce an electronic signal shown in Figure 7. The signal becomes amplified and converted to

a digital signal through an analog-to-digital converter. The signal transfers its information to the

computer and undergoes a Fourier Transform.

26
 Figure 7: FTIR schematic

FTIR displays the amount of light hitting the detector by using a broadband light detector

that observe all the wavelengths simultaneously. It doesn’t inform the user which wavelengths are

used, and it only displays the power of light reaching the detector. The power can change by

creating interference within the light through the interferometers. The interference patterns

illustrate every wavelength that exist within the light. The interference of the radiation between

the beams yields a signal that reflects the change of path length of the beams. The moveable mirror

can travel a certain distance based on the design of the spectrometer If the length between the

stationary mirror to the beam splitter and the distance between the beam splitter to the moveable

mirror are the same, the condition is known as “zero path difference.” In this situation, the light

doesn’t change, and the resulting beam matches the radiation source. Both beams are in phase, and

the maximum signal reaches the detector. The mirrors are adjusted to be the same distance apart.

The output would be a power vs distance graph. In practice, there is partial interface pattern, in-

phase pattern and out of phase pattern that occurs. The net signal reaching the detector is a cosine

wave. In nature, IR sources are polychromatic, which each wavelength generates a cosine wave.

The summation of all the cosine waves reaches the detector, and the interferogram beam is

27
generated. The interferogram holds the spectral information in the distance (m) domain. The

'
Fourier transform converts it to wavenumbers 3%6 domain.

To obtain clean sample spectra, environmental influences need to be minimized such as

water vapor and CO2 in the atmosphere. This process can be accomplished by measuring a

spectrum without a sample present. The raw signal undergoes a Fourier Transform into the IR plot

of light intensity vs wavenumber, and it becomes a reference spectrum. Then, a sample is inserted

into the beam path and the procedure repeats. The sample spectrum is divided into a reference

spectrum to obtain the FTIR spectrum. When adjusting the moving mirror by have of the

wavelength of the beam source, the two beams have wavelengths that are out of phase and interfere

destructively. There isn’t a signal that reaches the detector, or it has a minimal signal.

4.2 Ultraviolet-visible Spectroscopy

Sunlight, also known as white light, consists of a wide range of wavelengths in the

electromagnetic spectrum: ultraviolet, visible and infrared sections. Electromagnetic radiation is

commonly treated as a wave and is characterized by its frequency or wavelength. Each segment of

the spectrum has an energy that is proportional to its frequency. Equation 4.2.1 describes the

relationship, which is the energy of the photon of a certain wavelength.

G
E = hf, where f = H (4.2.1)
Certain instrumentations, such as spectrophotometer, can identify the amount of solute

within a substance. Specifically, a UV-Vis spectrophotometer can help determine the wavelength

or wavelengths of light that are absorbed by the compound if the molecule absorbs light in the

visible or ultraviolet region of the electromagnetic spectrum. Different molecules can absorb

different wavelengths of light, and the UV-Vis spectrophotometer operates as an optical

28
spectroscopy. UV-Vis spectroscopy is utilized in various industries, such as production, research

and quality control to classify and study different substances.

The UV-Vis spectrophotometer consists of a xenon or deuterium source. The source creates

a range of wavelengths of light, which directs a beam of light into a monochromator. The

monochromator is a device that resolves a wide band of polychromatic light radiation into narrow

band of monochromatic radiation. There are three types of monochromators: prisms, gratings and

filters. In many instruments, the monochromator consists of two slits separated by a diffraction

grating or prism. As the light passes through the first slit, it ensures all the photons are traveling

along parallel pathways. The light strikes the prism, and the light refracts to different directions.

Only one wavelength of light will make it through the second slit in the monochromator.

Afterward, the beam of light passes through the beam splitter, which separates the beam of light

into two equal and parallel beams of light. Then, the beam enters the sample compartment, which

consist of the sample and reference cells. Next, the beams of light hit each detector, a device that

transforms the beam of photons into electrical current. There are a variety of detectors, such as

photo tube, photomultiplier tube, barrier layer cell, thermocouple, barometer, etc. However, the

photomultiplier is the most common detector. The following components are the amplifier and

recorder. The amplifier amplifies the incoming signal from the detector, and the recorder provides

the readouts. All of this is shown in Figure 8.

Figure 8: Dual Beam UV-vis Spectrometer Schematic

29
 The current generated by each detector is the same, and therefore, the intensity is also the

same. If the sample has zero concentration, there is 100% transmittance. This signifies the ratio of

the intensity leaving the sample cell to the reference cell must equal to one. When solute is added

into the sample, the solute can absorb some of the light. The intensity of light exiting the sample

decreases, and the current from the detector also decreases. This leads to a ratio of intensities less

than 1. As more concentration increases, the ratio of the intensities decreases further. The

relationship between the percent transmittance and the concentration of the sample is exponential

shown in equation 4.2.2.

T = 10!I ∙[9] ∙ ℓ (4.2.2)

August Beer transformed this percent transmittance into absorbance by taking the negative

logarithm of the transmittance, which creates a linear function. The data is plotted in absorbance,

and it is easier to predict the absorbance through interpolating or extrapolating the collected data.

During the time when the sample is stored inside the cuvette, there exist a linear relationship

between the concentration and the amount of light absorbed sample, which is known as the Beer

Lambert Law shown in equation 4.2.3.

A= 𝜀 ∙ [c] ∙ ℓ (4.2.3)

A is absorbance, and it is displayed as the y-axis on the chart. 𝜀 is the molar absorptivity,

C is the concentration of the sample, and ℓ is the path length of the specific type of

spectrophotometer. The path length and the molar absorptivity are the only constant parameters in

the Beer Lambert Law. The molar absorptivity is a specific value for each substance. The

absorbance value is inversely proportional to the light transmitted through the sample. Therefore,

a higher concentration of the solute constitutes more light absorbed. The sample absorbs particular

wavelengths. The transmitted light results from the light that isn’t absorbed and passes through the

30
cuvette. The spectrograph displays the absorbance of the sample for a range of wavelengths within

the visible and ultraviolet region. A particular absorbance value will determine the concentration

that coincides with it.

When a molecule is exposed to light that contains an energy equal to a possible electronic

transition, the electron will be promoted to a higher energy orbital as the light energy is absorbed.

Figure 9, shows the various electronic excitations that an organic molecule can undergo.

Figure 9: Electronic excitations diagram

Of the six transitions, only the two with the lowest energy have energy values ranging from 200

to 800 nm. Energetic electrons will undergo a promotion and transit from the highest occupied

molecular orbit (HOMO) to the lowest unoccupied molecular orbital (LUMO) as depicted in figure

10.

31
 Figure 10: Energetic electrons transitions

The resulting state is known as an excited state. Only molecular moieties that absorb wavelengths

in the 200 to 800 nm spectrum are pi-electron functions and hetero atoms having non-bonding

valence-shell electron pairs. These groups are known as chromophores. The presence of

chromophores can be detected with the UV-Vis spectroscopy; however, it becomes problematic

for most instruments to provide absorption data below wavelengths of 200 nm. Luckily,

conjugation shifts the maximum absorption to longer wavelengths.

The UV-Vis Spectrophotometer detects whether the sample is a conjugated system because

it can absorb light. Conjugated systems are alternating double, single, double bonds. At times, it

can be triple, single, triple bonds, or alternating bonds. If the sample doesn’t absorb light, there is

a flat reading on the spectrograph of absorbance vs wavelength. If there is a conjugated system,

there is some absorption when UV light enters the cuvette. The conjugated system doesn’t absorb

every wavelength in the UV region of the electromagnetic spectrum. Instead, it absorbs at the

maximum wavelength, which is the highest peak on spectrograph.

32
4.3 Fluorescence Spectroscopy

When dealing with biological material, fluorescence spectroscopy is a good choice. It is

good to analyze plants, animal tissue and DNA. Fluorescence is 3 orders of magnitude better than

UV absorbance spectroscopy. Fluorescence is the process when a material emits light at one

wavelength when illuminated. A fluorescent material is called a fluorophore, and sometimes it is

called fluorescent dyes, labeling reagents or stains.

A fluorophore absorbs light at a lower wavelength followed by emission of light in a longer

wavelength. To understand how a molecule exchanges energy with the light, we can look at the

electromagnetic waves, and how it carries energy. Light can be understood as electromagnetic

waves with a wavelength and a speed (the speed by which the peak travels). Light can be described

as particles called photons. Each photon carries a small amount of energy that is related to the

speed and wavelength in equation 4.2.1.

The energy is given the Planck’s constant (ℎ), c is the speed of light, and the wavelength

is lambda. The energy of the photon is inversely proportional to the wavelength. This means, the

longer the wavelength, the less energy the photons carry. A Jablonski diagram shows the allowed

energy states that the molecule can be in. A molecule will be in the ground state can absorb a

photon with a certain wavelength and energy. The energy of the molecule gets raised to a higher

level. From this level, the molecule will relax a bit and in an excited state. Then the molecule can

relax to the ground state again through the emission of a photon.

Figure 11 shows a brief schematic view of a fluorometer. Typically, a broadband light

source such as a Xenon lamp is used in most instruments. The light then passes through an

excitation monochromator before being directed into the sample. The light emitted from the

sample is then collected at 90° by a detector after passing through the emission monochromator.

33
The electromagnetic spectrum is then scanned with the emission monochromator to determine

obtain the emission spectrum.

Figure 11: Fluorescence Schematic

4.4 Nuclear Magnetic Resonance

The NMR is composed of radiofrequency (RF) transmitter, magnet, receiver coil, sample

holder, sweep generator, amplifier, detector and recorder. The RF transmitter generates radio

frequency to the sample, which is transferred to the coil near the sample and the electromagnetic

radiation will be perpendicular to the receiver coil as well as the magnet field. Therefore, there

will be resonance in the sample. The magnet is located near the sample holder and produce the

magnetic field. The magnet’s strength determines he accuracy and quality of the instrument. The

resolution increases with an increase of field strength. There are three types of magnets that can

be used in NMR: conventional magnets, super conducting solenoids and electromagnets

(permanent). Each of the magnets have different frequencies. There is a relationship between the

magnetic field strength, 𝐵( and the frequency, v, and it can be determined using the Larmor

equation shown in 4.4.1.

34
 N
v = 3+O6 ∙ 𝐵( (4.4.1)

y is gyromagnetic ratio, and it is a constant, 267.53 radians/T. If the magnetic strength is known,

the frequency can be determined. The sweep generator works in conjunction with the sweep coil,

which alters the magnetic field by increasing or decreasing the magnetic field. It is a set of

Helmholtz coils located parallel to the magnetic faces.

The receiver coils wrap around the sample holder as seen in Figure 12. It will receive the

electromagnetic radiation and it will go into the amplifier, and it will amplify it by 10P times. Then

the signal will go to the detector to receive the signal, and it will finally reach the recorder or

oscilloscope.

RF
transmitter Radio frequency
receiver & amplifier
Sweep Coils Sweep Coils

Control Console
and
Recorder

Sample
Magnet Magnet
Receiver Coils

Sweep
Generator

Figure 12: General NMR Schematic

There are two types of NMR instrument: one is constant magnetic field and the other is

constant radio frequency transmitter. The constant radio frequency transmitter can increase or

decrease the magnetic field. In contrast, a constant magnetic field will allow the radio frequency

transmitter to vary. Therefore, the sweep coil or sweep generator will not be required.

The physical foundation of NMR spectroscopy lies in the magnetic properties of atomic

nuclei.55 According to the rules of quantum mechanics, the interactions between the external

35
magnetic field 𝐵) and the nuclear magnetic moment 𝜇, plays a vital role in energy level diagrams.

This is due to the magnetic energy of the nucleus. These energy restrictions led to certain discrete

eigenvalues 𝐸/ associated with eigenstates that an elementary particle can exist known as stationary

states.

For the purposes of this thesis, NMR was conducted for T1 measurements. Protons without

an external magnetic field move in all types of directions shown in Figure 13(A). T1 is a

longitudinal relaxation and follows first order kinetics given by equation 4.4.2.

!RTS
𝑀Q = 𝑀) 31 − 𝑒 & 6 (4.4.2)

𝑇' is a first order time constant in this equation, 𝑀Q is the magnetization along the z-axis along the

magnetic field 𝐵) and 𝑀) is the initial magnetization. 𝑇' is defined on the return to equilibrium in

the direction of magnetization along the z-axis as seen in Figure 13(C) occurring to the equation.

𝑇' relaxation is basically an energy flow amongst spins and their external environment seen in

Figure 13(B). The measure of energy transmitted from the nuclei is extremely small relative to

normal molecular kinetic energies. All energy emission in NMR must be stimulated through

encounter of the nucleus with another magnetic field fluctuating near the Larmor frequency.56 Just

like 𝐵) (𝑀Q ) field that initially excited the nuclei into resonance, the radio frequency fields causing

𝑇' relaxation must oscillate near the Larmor frequency in the transverse plane (𝑀UN ) along 𝐵' as

seen in Figure 13(D).

36
 Figure 13: NMR 𝑇' Relaxation

4.5 MTT assay

The 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) is a

colorimetric assay. The MTT assay measures the reduction of the yellow MTT reagent by an

enzymatic catalyzed reaction via the NADH Oxidoreductase or NADH Dehydrogenase. This will

convert NADH to NAD+ when the cells are viable as shown by the reaction seen in Scheme 2.

Scheme 2: NADH Oxidoreductase Reaction57

The MTT reagent enters the cells and passes into the mitochondria where it is reduced to a

formazan product observed as a purple color. If you have a well with no cells. No cells mean no

mitochondria. No mitochondria mean no enzyme. Therefore, the MTT reagent will not be

converted to form formazan. But, if you have cells, the more cell you have, the more mitochondria

you have. The more mitochondria, the more NADH oxidoreductase you have and there will be a

37
quantitative relationship with the conversion to formazan. It is an output of metabolic acidity this

MTT assay. The mitochondria are the driving force of metabolism of the cell. The more

mitochondria activity there is, the faster the conversion of this MTT will be to form formazan.

With any compound, if there is a cytotoxic or cytostatic effect, there will be a diminished

conversion of MTT to formazan.

4.6 Fluorescence Microscopy

Fluorescence microscopy is a great instrument for biological systems because it helps

understand different regions inside the cell.58 If the cell is present, the cell’s nucleus is marked

with a particular dye.59 The dye or chemical factor has a fluorescence property.60 The fluorescence

dye receives a particular wavelength, and it emits a different wavelength.60 In other words, a light

of lower wavelength is applied, and the dye responds by emitting light of a higher wavelength.61

In general, the emission light has a longer wavelength than the excitation light, and therefore the

emission light has a lower energy. The total amount of events in which light emission occurs and

some of the loss in energy can be expressed a ratio of quantum efficiency. Quantum efficiency is

an important characteristic of the dye, and the dye also depends on the environment.58 The intensity

of light emitted is another important parameter for a dye. The brightness is determined by how

well the dye absorbs the excitation light, as well as the fraction of the excited states returns to the

ground state on the emission of fluorescence. It is determined by both the quantum efficiency and

absorption coefficient. Another important factor of the dye is the time is takes between absorption

and the time it takes the dye to return to ground state from the excited state. This process is in the

order of nanoseconds, and it is a characteristic specific to the dye and the environment of the dye.

In fluorescence microscopy, there is a light source such as a tungsten-halogen lamp, LEDs,

lasers, mercury or xenon arc lamps.59 The light will travel to the excitation filter to eliminate certain

38
wavelengths, and it will allow only the wavelength that will excite the dye. The light will reflect

off the dichroic mirror, enter the objective and travel to the sample.59 The specimen contains the

chemical compound which shows the fluorescence. The fluorescence molecules interact with the

excitation energy.58 Only a particular wavelength of light can excite the fluorescent molecules.

The specimen absorbs light, and the absorption is a fast process, in the time scale of femto-scale.62

The electron is excited to a higher energy state, with a higher orbital, as seen on the Jablonski

diagram, and it will come down to the ground state and emit the fluorescence.61 The fluorescence

light is of a specific wavelength, and the photon will be of a higher wavelength with a lower energy

than the absorption energy. The emitted light will pass through the same objective, and then it will

encounter the same dichroic mirror.61 The dichroic mirror not only reflects a specific wavelength

of light, but it allows another wavelength to pass the through the mirror.61 After, the emitted light

will pass through the emission filter, which will block any scattered excitation light to obtain the

fluorescence on the detector or a CCD camera.59 This entire process is shown on Figure 14.

Figure 14: Fluorescence Microscopy Schematic

The two important factors that determine the quality of the image are resolution and

contrast.61 High resolution and contrast are always desirable.58 The aspect that makes fluorescence

microscopy different from a normal microscope is the filters. These filters discriminate the

39
excitation light from the emitted light.61 There are filters that absorb a certain range of wavelengths

from the light. These filters, based on absorption, are not great in practice because the spectrum is

broad, and it is prone to absorb a small amount of the emission light. Typically, interference filters

are commonly utilized in most instruments.61 These are thin alternating layers that have different

refractive index, so they are interfaces that can reflect the light. The thin and transparent layers are

about a half wavelength or wavelength of light apart with semi-reflective coatings in between.

When light hits the filter, part of the light will travel through and the rest will be reflected. The

reflected light will interfere with itself. If the reflected wavelengths have a phase difference equates

to 𝜋 plus an integer multiple of 2𝜋, it is known as constructive interference.59 That constructive

interference will allow the light pass through the filter. On the other hand, when the wavelengths

are different, there is destructive interference, and the light will be reflected away.59 The

manufacturer can design which wavelengths the filter should pass, and which wavelengths should

be refracted by computing how the layers will behave.

In fluorescence microscopy, there is a filter cube shown in figure 15 which includes the

excitation filter, a dichroic mirror and an emission filter.61 It is important to know the spectrum of

these filters to know which wavelengths are being reflected and transmitted. The dichroic mirror

is made to reflect everything that has a lower wavelength than a certain cut-off band and allow the

passage of higher wavelengths. The emission filter will allow higher wavelengths to pass but reject

the excitation light.58 The task is to select the correct filter that will match well with the dye that

will be utilized. As a general rule, it is desired to get all the photons to obtain a brighter image.

40
 Figure 15: Filter Cube

There are carousel or turret that contain filter cubes, and the user can select the appropriate

filter cube. The cubes are chosen to visualize the dyes that are of interest. In the computer, all the

images are combined to provide an image.

An aspect that needs to be taken into consideration is photobleaching.58 Fluorescence dyes

do not last a long time, and they have a limited number of cycles. Overtime, the fluorescence can

bleach and no longer occur.58 To minimize this effect, there are dyes that are resistant against

fading and do not bleach as fast relative to others. In the market, there are other dyes that have the

same excitation and emission properties but bleach at a slower rate. Each dye has a probable chance

that it will photobleach.58 By changing the environment of the dye, you can minimize the rate of

bleaching. For example, oxygen helps speed the photobleaching process, so it is ideal to remove

oxygen as much as possible from the sample. Using glycerol or using enzymatic systems can help

remove oxygen from the system. There are a lot of compounds that can be added to reduce the

bleaching effect.

41
4.7 X-Ray Diffraction (XRD)
The XRD can analyze thin film and powder samples.63 It can identify thin film materials,

detect defects, analyze crystal structures, etc.64 The XRD consist of a source made of tungsten

filament, which emits electrons.65 The applied voltage produces an electric field, and it accelerates

the electrons.65 The electrons are directed towards the x-ray target material such as copper.66 The

electrons collide the target’s atomic structure, causing secondary electrons to be removed from the

electron shell, and leaves holes in the electron shell.65 This unstable state causes electrons from

higher energies to occupy the lower energy shells, and it leads to releasing energy in the form of

x-rays.65 After, the x-rays travel to the primary optics, which contain solar slits and divergent slits.

The slits provide better resolution.65 Next, the beam travels to the secondary optics as shown in

Figure 16, where it receives the deflected rays.65 The monochromator filters the x-rays, and the

detector converts the x-rays into visible light, called a scintillation.65

Figure 16: XRD Basic Schematic

The XRD can perform its function by using Bragg’s equation, which helps calculate the

distance, d, between the layers of atoms or the scattering angle, 𝜃, of x-rays after it hits the crystal

lattice as seen in Figure 17. Equation 4.7.1, where 𝜆 is the wavelength of the x-ray.66

42
 2dsin𝜃 = n𝜆. (4.7.1)

Figure 17: Scattering and diffraction: The Bragg’s law

4.8 Circular Dichroism

Circular Dichroism is an absorption spectroscopy method that relies on the method of right

and left circularly polarized light absorption by the sample.67 It is used to examine biological

molecules.68 The broad majority of biological molecules are chiral.69 For instance, 19 out of 20

common amino acids that form proteins are chiral, as well as other higher structures of proteins

such as RNA and DNA.68 The Circular Dichroism spectrum of a protein, such as DNA, provides

a specific dichroic signature, and it can highlight the structural elements, as well as follow the

transformations.70 The wide application is in secondary structural elements of proteins such as 𝛼-

helix and 𝛽 sheet.69 A primary application is in the analysis of secondary structure or conformation

of macromolecules.69 Secondary structure is only a small portion of the capabilities of the circular

dichroism.71 Circular Dichroism can aid in observing the interaction between the secondary

structure and the environmental conditions or the interaction with other molecules, regardless of

if secondary structures are sensitive to temperature, pH or the environment.68 For example, it can

compare different macromolecules under different conditions, and it can provide the structure

43
analysis and can be used to verify if a protein is correctly folded.67 In addition, circular dichroism

can derive thermodynamic, kinetic and structural components of the macromolecules.68 The

benefit of using circular dichroism is that the recording time can be done in minutes.70

There are two types of polarized light: linear and circular polarized light (Figure 18).69

Linear polarized light occurs when the electric field vector oscillates in only one plane and in one

direction. It is polarized because all the other oscillations are cancelled.70 Meanwhile, circularly

polarized light occurs when the direction of the electric field vector rotates about its propagation

direction while the vector retains constant magnitude.69 Depending on the rotation, it can be

clockwise or counterclockwise, and they are referred to left-hand circular polarized or right-hand

circular polarized light.70 Circular polarized light consists of both left and right circularly polarized

light.69 When the sample absorbs both left and right circular polarized light, information about the

concentration will be provided.71

Figure 18: Unpolarized light with Circular and Linear Polarized Light

Circular Dichroism consist of a light source, a filter, a polarizer, a quarter phase plate, a

sample chamber and a detector.72 The light source emits electromagnetic radiation. The

44
electromagnetic radiation contains both an electric field and magnetic field, and both fields are

perpendicular to both each other and the propagation direction.69 The light will pass through a

filter wheel to form a monochromatic light of a single wavelength.67 After, it will pass through a

polarizer to obtain a linear polarized monochromatic light.73 When the light passes through the

PEM, the light becomes a circular polarized light, and it passes through the sample.67 An

appropriate plate will transform the linear polarized light into circular polarized light.67 The sample

absorbs the light and then the transmitted light passes through the detector.72 As the photon beam

passes through the sample, the sample is changing this polarization pattern and it is detected

(Figure 19).72 Circular dichroism is the difference in the spin angular momentum state of the

photon.

Figure 19: Circular Dichroism Schematic

The sample is optically active, and it is absorbing circular polarized light.67 This can be

extended to the Beer-Lambert Law, where the differential absorbance is going to be proportional

to the concentration and the length of the cuvette.73

∆A = 𝐴V -𝐴W = (∈V − ∈W ) ∙ C ∙ l (4.8.1)

𝐴V is left-hand circularly polarized and 𝐴W is right hand circularly polarized as demonstrated in

equation 4.8.1.71 The difference in polarization status calculated the difference in absorption.69

The difference in absorbance gives insight about the secondary structure.68 The epsilon terms are

45
the molar coefficient for the left circular polarization, ∈V , and right circular polarization light, ∈W .

The molar coefficient is also known as molar circular dichroism, and it measures the dichroism of

a particular sample, which is a function of the wavelength.73 Therefore, the light that will be used

for detecting the sample is of great importance.72 Meanwhile, C is the molar concentration, and l

is the path length in centimeters (cm).68

The limitations of using the circular dichroism spectrophotometer are determined by the

signal-to-noise ratio, which is limited by the photon shot noise.68 The contributing factors to the

signal-to-noise can be expressed as the following:

X
Y
=( Q ∙ ℓ ∙ 𝑡)).P (4.8.2)
Q is the detector quantum efficiency; ℓ is the light intensity and t is time of the measurement.

Based on equation 4.8.2, there are three methods to improve the signal-to-noise. One method is to

increase the intensity of the incident linearly polarized light.68 Another method is to increase the

amount of time collecting data points. Also, a third option is to increase the quantum efficiency of

the detector.

46
 Results

5.1 Synthesis of CQDs

The CQDs in this project utilizes fullerene (C60) as the carbon source mixed with lithium

borohydride (LiBH4) dissolved in tetrahydrofuran (THF) as the solvent in a round bottom flask in

a glovebox was stirred for 3hrs. The solvent is then removed under vacuum to produce a brownish

black powder labeled as prepared material. Afterward, the sample is annealed at 300℃ for 60

minutes resulting in a carbon quantum dot precursor with a black color appearance. The CQD

precursor, with no passivation shows no florescence when exposed to a UV light source. However,

once it is passivated in air, the CQD precursor had a reddish-brown color and showed fluorescence

when exposed to a UV light source.

5.1.1 Synthesis of 70:30 LiBH4-C60 CQDs

100

80

60
T%

40

20
Annealed sample
After air exposure
0
4000 3500 3000 2500 2000 1500 1000 500
-1
Wavenumber (cm )

Figure 20: FTIR of CQDs

The FTIR results seen in Figure 20 showed without air passivation, oxidation doesn’t

exist. A board alcohol (-OH) peak from 3200-3400cm-1. We also see a very strong board

carboxylic acid (-COOH) peak at 2700-3300 cm-1. Aromatic rings appear around 1600cm-1 and

47
1430-1500 cm-1 and can be weak to strong as in Figure 17. There is a carbonyl (C=O) group

between 1650-1780cm-1. Finally, a strong C-O peak shows is present at 1050-1250cm-1.

Excite @
260
200000 280
300
320
340
150000 360
Intensity

380
400
420
440
100000 460
480
500
520
540
50000 560

0
300 400 500 600 700 800
Wavelength (nm)

Figure 21: CQDs Emission Map

Fluorescence Spectroscopy measurements were also performed. Based on the emission

map on Figure 21, the highest excitation wavelength occurred at 500 nm.

1.2
> 200 nm Stokes Shift

1.0
Intensity (a.u)

0.8

0.6

Ex. Em.
0.4

0.2

0.0
300 400 500 600 700 800
Wavelength (nm)

Figure 22: CQDs Excitation and Emission Spectrum

48
 The excitation and emission spectra for the CQDs is shown in Figure 22, where the

excitation was fixed 520nm and the emission was fixed at 300nm.

35000

Fluorescence Intensity
30000

25000

20000
Excite at 400 nm
15000 Emission at 515 nm
10000

5000

0
0 100 200 300 400
Time (s)

Figure 23: CQDs Photostability Spectrum

Photostability studies were also tested on the CQDs for potential fluorescence dyes. The

emission intensity of the CQD at 151 nm remains constant for 400 s when excited at 400 nm. If

photo bleaching of the CQD is observed, a gradual decrease in the emission intensity over time

would occur. Photo bleaching is commonly observed in many fluorescence microscopy dyes.

5.1.2 Synthesis of 70:30 LiBH4-C60-GdCl3 CQDs

The synthesis of doped 70:30 LiBH4-C60 CQDs was similar to the prior method with a

minor change. The fullerene was mixed with lithium borohydride and gadolinium chloride

dissolved in tetrahydrofuran in a round bottom flask inside an inert glovebox and was stirred for

3hrs. The solvent is then removed under heat and vacuum to produce a brownish black powder

labeled as prepared material. Then, the as prepared material is annealed. The temperature profile

is a ramp to 300℃ over 90 minutes followed by soak at 300℃ for 60 minutes. The resulting carbon

quantum dot precursor had a black color. A schematic of the overall synthesis is seen in Scheme

3.

49
 homogenize

anneal
Gd-CQD
GdCl3 + LiBH4

Scheme 3: Synthesis Pathway of Gd-CQDs

The CQD precursor, with no passivation state shows no florescence when exposed to a UV

light source. However, it is passivated by air exposure, the CQD precursor had a reddish-brown

color and showed fluorescence when exposed to a UV light source.

120000
Excite @ (nm)
270
100000 290
310
330
80000 350
Intensity

370
390
410
60000 430
450
470
40000 490

20000

0
300 400 500 600 700 800
Wavelength (nm)

Figure 24: Emission Map of Gd-CQDs

Figure 24 shows the emission map of the Gd doped CQDs and is similar to that of the base

CQDs.

50
 GdCl3

CQD annealed -BH

bend
Gd:CQD annealed
CQD oxidized

%T
Gd:CQD oxidized

-OH
C=O bend
-OH -BH
C=C
stretch stretch Ar

4000 3500 3000 2500 2000 1500 1000

-1
Wavenumber (cm )

Figure 25: FTIR of Gd doped CQDs

Figure 25 shows five different spectra. The pure GdCl3 shows no well-defined peaks. The

annealed CQD and the Gd doped CQD samples are very similar. There is a -BH bend at 1000-

1500 cm-1 and a -BH stretch 2300 cm-1. The CQD oxidized sample shows -OH stretch at 3000-

3600 cm-1, -BH stretch reduced peak at 2300 cm-1, C=O peak at 1618 cm-1, Ar C=C peak at 1427

cm-1 and an -OH bend at 975.8 cm-1. The Gd doped CQD oxidized sample shows a reduced -OH

stretch peak at 3000-3600 cm-1, -BH stretch sharp peak at 2300 cm-1, C=O peak at 1618 cm-1, Ar

C=C peak is slight shifted at 1450 cm-1 and an -OH bend is also slightly shifted and slightly

broadened at 976 cm-1 The slight differences between the CQD and the Gd doped CQD in the

oxidized state show slight differences in the –OH stretch and bending regions. However, further

studies are need to determine if the slight differences are due to the incorporation of Gd into the

CQDs.

51
 Annealed GdCl3 -LiBH4 -C60

! !! ! ^ ^ As prep GdCl3-LiBH4-C60
! ! ! ^! !
!! ^
#
! !
*! ! Hand mix GdCl3-LiBH4-C60
#
# * ! *
*! !! * ! !*!
# ! !
* !# *
# * * * * ** **
*
*
* GdCl3
*
* * * * *
* * * * ** * *

10 20 30 40 50 60
Degrees (2q)

Figure 26: XRD on Gd doped CQDs

Figure 26 shows four different spectra. The GdCl3 spectra shown in black was used to

compare all Gd containing CQDs and shows all the GdCl3 peaks (*). The hand mixed shown in

red shows a mixture of all three compounds (GdCl3, LiBH4 and C60) mixed together and shows

the peaks pertaining to, LiBH4 (!) and C60 (#). The as prep GdCl3 doped CQDs is shown in blue

and clearly shows the formation of LiCl (^) and LiBH4 peaks. The annealed Gd doped CQDs

shown in purple tells us that we formed a solid solution since all the LiCl peaks disappear and only

very tiny peaks of LiBH4 are shown.74

5.1.3 Synthesis of 70:30 LiBH4-C60-EDC/NHS-Trp CQDs

Studies have shown various amino acids were introduced on the surface of carbons dots

by EDC/NHS coupling.

52
 Scheme 4: Synthesis Pathway of Chiral Functional CQDs

The overall synthesis pathway summary of functionalizing our CQDs is shown in scheme

4. 10.02mg CQDs made in section 5.1 was placed in a vial to passivate for 24hrs. 9.0mg of water,

100mg EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 100mg NHS

(N-hydroxysuccinimide) was added to the vial and mixed for 2 hours shown in Scheme 5.

Scheme 5: EDC/NHS crossing coupling reaction mechanism

With the addition of EDC/NHS precursor, 100mg L-Trp was slowly added to the reaction

mixture and left stirring for 24hrs as shown in scheme 6. Setup a 24-hour dialysis. We removed

solvent to collect 2.5mg of L-Trp CQD.

53
 Scheme 6: Tryptophan insertion mechanism

The 10.05mg CQDs made in section 5.1 was placed in a vial to passivate for 24hrs. 9.0mg

of water, 100mg EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and

100mg NHS (N-hydroxysuccinimide) was added to the vial and mixed for 2 hours. Added 100mg

D-Trp slowly to the reaction mixture and left stirring for 24hrs. After a 24-hour dialysis, the

solvent was removed to 2.7mg of D-Trp CQDs was collected.

5.1.4 Synthesis of Lawsone CQDs

Scheme 7: Synthesis pathway of generating lawsone CQDs

Lawsone was examined as a possible inexpensive plant-based material as a carbon source

for CQDs. Lawsone was mixed with potassium periodate and ethanol to undergo a solvothermal

54
reaction at different times (1, 12 and 24 hours) to yield lawsone derived CQDs as shown in scheme

5.

30000
Excite @ Excite @
4000 350 nm 350 nm
375 nm 25000 375 nm
425 nm 425 nm

Intensity
3000 20000
Intensity

15000
2000
10000
1000
5000

0 0
300 400 500 600 700 800 300 400 500 600 700 800

Wavelength (nm) Wavelength (nm)

Figure 27: 0 Hour Emission Spectra Figure 28: 1 Hour Emission Spectra

Figure 27 shows that pure lawsone (0 hour) has only minor, low intensity emission peaks

when compared to the hydrothermal treated samples. The sharp emission peaks in the spectrum

that change wavelength based on the excitation wavelength, are due to Raman scattering of the

solvent and not due to the emission from lawsone. The sharp Raman scattering peaks are attributed

to ethanol and possibly water and its shifts as the excitation wavelength is changed. Raman

scattering is typically observed when the sample shows weak emission. Figure 28 shows a big

significance compared to Figure 27. The emission intensity increases upon hydrothermal

treatment with the formation of a peak at 400 nm and two additional peaks at 525 nm 575 nm.

This indicates the possible formation of a larger organic polymeric structure such as a CQD.

55
 35000
Excite @ Excite @
350 nm 160000 350 nm
30000
375 nm 375 nm
425 nm 425 nm
25000
Intensity

120000

Intensity
20000

15000 80000

10000
40000
5000

0 0
300 400 500 600 700 800 300 400 500 600 700 800

Wavelength (nm) Wavelength (nm)

Figure 29: 12 Hour Emission Spectra Figure 30: 24 Hour Emission Spectra

As hydrothermal time increases to 12 hours, the overall intensity of the emission peaks increase

and the 525 and 575 nm peaks become more intense relative to the 400 nm peak. Raman scattering

is not observed due to strong emission from the sample. 24 hours of hydrothermal treatment is

shown in Figure 30. The intensity of the emission further increases, however, the well-defined

peaks at 400, 525, and 575 nm are broadened. This indicates a collapse or possible destruction of

the structure of the polymeric CQD material during the process. Further investigation is needed

to understand the cause of this behavior.

100000
Lawsone
1 hr
80000 12 hr
24 hr
Intensity

60000

40000

20000

0
300 400 500 600 700 800

Wavelength (nm)
Figure 31: 375 nm excitation spectra

56
Figure 31, shows the emission spectrum at a fixed excitation wavelength of 375 nm for the

different hydrothermal treatments. There is no significant emission at the zero-hour sample (pure

Lawsone). As the hydrothermal treatment time is increased, the overall intensity of the emission

resulting from the lawsone derived CQDs increases. This clearly indicates that hydrothermal

treatment time has a major influence on the Lawsone derived CQDs. Further studies are needed

to understand the anatomy and mechanism of the CQDs formed from lawsone

5.2 Cell Toxicity

Biochemical evidence indicates that MTT is mainly reduced in the cytoplasm by NADH

oxidoreductases associated to the endoplasmic reticulum75, endosome/lysosome vesicles76, and

plasma membrane77. Measurement of the absorbance of formazan via 96-well plate assay was

proposed for assessing the effects of CQDs inhibition on the cell mechanism cytotoxicity in cancer

cells. A way to measure cell toxicity is by fluorescence plate reader using a 96 well plate reader.

Figure 32: Quantify effects inhibit on cell metabolism spectrum78

Once the absorbance was collected at two distinct wavelengths shown in Figure 32, the

data points are corrected by equation 5.2.1 and plotted:

(\'() !\*+) )
% 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = \ × 100
,!--. ,!01-!2 345. (5.2.1)

57
Most of the data were done on independent triplicate runs except for pure Gd. All the MTT assay

analysis were done Prism 9 software to do plots and perform statistics. Dunnett’s multiple

comparison 1-way ANOVA test was performed for all the data collections.

5.2.1 MTT Assay of 70:30 LiBH4-C60 CQDs

Dialyzed CQDs
ns ns

ns
150
ns
150
ns

100

% Viability
100
% Viability

50
50

0 0
Control 50ug/mL 200ug/mL 500ug/mL Control 50ug/mL 200ug/mL 500ug/mL
Concentration CQDs (ug/mL) Concentration CQDs (ug/mL)
Figure 33: Comparison to the Control Figure 34: Comparison to the lowest
concentration (50𝜇g/mL)

Two different comparisons for the dialyzed CQDs were performed. In Figure 33, the

control was compared to the different concentrations (50𝜇g/mL, 200𝜇g/mL, & 500𝜇g/mL). As

determined by Dunnett’s multiple comparison 1-way ANOVA test, dialyzed CQDs did not

significantly impact cell viability of the cancer cells based on the MTT assays. In Figure 34, the

cell viability is compared to the samples that contained the lowest concentration of CQDs (50

𝜇g/mL)

58
 Undialyzed CQDs
ns ✱
200
✱✱ ✱✱✱✱
200
ns 150

% Viability
150
% Viability

100
100
50
50

0
0 Control 50ug/mL 200ug/mL 500ug/mL
Control 50ug/mL 200ug/mL 500ug/mL
Concentration CQDs (ug/mL)
Concentration CQDs (ug/mL)

Figure 35: Comparison to the Control Figure 36: Comparison to the lowest
concentration (50𝜇g/mL)
In Figure 35, once again, the control was compared to the different concentrations

(50𝜇g/mL, 200𝜇g/mL, & 500𝜇g/mL). As determined by Dunnett’s multiple comparison 1-way

ANOVA test, dialyzed CQDs did not affect the viability of the cancer cells. However, there was

a significant different between the control and the 200𝜇g/mL with a p-value = 0.003. We also did

a comparison based on concentrations only Figure 36 and compared it to the lowest concentration

and once again there was a large significant between the 200𝜇g/mL verses the 50𝜇g/mL with a p-

value < 0.0001 and a slight significant between 500𝜇g/mL vs the control with a p-value =0.0135.

Regardless of these results, ultimately the cancer cells did not die since it clear shows more than

50% viability survival on both Figures 35 and 36.

5.2.2 MTT Assay of 70:30 LiBH4-C60-GdCl3 CQDs

ns
ns
200
ns
200 ns
ns
150
% Viability

150
% Viability

100
100

50 50

0 0
Control 50ug/mL 200ug/mL 500ug/mL Control 50ug/mL 200ug/mL 500ug/mL
Concentration Gd-CQDs (ug/mL) Concentration Gd-CQDs (ug/mL)

Figure 37: Comparison to the Control Figure 38: Comparison to the lowest
concentration (50𝜇g/mL)

59
 In Figure 37, the control was compared to the different concentrations (50𝜇g/mL,

200𝜇g/mL, & 500𝜇g/mL). Based on the data, Gd-doped CQDs did not affect cell viability. We

also did a comparison based on concentrations only and compared it to the lowest concentration

and once again, no significance difference was observed in Figure 38.

5.2.3 MTT Assay of GdCl3

✱✱✱ ✱✱✱

ns 150 ns
150 ns

100

% Viability
100
% Viability

50
50

0 0
Control 50ug/mL 200ug/mL 500ug/mL Control 50ug/mL 200ug/mL 500ug/mL
Concentration Gd (ug/mL) Concentration Gd (ug/mL)

Figure 39: Pure Gd MTT assay comparison Figure 40: Pure Gd MTT assay comparison
to control to lowest concentration (50𝜇g/mL)

The Gd source (GdCl3) was also evaluated by MTT assay to make a clear indication

whether gadolinium was inside the CQD or on the surface. In Figure 39, the control was compared

to the different concentrations (50𝜇g/mL, 200𝜇g/mL, & 500𝜇g/mL). Based on the data, there is a

significant difference on the highest concentration dosage compared to the control showing cell

death with a p-value = 0.001. A comparison amongst the concentrations only and compared it to

the lowest concentration and once again, there was a huge significance different on the highest

concentration dosage seen in Figure 40 with a p-value = 0.001.

5.3 Fluorescent Microscopy

Previous studies have done CQDs images and showed promising results. The C60 based

CQDs were then evaluated as fluorescence microscopy dyes with the MDA-MB-231 cancer cell

line.

60
5.3.1 LiBH4-C60 CQDs

Table 1: Initial Optimization Experiments for Fluorescence Microscopy

DIC DAPI CFP EGFP TaYFP mRF12

Negative
Control

MDA-MB-231
Cells with
CQDs

From the results shown on Table 1, we saw promising results with our CQDs and MDA-

MB-231 cells lines when they were in the trypsinized state. The negative control looked like

photobleaching occurred or the light from the monitor was a disturbing factor since the room

wasn’t completely dark. However, the cells with CQDs, we clearly saw a sharp fluorescence where

the CQDs were localized. The big question raised if the cells were on the surface or inside the

membrane. To answer the question, a more advanced fluorescence microscope was utilized.

61
Table 2: New Fluorescence Microscope Images

DIC DAPI CFP EGFP TaYFP mRF12

Negative
Control
(10X)

MDA-MB-231
Cells with
CQDs
(10X)

MDA-MB-231
Cells with
CQDs
(100X)

From Table 2, the advanced fluorescence microscope showed very promising results for

the cancer cells in their nature (not trypsinized) state. We plated the cells in an 8 well microscope

slide. After 15 hours, we placed the slide on the microscope stage. We clearly saw the negative

control had no fluorescence. Conversely, cells with CQDs showed very sharped fluorescence with

the CQDs inside the cells. We ran Z-stack to verified if the cells were on the surface or inside the

membrane.

From Table 3, three different foci planes were selected from the z-stack and shows that the

CQDs were eventually inside the cell membrane. The first row shows the fluorescence of the cells

with CQDs blurry and out of focus with the focal plane outside of the cell. The second row shows

a focal plane that is inside the cell, but not at the exact focal plane where the CQDs are located

inside the cell. This is the cause of the increase in emission intensity in the different channels.

The last row is a focal plane that is inside the cell where the CQDs are localized with significantly

enhanced resolution. This indicates that the CQDs are localized inside the cells and not on the

surface.

62
Table 3: MDA-MB-231 Cells with three different focal planes

DIC DAPI CFP EGFP TaYFP mRF12

5.4 Circular Dichroism

Chiral quantum dots are a class of nanomaterials with chiral and fluorescence properties

and have been gaining lots of interest due to its fluorescence properties and cell regulation. After

the synthesis of L/D-Trp functionalized CQDs, circular dichroism and FTIR measurements were

performed.

5.4.1 LiBH4-C60-EDC/NHS-Trp CQDs

8
L-Trp
L-Trp:CD
6 D-Trp
D-Trp:CD
4
CD (mDeg)

-2

-4
200 210 220 230 240 250 260

Wavelength (nm)

Figure 41: Chiroptical properties of tryptophan enantiomers and their respective chiral CQDs

Based on our results seen in Figure 41, L- and D- Trp conjugated CQDs produced the

corresponding mirror image CD signature. The CD spectra of free L- and D- Trp showed, as

63
expected, near symmetrical spectra with a positive and negative effect, respectively, at 210-230

nm. The sign of CD signature from L-/D- Trp CQDs were similar to their parent L-/D- Trp,

respectively, with a slight shift of the peak maxima at 221 nm and minima at 223 nm.

L-TRP
Absorbance

L-TRP:CD

CD

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure 42: FTIR of Enantiomeric CQDs

The FTIR of the sample is shown in Figure 42. Based on the results, there is a sharp

primary amine peak at 3400cm-1 and a -C=O stretch at between 1500-1700 cm-1 for the pure

tryptophan sample. However, as we observe the functionalized L-Trp CQD, the primary amine is

weak and merges with a weak broad -OH hump. The carbonyl (-C=O) stretch broadens between

1500-1700cm-1 and an amide (-CN) stretch is present at 1450cm-1. When compared to the base

CQD, there is a large broad (-OH) with (-COOH) -peaks between 3300-3700cm-1. We believed

we successfully functionalized a chiral CQDs.

5.5 Nuclear Magnetic Resonance T1 Relaxation

Initially, T1 were done on NMR. However, we wanted to replicate the runs to confirm if

our results are real using a benchtop Alegre MRI.

64
5.5.1 LiBH4-C60-CQDs
Previous results showed CQDs had promising results as seen in Figure 43. The longitudinal

relaxation (r1) of the CQDs prepared under optimal conditions was measured at 3.6s to 0.047s.

Table 5: Initial CQDs NMR Results

25
concentration [mmol/L] 1/T1 (s) T1 (s)
0 0.27700831 3.610 20
0.02 1.44092219 0.694

1 / T1 (s-1)
0.025 5.37634409 0.186 15
0.05 8 0.125
0.075 14.5485232 0.047 10
CQD
y = 26.6x - 0.99
Table 6: Gadoderitol NMR Results
5 Gadaderitol
y = 1.5x + 0.32
concentration [mg/mL] 1/T1 (s-1 ) T1 (s)
0 0.28 3.61 0
0.11 0.61 1.65
0.22 0.66 1.51 0.0 0.2 0.4 0.6 0.8 1.0 1.2
0.34 0.69 1.44 Concentration (mg / mL)
0.45 0.87 1.15
0.56 1.33 0.75 Figure 43: NMR Initial CQD Results
0.67 1.28 0.78

Commercially available MRI agent, Gadoderitol, was tested to illustrate the MRI

performance of our CQDs. The r1 relaxivity shows a superior enhancement of relaxation as the

concentration of CQD increased in Figure 43. We also ran MRI to confirm if our CQDs data was

real.

Table 7: MRI CQDs Results

concentration [ug CQD/mL] T1 (s-1) 1/T1 (s) T2 (s) 1/T2 (s-1)

0 3.01 0.33 2.70 0.37
200 2.95 0.34 2.71 0.37
400 3.12 0.32 2.64 0.38
800 3.1 0.32 2.81 0.36

Both T1 and T2 measurements were taken on the MRI. Unfortunately, we did not see an effect

on the T1 or T2 for the base CQD (Gd free). We also didn’t see the results compatible with the

previous experiments obtained by NMR.

65
5.5.2 LiBH4-C60-GdCl3 CQDs

We also did a previous NMR on Gd-CQDs and showed promising results as seen in figure

34. The longitudinal (r1) of the Gd-CQDs prepared under optimal conditions was measured at

Table 8: Initial Gd NMR Results 1.8

1.6

-1
concentration [mg/mL] 1/T1 (s ) T1 (s) 1.4
1.2

0.11 0.61 1.65

1/T1 (s-1)
1 y = 4.4688x + 0.1628
R² = 0.9979
0.22 0.66 1.51 0.8

0.6
0.34 0.69 1.44 0.4

0.45 0.87 1.15 0.2

0
0.56 1.33 0.75 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Gd3+ [mg/mL]
0.67 1.28 0.78
Figure 44: Initial Gd NMR Results

From the data on table 8 and Figure 44, the r1 relaxivity shows a superior enhancement of

relaxation as the concentration of Gd3+ increased. We also ran MRI to confirm if the Gd3+ data

was consistent.

Table 9: MRI Gd-CQDs T1 & T2:

+3
concentration [ug Gd /mL] concentration [mM Gd ]
+3
T1 (s) 1/T1 (s-1) T2 (s) 1/T2 (s-1)
0 0 3.01 0.33 2.70 0.37
0.90 0.00572 0.560 1.79 0.503 1.99
2.69 0.0171 0.277 3.61 0.237 4.22
6.75 0.0429 0.173 5.78 0.151 6.62
15.73 0.1 0.077 12.99 0.067 14.93

Both T1 and T2 measurements were taken on the MRI and showed a clear decreased in the

presence of the Gd-CQD.

66
 Bioimaging Implications and Conclusion

The aim of this thesis was to functionalize our CQD for bimodal purposes, particularly for

fluorescence microscopy and as MRI contrast agents. We were able to observe the

photoluminescent properties and stabilize our CQDs for potential fluorescence microscopy dyes.

We were also able to observe our CQDs are inside the MDA-MB-231 cancer cells. Fluorescence

microscopy suggest that the CQDs are localized in the nucleus of the cell, however, further studies

are needed to confirm.

We performed cell viability studies using MTT assay and found that our CQDs and Gd-

CQDs did not affect cell viability. However, pure Gd showed cell death at the highest

concentration (500𝜇g/mL). This demonstrated that the Gd was encapsulated in the CQD and is

close to the surface to shorten T1 and T2. To guarantee our hypothesis, XPS would need to be

performed to conclude this thesis and determine the chemical environment around the Gd.

We were successful in doping our CQDs with Gd3+. We conducted NMR studies for MRI

objectives by running T1 relaxations. Initially, T1 showed superior enhancement on r1 with our

CQDs compared to the commercially available MRI contrasting agent, Gadoderitol. Conversely,

we also ran trials via a benchtop MRI and found no significance in enhancement. We also

performed NMR studies for MRI purposes for the Gd-CQDs and once again found enhancements

on r1. We also ran our trials via MRI and once again found enhancements on T1 and T2.

We can also functionalization our CQDs by adding chiral groups via cross-coupling

reactions using EDC/NHS. This thesis shows one amino acid (tryptophan), but we can attach other

amino acids (e.g., proline, cysteine, histidine, etc.) to the CQD. We were successfully able to

conduct circular dichroism and confirmed they were similar to the parent signature. One other

aspect to consider is to see if sugars can be functionalized to the CQDs.

67
 Finally, we can also functionalize our CQDs using a sustainable carbon source. This thesis

shows Lawsone (henna plant), which is a natural sustainable carbon source for their production,

as an interesting candidate for the production of CQDs

68
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