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Functionalization of nanodiamond surface for magnetic sensing application

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Functionalization of nanodiamond surface for magnetic sensing application

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Functionalization of Nanodiamond Surface for Magnetic Sensing Application

by

Rana Dib Akiel

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

December, 2016

© Copyright by
Rana Dib Akiel
2016
III

Dedication
I dedicate this work to my husband, Dori Akiel, for his unconditional love and support
throughout my education, starting from my Bachelor’s degree and ending with my PHD.
It is your constant pushing me forward that got me where I am today. Your great sacrifices
and encouragements kept me going all these 10 years. You have an equal share in this
work. I am very grateful for having you in my life.
I also dedicate this work to my two beautiful daughters, Yara and Sarah Akiel. It is you
being independable and responsible that allowed me the time to spend on my work. I
couldn’t have asked for a better behavior than you two showed. I am so proud of you.
You girls are my rock stars. I know that my pursuit of this degree have taken many hours
away where I could have spent them with you, but I am hoping that I have set a good
example for you to follow when you grow-up.
Finally, I dedicate this work to my family. Words cannot express how grateful I am to
my mother, Amal Faraj, my father, Ibrahim Dib, my mother-in-law, Helene Akrouche, and
my father-in-law, Khalil Akiel, for your love and support. Your prayers for me was what
sustained me thus far.
IV

Acknowledgment
I would like to express my sincere gratitude to my PHD advisor, Professor Susumu
Takahashi. I thank him for the opportunity he has presented me when he allowed me to
join his group. Professor Takahashi is a great mentor that has always been there to
discuss and help with my experiments and analysis. It is his guidance and support that
allowed me to grow as a research scientist. During my graduate years in his group, I have
learned the essence of carrying experiments, analysis, and most importantly I have
developed a great sense of problem solving. I truly appreciate the love of science he has
in him. His contribution to the scientific world keeps on growing year after year.
I greatly appreciate the support received through the collaborative work undertaken
with the Qin’s group at USC. In particular, I would like to thank Professor Peter Qin for his
scientific advice and the many helpful discussions and suggestions that he has provided
me. I truly appreciate the great scientist he is and his broad knowledge. I also would like
to thank Dr. Xiojun Zhang for his support and help in the experiments and measurements.
Next, I would like to acknowledge my group members. A special thanks to Dr. Ekaterina
E. Romanova, a former post-doc in our group who has provided me with tremendous
guidance during my first year of graduate research. My former colleague, Dr. Franklin
Cho, and my present group members, Chathuranga Abeywardana, and Viktor Stepanov
have all extended their support in many different ways. I gained a lot from them, through
their scholarly interactions and their suggestions at various points of my research
program.
I also would like to present my gratitude to my committee members, Professor Stephan
Bradforth, Professor Peter Qin, Professor Fabian Pinaud, and Professor Mark Thompson
V

(listed in alphabetic order) for their time and support throughout the screening,
qualification, and defense processes.
Finally, I would like to thank Professor Hanna Reisler for her support as an advisor
during the first year of my graduate work and also for her advice and guidance during my
qualification exam process. Last but not least, I would like to present my appreciation for
the staff and faculty members of the Department of Chemistry who have nominated to
receive the various awards that I have been granted at USC, namely the 2013 Teaching
Award, the Department of Chemistry Advanced Fellowship for summer 2015, the Stauffer
Endowed Fellowship for 2105-2016, and the the Micheal J. Dulligan memorial award
2016.

VI

Table of Contents
Dedication ..................................................................................................................... III
Acknowledgment.......................................................................................................... IV
Table of Contents ......................................................................................................... VI
List of Figures ............................................................................................................... X
List of Schemes .......................................................................................................... XV
List of Abbreviations ................................................................................................. XVI
List of Symbols ....................................................................................................... XVIII
List of Physical Constant ......................................................................................... XIX
Abstract .......................................................................................................................... 1
Chapter 1 Introduction on Nanodiamond .................................................................... 2
1.1 Methods for ND synthesis ........................................................................................................ 3
1.2 Properties of ND ........................................................................................................................... 4
1.2.1 Nitrogen-vacancy centers .............................................................................. 4
1.2.2 Biocompatibility ............................................................................................. 7
1.3 Surface modification of NDs ................................................................................................... 7
1.3.1 Surface homogenization of ND ...................................................................... 9
1.3.2 Surface coating of NDs................................................................................ 12
VII

1.3.3 Functionalization of ND with target molecule ............................................... 14
Chapter 2 Electron Paramagnetic Resonance Spectroscopy ................................. 16
2.1 Magnetic interactions .............................................................................................................. 16
2.1.1 Zeeman interaction ...................................................................................... 17
2.1.2 Zero-field splitting ........................................................................................ 19
2.1.3 Nuclear Zeeman interaction ........................................................................ 20
2.1.4 Hyperfine coupling ....................................................................................... 20
2.1.5 Nuclear quadrupole interaction .................................................................... 21
2.1.6 Dipolar interaction ....................................................................................... 21
2.2 Principles of continuous wave EPR spectroscopy .................................................... 22
2.3 Simulation of EPR spectra.................................................................................................... 25
2.3.1 Solid state regime ........................................................................................ 25
2.3.2 Slow motion regime ..................................................................................... 31
2.4 Characterization of paramagnetic impurities in ND using high-frequency CW
EPR ........................................................................................................................................................... 35
Chapter 3 Dynamics of molecules undergoing Brownian motion .......................... 37
3.1 Langevin equation .................................................................................................................... 37
3.2 Free Brownian motion ............................................................................................................. 38
3.3 Rotational Correlation function and correlation time ................................................ 39
3.4 Simulation procedure .............................................................................................................. 41
VIII

Chapter 4 Surface Chemistry of Diamond to Introduce Functional Groups .......... 44
4.1 Silanization technique to introduce azide groups to ND surface ........................ 44
4.1.1 Preparation of ND surface for silanization ̶ acid cleaning and borane
reduction .................................................................................................................. 45
4.1.2 Functionalization with azide through silanization ......................................... 47
4.2 Copper click reaction applied to NDs .............................................................................. 49
4.2.1 Synthesis of alkyne functional nitroxide ....................................................... 50
4.2.2 Copper click reaction of N3-ND with TEMPO-alkyne ................................... 51
4.3 FTIR characterization .............................................................................................................. 53
4.3.1 Characterization of the azide functional NDs ............................................... 53
4.3.2 Characterization of copper click reaction ..................................................... 57
4.4 Characterization using EPR ................................................................................................. 58
4.4.1 Characterization of N3-ND ........................................................................... 60
4.4.2 Characterization of TEMPO-ND .................................................................. 62
Chapter 5 Nitroxide-Functionalization of ND Surface Using Copper-Free Click
Chemistry ..................................................................................................................... 64
5.1 Copper free click chemistry applied to ND.................................................................... 65
5.1.1 Optimization of reaction conditions .............................................................. 66
5.2 Copper free click of N3-ND and TEMPO ........................................................................ 69
5.2.1 Synthesis of TEMPO-DBCO ........................................................................ 69
5.2.2 Reaction between TEMPO-DBCO and N3-ND ............................................ 70
5.3 EPR results and analysis ...................................................................................................... 70
IX

5.4 Simulation of restricted Brownian motion ...................................................................... 74
Chapter 6 DNA-Functionalization of ND Surface ...................................................... 78
6.1 Copper free click reaction applied to DNA .................................................................... 80
6.1.1 Synthesis of DBCO tagged DNA ................................................................. 80
6.1.2 Click chemistry between S1-DBCO and azide functional TEMPO .............. 81
6.1.3 Results ........................................................................................................ 82
6.2 Attachment of S1-DBCO to N3-ND and multiple hybridization of different
complimentary strand ................................................................................................................................. 85
6.2.1 Methods....................................................................................................... 85
6.2.2 Results ........................................................................................................ 87
6.2.3 EPR analysis ............................................................................................... 93
Chapter 7 Conclusion ................................................................................................. 99
Appendix .................................................................................................................... 100
A.1 Normalization of the FTIR spectra in the case of copper click reaction ........ 100
A.2 Calibration curve of intensity of EPR vs. concentration of TEMPO ................ 102
A.3 Adsorption Studies of TEMPO on the surface of ND ............................................. 104
A.4 Verification of the wash procedure for removing DNA strands non-specifically
associated with ND ................................................................................................................................... 106
Reference ................................................................................................................... 108
X

List of Figures
Figure 1.1. NV center structure and energy levels. (a) Structure of NV center in diamond.
The black circles represent the carbon atoms, the red circle represents the nitrogen atom,
and V represents the vacancy. (b) Electronic energy levels of NV centers. The green
arrow represents optical excitation, the red wiggly arrow represents FL decay with
transition energy of 638 nm, and the dashed arrows represent non-radiative decay to
metastable states. ........................................................................................................... 6
Figure 1.2. Surface functional groups of ND. The surface of ND can be terminated by a
variety of surface functional groups originating form the production process. ................. 8
Figure 1.3. Homogenization of the ND surface. Different homogenization techniques
have been developed. Oxidation results in a surface rich with COOH groups.
Hydroxylation results in a surface rich with OH groups. Hydrogenation results in surface
homogeneous with hydrogen termination. Thermal treatment or irradiation of ND with high
energy beam results in the removal of all functional groups with the formation of thin
graphitic layer. ............................................................................................................... 11
Figure 2.1. Energy levels of 1/ 2 S = system. ................................................................ 18
Figure 2.2. Energy levels of 1 S = system. (a) Energy level splitting of S=1 system caused
by magnetic field in the absence of ZFS. (b) Energy level splitting of S=1 system caused
by magnetic field in the presence of ZFS. ..................................................................... 19
Figure 2.3. Energy levels of 1/ 2 S = and 1 I = system. ................................................ 21
Figure 2.4. Modulation field technique. ......................................................................... 24
Figure 2.5. CW EPR simulation of P1 centers. (a) represent the energy levels of P1
centers as a function of magnetic field. The arrows represent the allowed transitions when
XI

the frequency of the applied microwave is 230 GHz. The simulation spectrum shows one
possible orientation of P1. (b) the simulation of P1 center considering all possible
orientations. The inset represents the absorption spectra (blue) obtained by the addition
of all orientations (red). .................................................................................................. 30
Figure 2.6. Simulation of nitroxide radicals using slow motion regime with different
correlation times. We see that as the correlation time increases, uneven broadening of
the EPR peak occurs. For very slow correlation time, new features appear. ................ 34
Figure 2.7. Spectral analysis of 100 nm-ND EPR signals. 230 GHz EPR of the NDs is
shown by the solid blue line. The spectrum of the P1 centers was simulated using
, 1 I = , gx,y = 2.0024, gz = 2.0024, and the
14
N hyperfine coupling 82
x y
A A = = MHz,
114
z
A = (magenta). 1/2 S = spins were simulated with 1/2 S = ,
,
2.0029 0.0001
xy
g= ±
and 2.0027 0.0001
z
g= ± (violet). The green dotted line is the sum of the N and 1/ 2 S =
EPR spectra. The simulations were performed using Easyspin.
66
................................ 36
Figure 3.1. Correlation function simulation of free rotational Brownian motion. (a) and (b)
are the trajectories of particles of radius 25 and 50 nm respectively. (b) shows the results
of the correlation function as a function of time along with the exponential fit. .............. 43
Figure 4.1. FTIR spectra of NDs. FTIR signals of 100 nm NDs after acid treated, borane
reduction, silanization with bromide, and azide functionalized ND. ............................... 55
Figure 4.2. Results of the stability studies. blue represents the experimental data. The
red represents the multi-peak Lorentzian fits. The green traces represent the Lorenztian
fit of each peak separately which addition results in the red trace. The ratio between N3
and C=O was determined using the green traces corresponding to each group. .......... 56
1/2 S =
XII

Figure 4.3. FTIR results of copper click chemistry between N3-ND and TEMPO-alkyne.
(a) FTIR spectra of N3-ND and TMEPO-ND. (b) FTIR spectra of azide moieties at 2100
cm
-1
. .............................................................................................................................. 57
Figure 4.4. Aqueous sample holder. (a) Electric and magnetic field components of
microwave in the sample holder. The sample is positioned on the aluminum tape. (b)
Schematics for aqueous sample design. The top and bottom caps are made of Teflon.
Doted lines indicate threads and volume for screws that are used to tighten top and
bottom caps. .................................................................................................................. 59
Figure 4.5. EPR analyses of the N3-ND sample. (a) 115 GHz EPR of N3-ND and (b) X-
band EPR of N3-ND are shown by the solid blue lines. (a) and (b) also show the
contributions of P1 centers (magenta) and the 1/ 2 S = spins (purple) obtained by
simulating the experimental EPR spectra. The dotted green lines overlaid with the
experimental data shows the sum of the N and 1/ 2 S = contributions. The simulations
were performed using Easyspin.
66
................................................................................ 61
Figure 4.6. CW EPR results on TEMPO-ND. (a) 115 GHZ EPR results of TEMPO-ND.
(b) X-band EPR of TEMPO-ND. .................................................................................... 63
Figure 5.1. FTIR results for copper free click chemistry on N3-ND. (a) FTIR spectra of N3-
ND and DBCO-ND. The spectra were normalized by the sample weight. (b) Attachment
efficiency determined based on intensity changes in the N3 FTIR peaks. To allow proper
comparison, each data point was scaled by normalizing the amount of ND to 0.3 mg and
the reaction volume to 100 µL. ...................................................................................... 68
Figure 5.2. EPR analyses of the TEMPO-ND sample. (a) 115 GHz EPR data. The
experimental EPR data of nitroxide radicals is magnified by 4 times. The solid blue line is
XIII

experimental data, and the green and magenta solid lines in inset are simulated EPR
spectra of ND and nitroxide radical. The red dotted line shows the sum of the simulated
spectra. (b) X-band EPR data are shown by the solid blue lines. The red dotted lines
represent the simulation using same parameters as (a). Samples (a) and (b) were
suspended in phosphate-buffered saline pH 7 at a concentration of 0.1 mg/µL and the
spectra were recorded at room temperature. ................................................................ 72
Figure 5.3. EPR simulation of free TEMPO and of TEMPO tethered to ND. ................ 73
Figure 5.4. Motions of grafted nitroxide. i) Global rotational tumbling of nitroxide. This
motion is dictated by the rotational tumbling of ND. It is the same for TEMPO-ND and the
nitroxide-S2-S1-ND. ii) internal motion in the case of TEMPO-ND. ............................... 76
Figure 5.5. Cone model used for the simulation of tethered nitroxide. ......................... 76
Figure 5.6. Results of the simulation of the tethered motion. (a) Simulation of tethered
motion using cone semi angle of 60
o
. (b) Simulation with cone semi-angle of 30
o
. (c) and
(d) results of the correlation time obtained form (a) and (b) respectively. (e) The results
of correlation function simulation of free molecules of radius 0.1 nm. ........................... 77
Figure 6.1. Anion-exchange HPLC of DNA-DBCO. The traces show precursors (top:
DBCO-NHS; middle: DNA S1-NH2) and DBCO-reacted products (bottom). ................. 83
Figure 6.2. EPR spectrum of S1-TEMPO. 𝜏𝜏 c was determined to be 0.57±0.04 ns based
on EPR lineshape simulation (solid blue line). The inset shows EPR data of a free
nitroxide, which gives 𝜏𝜏𝜏𝜏 = 0.03±0.01 ns under the same experimental conditions.
Lineshape simulations were performed using Easyspin with the isotropic motion model
and the TEMPO spin Hamiltonian ( 1/ 2 S = , 2.0085
x
g = , 2.0059
y
g = , 2.0021
z
g = ,
6.5
x
A = MHz, 5.6
y
A = MHz, 37
z
A = MHz). ............................................................... 84
XIV

Figure 6.3. FTIR results of S1-ND. (a) The spectra were normalized by the sample weight
as describe in Sect. A.2. (b) ND-weight-normalized FTIR signal at the 2100 cm
-1
region.
...................................................................................................................................... 88
Figure 6.4. Repeated hybridization with DNA grafted on ND surface. (a) EPR spectrum
of DNA-tethered ND sample (S1-ND) before (top) and after (center) hybridization with
R5(p4)-labeled complementary strand (R5(p4)-S2-S1-ND) and EPR results measured
after adsorption studies (bottom). The microwave frequency was ~9.33 GHz. (b) The
results of the 1st hybridization of R5(p4)-S2 with S1-ND, EPR spectrum of S1-ND after
denaturing, and the 2nd hybridization of the R5(p4)-S2 with the S1-ND obtained after
denaturing. (c) EPR spectrum of different S2 strand hybridized to S1-ND. The top
spectrum is the hybridization of R5(p4), the middle spectrum is the hybridization of
R5(p17)-S2 and the bottom spectrum is the R5a(p4)-S2. The R5a(p4)-S2 is the result of
a third hybridization on the same S1-ND from (b). ........................................................ 92
Figure 6.5. EPR results along with the simulations of (a) R5(p4)-S2-S1-ND, (b) R5(p17)-
S2-S1-ND, and (c) R5a(p4)-S2-S1-ND. In all images the green spectra correspond to the
ND simulation, the magenta spectra correspond to nitroxide simulation, the red spectrum
is the addition of ND and nitroxide, and the blue spectra are the experimental results. 97
Figure 6.6. Motions of nitroxide in S2-S1-ND. i) Rotation tumbling of nitroxide dictated by
the rotational tumbling of ND. ii) DNA internal motion dictated by 10 torsional rotation of
the bonds attaching DNA to ND. ii) internal motion of nitroxide dictated by three torsional
rotation of the bonds attaching nitroxide to DNA. .......................................................... 98

XV

List of Schemes
Scheme 4.1. General scheme of functionalization of ND with azide. ............................ 45
Scheme 4.2. Hydroxylation of ND surface. ................................................................... 46
Scheme 4.3. Silanization. ............................................................................................. 48
Scheme 4.4. Azide substitution of Br in Br-ND ............................................................. 49
Scheme 4.5. General scheme for copper click reaction................................................ 49
Scheme 4.6 TEMPO-alkyne synthesis. (a) Reaction of 4-hydroxy-TEMPO with propagyl
bromide to form TEMPO-alkyne. (b) Mechanism of the reaction. ................................. 51
Scheme 4.7. The reaction mechanism of the copper click chemistry. .......................... 52
Scheme 5.1 Copper free click between DBCO and ND. ............................................... 67
Scheme 5.2. Synthesis of TEMPO-BDCO. ................................................................... 69
Scheme 6.1. Reusable sample stage. .......................................................................... 79
Scheme 6.2. Synthesis of S1-DBCO. ........................................................................... 81
Scheme 6.3. Copper free click chemistry between S1-DBCO and TEMPO-N3. ........... 81
Scheme 6.4 Copper free click between S1-DBCO and N3-ND. ................................... 86
Scheme 6.5. Nitroxide structures and DNA sequence. (a) R5 structure. (b) R5a structure.
(c) S2-S1 duplex tethered to ND. Arrows indicate the backbone phosphate sites at the S2
strand at which spin labels have been attached. ........................................................... 87

XVI

List of Abbreviations
CVD Chemical Vapor Deposition
CW Continuous Wave
DND Detonation Nanodiamond
EPR Electron Paramagnetic Resonance
FTIR Fourier Transform Infrared
HPHT High Pressure High Temperature
IR Infrared
ND Nanodiamond
NV Nitrogen-Vacancy
PEG Polyethylene Glycol
QD Quantum Dots
SLE Stochastic Liouville Equation
TEMPO 4-hydroxy- 2,2,6,6-Tetramethylpiperidine 1-oxyl
ZFS Zero Field Splitting

XVII

A
hyperfine tensor
1
B oscillating magnetic field produced by the microwave excitation
0
B externally applied magnetic field
D
traceless D-tensor characterized
g
electron g-tensor
g
electron g-value
2
() g τ
autocorrelation function
H
general spin Hamiltonian
EE
H electron-electron dipolar interaction in spin Hamiltonian
ZE
H electron Zeeman interaction term in spin Hamiltonian
HF
H hyperfine interaction term in spin Hamiltonian
NZ
Η nuclear Zeeman interaction term in spin Hamiltonian
NQ
Η nuclear quadrupole interaction term in spin Hamiltonian
ZF
H zero field splitting term in spin Hamiltonian
ˆ
() H Ω
orientation dependent total spin Hamiltonian
ο
H isotropic Hamiltonian
1
() H Ω the orientation dependent Hamiltonian
I
nuclear spin operator in vector form whose elements are , ,and
xy z
II I
I nuclear spin value ( / 2 n where n is a positive integer)
() L Ω Liouville operator associated with the total spin Hamiltonian
l
m nuclear spin quantum number
s
m electron spin quantum number
Q nuclear quadrupole tensor
S
electron spin operator in vector form whose elements are
, , and
xy z
SS S
S electron spin value ( / 2 n where n is a positive integer)
ν Microwave frequency
c
τ correlation time associated with the dynamics of nitroxides
Ω

complete set of random variables which represent Euler angles
( , ) t ρ Ω
density matrix associated with a particular value of Ω
XVIII

List of Symbols

() ΓΩ rotational diffusion operator and depend on the motion model
XIX

List of Physical Constant
h
34
6.62606957 10
−
⋅
2 -1
kg m s or J s ⋅⋅ ⋅
Plank constant
B
k
23
1.3806488 10
−
⋅
2 -2 -1 -1
kg m s K or J K ⋅ ⋅⋅ ⋅
Boltzmann constant
B
µ
-24 2 2 -1 1
9.27400968 10 kg m s T or J T
−−
× ⋅ ⋅⋅ ⋅
Bohr magneton
0
µ
-7 -2 -1
4 10 N A or T m A π × ⋅ ⋅⋅
Permeability of free space

1

Abstract
Nanodiamonds (NDs) are a new and attractive class of materials for sensing and
delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable
in these applications, yet reported approaches for covalent modification with biological
macromolecules are still limited, and characterizing behaviors of ND-tethered bio-
molecules is difficult. The approach we apply in our group employs click chemistry and
electron paramagnetic resonance (EPR) to graft and characterize different target
molecules on the ND surface.
This dissertation will focus on describing the techniques used to efficiently graft small
molecules and large biological molecules in particular DNA to the ND surface as well as
the study of the dynamics of ND-grafted molecules through EPR analysis. In Chapter 1,
an introduction to ND describing the methods of production, the properties, and the
different functionalization approaches will be given. In Chapter 2, an insight about the
EPR principles along with the different simulation approaches used throughout the
dissertation is presented in addition to EPR characterization of paramagnetic impurities
in NDs. Chapter 3 describes the Brownian motion simulation which will be used to
understand the motion of the tethered molecules. In Chapter 4, grafting of nitroxide
radicals on the surface of NDs using Cu(I)-catalyzed azide/alkyne. In Chapter 5, the
investigation of the dynamics of the nitroxide radicals grafted on ND surface using EPR
analysis is presented. Finally, in Chapter 6, the use of copper-free click chemistry to
covalently attach DNA strands at ND surfaces and the employment of site-direct spin
labeling and EPR spectroscopy to study the ability of the tethered DNA strands to undergo
repetitive hybridizations is showed.
2

Chapter 1
Introduction on Nanodiamond

Nanoparticles are emerging materials revolutionizing many fields including biology,
chemistry, materials science, medicine and physics. Nanodiamonds (NDs) are diamond
crystals with sizes ranging from hundreds to a few nanometers. In a majority of cases,
NDs contain nitrogen-vacancy (NV) centers, which are atomic-scale fluorescent (FL)
defects embedded within a diamond lattice. NDs are bio-compatible and possess
excellent chemical, mechanical, and photo stability, therefore are emerging as a
promising class of agents for applications such as biological imaging and drug delivery.
1-
8
In addition, single NV centers can be monitored individually using FL detection methods,
and are extremely sensitive to their surrounding environments, such as variations in
magnetic and electric fields as well as temperature.
9-16
As such, NV centers, either in ND
or in bulk diamond, are one of the leading candidates in nano-sensor development.
13-15,
17

A type of diamond is classified by impurity contents existing in the diamond lattice. For
example, type-I diamond (type-Ia and type-Ib diamond) contains a high concentration of
nitrogen impurities. In type-Ia, the concentration of nitrogen impurities is typically >3000
ppm and the nitrogen impurities exist in pairs or aggregates. On the other hand, in type-
Ib, the concentration of nitrogen impurities is commonly 10-300 ppm and a majority of
nitrogen impurities exist in the form of paramagnetic single substitutional defects (called
P1 centers).
18
Typically, natural diamond crystals often belong to type-Ia diamond while
3

synthetic diamond crystals belong to type Ib. Moreover, there is Type-II diamond (type-
IIa and type-IIb) containing much smaller amounts of nitrogen impurities. For example,
type-IIa diamond has the concentration of nitrogen impurities to be < 10 ppb. Type-IIb
diamond has a small amount of nitrogen impurities, but also contains boron impurities,
which makes it as a p-type semiconductor. type-I and type-IIa diamond are often used for
applications of biological imaging and nano-scale sensing.
1-8, 13-15, 17, 19
Type IIb diamond
crystals are used in electro-analysis, capacitors, and batteries.
20-21

1.1 Methods for ND synthesis
There are three main types of ND productions: 1) NDs obtained by detonation of
explosives that are rich in carbon, commonly called detonation nanodiamond (DND), 2)
NDs produced by the grinding of micron size diamond crystals produced by high-pressure
high-temperature technique (HPHT NDs), and 3) NDs produced by chemical-vapor
deposition (CVD). Two main NDs are widely used in research laboratories, DND and
HPHT NDs. While the size of DNDs can be a few nanometers (~2 nm), their FL is
unstable. In contrast, the HPHT NDs exhibit excellent photostability, but the size of HPHT
NDs tends to be much larger (≥30 nm). Therefore, applications such as targeted drug
delivery or delivery of biological molecules employ DND, and applications such as
biological imaging and quantum optics, which require photo-stability, often use HPHT
NDs. NDs used in this work are based on HPHT diamond. Therefore, for the rest of the
dissertation, we consider HPHT NDs.
Typically, HPHT diamond crystals are synthesized by mixing a source of carbon such
as graphite powder with a metal alloy catalyst and a source of nitrogen. The mixture is
then compressed under high pressure and high temperature resulting in the conversion
4

of sp
2
layers to sp
3
layers. The final step of fabrication is acid purification of the recovered
samples to remove any remaining graphite and metal catalyst. To obtain the nanometer
sized diamonds, HPHT diamond crystals are subjected to mechanical grinding using ball
milling techniques. The different particle sizes are sorted using different separation
techniques such as gradient centrifugation or simple grid separation. Furthermore, ND
samples are purified by treatment with harsh acid conditions.
1.2 Properties of ND
1.2.1 Nitrogen-vacancy centers
Within the lattice of diamond, there exist impurities called NV centers. NV centers are
atomic size defects where a single substitutional nitrogen is adjacent to a vacancy (Figure
1.1(a)). A NV center has gained much attention in recent years due to its unique optical
and magnetic properties. The NV center consists of 6 electrons forming the spin triplet
state ( 1 S = ). The energy levels of the NV centers are shown in Figure 1.1(b). It consists
of the ground and excited states and the metastable singlet states. Both the ground and
excited states are the 1 S = triplet states, thus there are three sublevels ( 0
s
m = and
1
s
m = ± ). At zero magnetic field (
0
0 B = ), 1
s
m = ± are degenerate, but the degeneracy
between the 1
s
m = ± and 0
s
m = states is lifted due to the magnetic dipole interaction
between the lone pair electrons. The energy separation between the 1
s
m = ± and 0
s
m =
sublevels (called zero-field splitting (ZFS)) are 2.87 GHz and 1.42 GHz for the ground
and excited states respectively. The transition between the ground and excited states (
E ∆ ~ 638 nm) can be driven through a laser excitation. This optical transition brings
ground states electrons to the excited states with conservation of their spin states.
5

Namely, the electron on the 0
s
m = ground state is excited to the 0
s
m = excited state
and the optical excitation pumps the 1
s
m = ± electron on the ground states to the 1
s
m = ±
excited states. On the other hand, the relaxation process depends on the spin states. The
electron on the 0
s
m = excited state decays to the 0
s
m = ground state with emission of
FL while the electron on the 1
s
m = ± states preferably decays to the metastable states,
then relaxes to the 0
s
m = ground state (non-radiative relaxation). As a result, the spin
population is transferred from the 1
s
m = ± state to the 0
s
m = state with the optical
excitation (initialized to the 0
s
m = state) and the FL measurement allows to readout the
spin state of a single NV center. Moreover, it has been shown that the FL signal emitted
by the NV centers is highly stable, showing no signs of photobleaching.
22
Furthermore,
when we apply resonant microwave field to electron paramagnetic resonance (EPR)
transitions of a NV center, EPR of the single NV can be observed with decrease of the
FL, namely optically detected magnetic resonance (ODMR) of the single NV center.

6

Figure 1.1. NV center structure and energy levels. (a) Structure of NV center in diamond.
The black circles represent the carbon atoms, the red circle represents the nitrogen atom,
and V represents the vacancy. (b) Electronic energy levels of NV centers. The green
arrow represents optical excitation, the red wiggly arrow represents FL decay with
transition energy of 638 nm, and the dashed arrows represent non-radiative decay to
metastable states.

7

1.2.2 Biocompatibility
As mentioned, ND is an excellent candidate for applications such as FL labeling for
biological imaging and targeted drug delivery. The suitability of biological applications
depends largely on the biocompatibility of the NDs. When compared to FL
semiconductors such as quantum dots (QDs), NDs show better nontoxicity mainly
because of the carbon composition. Due to their heavy metal composition, QDs
(especially uncoated) have a potential to react with biological systems, for example,
functional groups in biological tissues generating oxygen free radicals, thus damaging
DNA, RNA and proteins. Many studies have been performed to assess the safety of NDs.
In vitro studies on NDs such as, cell viability and genotoxicity conducted on different cell
lines, have demonstrated cellular uptake of ND particles with no effect on cell survival or
protein expression.
23-28
Furthermore, in vivo studies found that, whether administered
intratracheally, subcutaneously, orally, or intravenously, NDs neither induce inflammatory
responses, nor alter the growth or damage internal organs, and in few days it clears out
from the system.
29-33

1.3 Surface modification of NDs
In the ND applications, methods for surface modification are highly desirable, as they
allow tailoring of the ND surface for specific tasks, for example, carrying drug for targeted
delivery, FL tagging of biological molecules or magnetic sensing. The surface properties
of NDs depend on the fabrication process. Whether it is a DND or a HPHT ND, the
purification steps are generally executed in strong oxidizing media, resulting in the
formation of carbonyl such as ketones, lactones, anhydrides, esters and carboxyl groups
(Figure 1.2). In addition to the carbonyl, primary, secondary, and tertiary hydroxyl groups
8

are also observed on the ND surface which originate from the presence of water in the
production process. The presence of these functionalities makes it possible to modify the
surface of the ND with a variety of groups that can be used in subsequent reactions.

Figure 1.2. Surface functional groups of ND. The surface of ND can be terminated by a
variety of surface functional groups originating form the production process.
9

1.3.1 Surface homogenization of ND
To be able to control the amount of target molecules that are grafted on the ND
surface, it is desirable to homogenize the surface of ND with suitable functional groups
that are used in subsequent reactions. Many methods have been developed and are
illustrated in Figure 1.3.
One of the most frequently used groups, specifically for biological applications, is
COOH group. Carboxylation was developed in an attempt to increase the COOH surface
group on ND, and is achieved by treating the ND with strong oxidizing agents such as, a
mixture of H2SO4, HNO3, and HClO4 in equal amounts, a mixture of concentrated H2SO4
and HNO3 in 9:1 ratio, piranha water (which is a mixture of H2SO4 and hydrogen
peroxide), and supercritical water. The oxidation increases the number of COOH groups,
however it is not sufficient to render the surface of the diamond entirely homogeneous
because some groups, mainly ketones, are resistant to the harsh acid conditions. In
addition to increasing the number of COOH group, the acid treatment is often used to
remove residual impurities as well as to remove sp
2
layers on the surface.
Another group that is useful for subsequent reactions to tether target molecules is
hydroxyl group. Most of the oxygen containing groups can be reduced to hydroxyl in the
presence of a reducing agent. Krueger et al. successfully employed borane reduction.
34

This method is able to reduce carboxyl, ketone, and anhydride to hydroxyl groups, but
not ester and lactones, resulting in partial homogenization of the surface. By employing
a combination of carboxylation followed by hydroxylation, we can achieve optimal surface
homogenization as all the surface groups are converted to hydroxyl at the end of the
process. Other approaches have also been developed to achieve total homogenization
10

of the surface of ND with OH such as reduction using LiAlH4, Fenton reagent, or
mechanochemical treatment of the ND in water.
35-38

Furthermore, hydrogenation has been developed to homogenize the ND surface with
hydrogen groups. This method requires Harsh reducing condition to replace all oxygen
groups by hydrogen. Hydrogenation is achieved by placing the ND in a microwave plasma
reactor with a flow of hydrogen gas and elevated temperatures.
39-40

Yet another method is to remove all existing surface groups either by thermal
treatment or by irradiating the sample with high energy beam.
41-42
Both cases lead to the
formation of π -bonds between two adjacent carbons on ND surface increasing the
graphitic layer. This method renders the ND particles unsuitable for biological applications
because of the non-biocompatibility of graphite.

11

Figure 1.3. Homogenization of the ND surface. Different homogenization techniques
have been developed. Oxidation results in a surface rich with COOH groups.
Hydroxylation results in a surface rich with OH groups. Hydrogenation results in surface
homogeneous with hydrogen termination. Thermal treatment or irradiation of ND with high
energy beam results in the removal of all functional groups with the formation of thin
graphitic layer.

12

1.3.2 Surface coating of NDs
The homogenized diamond, whether it has COOH groups, OH groups, H termination,
or graphite, can be used for subsequent functionalization to attach the target molecule.
For example, COOH can be coupled to primary amine containing molecules in the
presence of a coupling agent such as carbidiimide, and DCC/NHS.
2, 43-44
OH groups can
also participate in a variety of reactions, such as, formation of esters when reacted with
carboxylic groups, formation of ethers when reacted with alkyl halides in the presence of
NaH, and opening of epoxide when mixed with glycidol.
38, 45-46
When one uses the
homogenized diamond described above, potential problems are low colloidal stability in
biological media and tendency to adsorb a wide range of molecules from small organic to
very large bio-macromolecules. To avoid those potential issues, coating techniques have
been employed successfully. Two types of coating are widely used with ND,
polyethylenglycol (PEG) and silica coating.
The coating with PEG or PEGylation has been investigated extensively and has been
applied successfully to many types of nanoparticles.
47-49
The PEGylation has many
advantages such as, increasing blood circulation time of nanoparticles and in the case of
ND improving colloidal stability under physiological conditions. The commercial
availability of a wide range of PEG having their two ends terminated with orthogonal
functional groups makes them great candidate not only for coating but also for introducing
different functional groups that can be used with subsequent modifications facilitating the
tailoring of target molecules. PEG can be covalently attached to the surface of NDs via
the formation of an amide or an ester linkage.
50-52
The PEGylation of ND is most suitable
for applications such as targeted drug delivery and cellular biomarkers. However, the
13

need for long PEG linkers for efficient coating hampers the employment of PEGylation in
our applications of magnetic sensing which require that the target molecule placed in
close proximity from the ND surface. Therefore, the coating with silica layer (or
silanization) is considered to be more advantageous for the magnetic sensing.
Silica is well-known to be non-toxic, biocompatible, and optically transparent. In
addition, the availability of functionalized silanes allows the introduction of a variety of
surface groups suited for coupling with different targets. The traditional silanization uses
the Stöber method, in which silica layers can be formed by the hydrolysis and
polymerization of silyl ether under basic conditions in ethanol/water solution.
53
One
potential problem in the reaction is that NDs may aggregate under the reaction conditions.
In addition, alkoxy-silane agents self-polymerize in aqueous media leading to the growth
of a thick silica layer surrounding ND. Bumb et al. were able to overcome these problems
by developing a new salinization technique in which the reaction is carried out inside
liposome-vesicles. By controlling the size of the vesicles they demonstrated the ability to
obtain monodispersed ND particles coated with single silica layer.
54
Another way to
prevent ND aggregation and achieve single layer coating is to perform the silanization
using a silane in which the alkoxy groups are substituted with chlorine. In this case,
instead of hydrolysis/condensation reactions occurring in the Stöber process, the reaction
proceeds following a nucleophilic substitution. This type of reaction is performed in
anhydrous organic media in which the ND particles are well dispersed and the silanes are
not prone to self-polymerization.
55

14

1.3.3 Functionalization of ND with target molecule
Any potential organochemistry can be used for surface modification of ND as long as
the latter is homogenized with a suitable functional group. The most common reaction
used in ND applications is the formation of amide linkage with the target molecule. For
this chemistry the surface of ND has to be homogenized either with amine (-NH2) or
COOH. For example, Teeling-Smith et al. used PEGylation to introduce amine groups to
the ND surface which was further reacted with amine reactive biotin conjugated to
streptavidin. The streptavidin was then used to attach a biotin modified DNA to the surface
of ND.
56
Bumb et al. also attached DNA to the ND surface using streptavidin, but instead
of PEGylation, they introduced amine groups to ND via silanization.
54
On the other hand,
Chang et al. introduced COOH groups on the ND surface via PEGylation, then they
reacted COOH groups with the primary amine of streptavidin forming an amide linkage.
The streptavidin then was used to attach a variety of biotinylated proteins to the ND
surface.
1

Another versatile chemistry used for grafting target molecules to the ND surface is
click chemistry. Click chemistry is a widely used technique in biological systems due to
its specifity, occurring only between an azide and an alkyne, tolerating a large number of
functional groups. The click chemistry concept currently is one of the most versatile
methods for grafting molecules on nanoparticle surfaces and has been successfully
applied to various nanoparticles, such as Au,
57
CdSe,
58
Fe2O3,
59
SiO2,
55
and diamond.
60-
61
The main advantage of the click chemistry and its application on surfaces is high
efficiency, the avoidance of high temperatures as well as tolerance towards many
functional groups. Furthermore, the progress of the reaction can be monitored by Fourier
15

transform infrared spectroscopy (FTIR) because the azide group has a characteristic
peak at ~2120-2160 cm
-1
.
Barras et al. introduced azide groups by reacting amine terminated NDs with azido-
benzoic acid, then performed copper click reaction with a variety of alkyne containing
compounds. Meinhardt et al. introduced azide or alkyne to the surface of ND through
arylation. They first homogenized the surface of ND with graphitic sp
2
layers and reacted
them with diazonium salt. Then, they demonstrated click chemistry with small molecules
as a proof of principle.
61
Recently, Rehor et al. double coated the ND using silca as a first
coat then azide terminated PEG as a second coat. Then, through copper click chemistry,
they were able to functionalize the ND surface with either peptides, coumarin or
fluorescein.
62

16

Chapter 2
Electron Paramagnetic Resonance Spectroscopy

EPR is a spectroscopic technique based on the absorption of microwave by
paramagnetic species either placed in a magnetic field or having non-degenerate energy
levels due to electronic properties. EPR is a powerful technique to probe spin structure
and dynamics of unpaired electrons. Giving the sensitivity of the EPR lineshape to the
changes in the molecular motion and the nature of the environment, EPR is widely used
to investigate dynamical behaviors and microenvironments of various molecules including
free radicals and biological molecules.
63-65

In this chapter, Hamiltonian for magnetic interactions used in EPR analysis will be
reviewed. Then, methods to simulate EPR spectra of powder samples and solution
samples will be discussed. Finally, EPR analysis of paramgnetic impurities in NDs will be
presented.
2.1 Magnetic interactions
The Hamiltonian of a spin system is given by
ZE
H describes the interaction between electron spin and the applied magnetic field and
is called Zeeman interaction.
ZF
H describes the interaction between electron spin in a
system possessing more than one unpaired electron ( 1/ 2 S = ) and is called zero-field-
splitting.
NZ
Η is the interaction between nuclear spins and applied magnetic field.
NQ
H is

ZE ZF NZ NQ HF EE
= ++ + + + H H H H H H H
(2.1)
17

the interaction between nuclear spins in a system with 1/ 2 I > .
HF
H is the hyperfine
coupling describing the interaction between electron spin and nuclear spins.
EE
H is the
dipolar interaction between two electron spins.
2.1.1 Zeeman interaction
Due to spin and angular momentum, electrons possess magnetic moments. The
interactions between the electron magnetic moments and the applied external magnetic
field (
0
B ) is called Zeeman interaction. The spin Hamiltonian of the Zeeman interaction
in the dimension of Hertz (Hz) is given by,

T
ZE 0
B
. . .
h
µ
= H B gS ,
(2.2)

where
B
µ is the Bohr magneton, h is the Plank constant,
0
T
B is the external applied
magnetic field with components
0
x
B ,
0
y
B , and
0
z
B . g is the g-tensor with components
ij
g
( , , , i j x yz = ). S is the electron spin operator with components
x
S ,
y
S , and
z
S . For
convenience, we choose the x, y, and z axes to coincide with the g-tensor principle axes.
Hence, the g-tensor is transformed into its diagonal form

diag
0 0
00
00
x
y
z
g
gg
g


=



.
(2.3)

Substituting the components of B , S and g in Eq. (2.2), the Hamiltonian of the Zeeman
interaction becomes

ZE 0 0 0
0 0
( )0 0
00
xx
x y z B
yy
zz
gS
BB B g S
h
gS
µ
  
  
= ⋅ ⋅⋅
  
  
  
H .
(2.4)

18

Considering a magnetic field (
0
B ) applied in the molecular axis (z-axis) and a g-tensor
that is isotropic, the Zeeman Hamiltonian becomes

ZE 0
B
z
H B gS
h
µ
= ⋅ ⋅⋅ .
(2.5)

The component of
z
S denoted by
s
m can take any value ranging from S − and S + with
integer steps. Therefore, the number of states resulting from the applied magnetic field
is 21 S + . The eigenvalues of the Zeeman Hamiltonian are

ZE 0
B
s
E B g m
h
µ
= ⋅ ⋅⋅ . (2.6)

For example, a system with one unpaired electron ( 1/ 2 S = ) can have only two values
of
z
m , namely 1
z
m = ± . If no magnetic field is applied, the two states are degenerate.
When the system is placed in a magnetic field, the energy difference between the two
levels is (Figure 2.1),

B
0
E gB
h
µ
∆= ⋅⋅ . (2.7)

Hence the separation between the energy levels depends on the strength of the magnetic
field.

Figure 2.1. Energy levels of 1/ 2 S = system.
19

2.1.2 Zero-field splitting
In the case where the system contains more than one unpaired electron, the spin-
interactions may be considered and denoted by ZFS. The Hamiltonian is given by

T
ZF
= ⋅⋅ H S DS ,
(2.8)

where D is the traceless and symmetric D-tensor characterized by two parameters, D
and E , and given by

1
00
3
1
00
3
2
00
3
DE
DE
D
 
−+
 
 
 
= −−
 
 
 
 
 
D
.
(2.9)

Therefore, the zero-field splitting Hamiltonian can be written as

2 22
ZF
1
( 1) ( )
32
z
E
H D S SS S S
+−
 
= ⋅ − + + +
 
 
. (2.10)

As shown in Figure 2.2(a) and (b), the spin-interaction causes the splitting of states
even when magnetic field is absent.

Figure 2.2. Energy levels of 1 S = system. (a) Energy level splitting of S=1 system caused
by magnetic field in the absence of ZFS. (b) Energy level splitting of S=1 system caused
by magnetic field in the presence of ZFS.
20

2.1.3 Nuclear Zeeman interaction
In addition to unpaired electrons, if an atom has a non-zero nuclear spin angular
moment (e.g.
14
N with
N
1 I = ), the interaction between the magnetic moment and applied
magnetic fields (called nuclear Zeeman interaction (NZ)) may be considered. The
Hamiltonian is given by

T n
NZ ο
n
..
g
h
µ ⋅
= H BI , (2.11)

where
n
µ is the nuclear Bohr magneton, and I is the nuclear spin angular momentum
with components
x
I ,
y
I , and
z
I .
n
g is the nuclear g-factor.
2.1.4 Hyperfine coupling
The interaction between the electron magnetic moment and the nuclear magnetic
moment is known as the hyperfine coupling (HF). The Hamiltonian is given by

T
HF
= ⋅⋅ H I AS , (2.12)

where, I is the nuclear spin angular momentum with components
x
I ,
y
I , and
z
I , and
A is the hyperfine tensor with components in its principle axis given by

00
00
00
x
y
z
A
A
A
 
 
=
 
 
 
A .
(2.13)

In the principle axis of the hyperfine coupling, the Hamiltonian takes the following form

00
( )0 0
00
xx
x y z y y
zz
AS
H II I A S
A S
  
  
= ⋅⋅
  
  
  
HF
. (2.14)
21

For a system with 0 I > , in addition to the splitting caused by the Zeeman interaction
(considering no ZFS), each state is further split into 21 I + states by HF. Therefore, a
system with 1/ 2 S = and 1 I = has a total of six states (Figure 2.3).

Figure 2.3. Energy levels of 1/ 2 S = and 1 I = system.

2.1.5 Nuclear quadrupole interaction
In a system with 1/ 2 I > , the interactions between different nuclear spins is called
nuclear quadrupole (NQ) interaction and is given by

T
NQ
= ⋅⋅ H I QI , (2.15)

where Q is a traceless and symmetric tensor. Note that similar to ZFS, the nuclear
quadrupole can lift the degeneracy of the energy levels in the absence of magnetic field.
2.1.6 Dipolar interaction
The dipolar interaction describes the interaction between two spin systems that are
separated by a distance ( R ) from each other. The Hamiltonian is given by

EE 1 2
D = ⋅⋅ H SS , (2.16)

22

where
1
S and
2
S are the spin operators of each of the systems. D describes the dipolar
coupling and depends on the distance vector ( R ) between the two spins and the angle θ
between R and the direction of the applied magnetic field,

22
2 0e B
3
(1 3cos )
4
g
D
R
µµ
θ
π
= −
.
(2.17)

2.2 Principles of continuous wave EPR spectroscopy
The CW EPR spectroscopy probes transitions between electronic spin states. In a
typical EPR instrument, the microwave is directed through a waveguide or quasioptics,
into a sample located at the center of magnetic fields. EPR signals are measured by
detecting microwave responses from the sample. Because most of EPR systems has
frequency-dependent components (e.g. waveguide, cavity etc.), CW EPR experiments
are usually carried out with a fixed microwave frequency and by sweeping the magnetic
field. To increase the signal-to-noise ratio, a magnetic field modulation technique
combined with phase detection is employed. As illustrated in Figure 2.4 as the magnetic
field oscillate from Bm1 to Bm2 with certain frequency, the detector will sense a current that
oscillates from i1 to i2 with the same frequency as the magnetic field and with certain
amplitude that is proportional to the slope of the absorption line between Bm1 and Bm2.
Because the phases of the rising part and the falling part of the absorption line are
inversely related, the obtained signal is the first derivative. This is only true when the
absorption line is linear within the amplitude of the oscillating magnetic field.
Consider a simple spin system with an isotropic g-value ( g) that is placed in a
magnetic field applied along the z-axis of the atom. Hamiltonian of the system consists of
the Zeeman term given by Eq. (2.4) and Eq. (2.5), respectively. The polarization of the
23

magnetic component of the microwave is perpendicular to the applied magnetic field.
Hence, we here consider the magnetic field associated with the microwave along the x-
axis with
11
cos( )
x
BB t ω = where 2 ω πν = is the angular velocity of the oscillating
microwave radiation with a frequency ν . The resulting Hamiltonian of the system is

ZE
' HH H = + , (2.18)
where
B
1
' cos( )
x
H gB S t
h
µ
ω = ⋅⋅ ⋅ ⋅ . (2.19)

Because
1
B is much smaller than
0
B , ' H can be treated as a time dependent
perturbation that induces the transition between states α and β which are created
by the spin Hamiltonian. The probability of the transition induced by ' H is given by

2
22 2
B1 3
1
()
2
x
P gB S E
αβ
µ α βδ ω
π
 
= ⋅⋅ ∆−
 
 
h
h
(2.20)

where EE E
αβ
∆= − , and δ is the Dirac delta function which take a non-zero value only
when E ω ∆= h . By substituting the delta function to a EPR lineshape function (L(∆E)), the
transition probability representing the EPR intensity is given by,
)) , ( (
0
2
B B E L S S P ∆ + ∝
− +
β α
αβ
. (2.21)

As a result, transition due to the application of the microwave radiation will occur when
the frequency of the microwave field matches the difference in energy between states
that satisfy the selection rule 1
s
m ∆= ± . For a system associated with hyperfine coupling,
the allowed EPR transition occurs between levels with 1
s
m ∆= ± and 0
I
m ∆= .
24

Figure 2.4. Modulation field technique.

25

2.3 Simulation of EPR spectra
All simulations presented throughout this document were performed using Easyspin
which is a Matlab based program available for free download.
66
Mainly two functions of
Easyspin were used. The solid state regime was used to simulate paramagnetic
impurities in diamond, for example, P1 center and surface impurity on diamond (Sect.
2.3.1). The slow motion regime was used to simulate the spectra of nitroxide radicals.
The slow motion regime simulates EPR spectrum by calculating the spin dynamics with
the Stochastic-Liouville equation formalism and it is used to simulate radical that are
placed in solutions (Sect. 2.3.2).
67-72
In the next two section, I will review the foundations
of the simulation for both regimes.
2.3.1 Solid state regime
CW EPR simulation in the solid state regime will be illustrated using P1 centers in ND.
P1 centers are paramagnetic defects inside the diamond lattice where a single carbon is
substituted with a nitrogen atom. The P1 center is a paramagnetic impurity with 1/ 2 S =
and 1 I = . Nanodiamonds are powder samples where each particle is randomly oriented,
resulting in a uniform orientational distribution of the P1 centers. There are three steps to
simulate the powder spectrum: 1) Specify the basis functions to build the operators that
are used to construct the spin Hamiltonian, which is then diagonalized to obtain the
different energies associated with different states. 2) Calculate the transitional probability
used to determine the intensity of the EPR spectrum. 3) Apply linewidth to account for
homogeneous and inhomogeneous broadening.
Considering only one orientation, the spin Hamiltonian of P1 center includes both
electron and nuclear Zeeman interactions, and the hyperfine coupling
26

ZE HF
= + H H H , (2.22)
where
( )
B
ZE 0 0 0
xy z
x x y y zZ
B g S B g S B g S
h
µ
= ⋅⋅+⋅⋅+⋅⋅ H , (2.23)
and

HF x xx y y y z z z
I A S I A S I A S = ⋅ ⋅ + ⋅ ⋅ +⋅ ⋅ H , (2.24)

where,
0
x
B ,
0
y
B , and
0
z
B are the components of magnetic field applied in the eigenframe
of the system, and they are computed by applying a rotation using the Euler’s angels. The
simulation starts with constructing the energy diagram resulting from the application of
the magnetic field. Therefore, one should first specify the basis functions. For
convenience, we will consider the following Zeeman-basis,

1 1/ 2, 1
2 1/ 2, 0
3 1/ 2, 1
4 1/ 2, 1
5 1/ 2, 0
6 1/ 2, 1
sI
sI
sI
sI
sI
sI
mm
mm
mm
mm
mm
mm
= = =
= = =
= = = −
= = −=
= = −=
= = −= −
. (2.25)

Then, the electron spin operators are 66 × matrices and given by,

000 1 00 0 1 0000
0000 1 0 1 0 1 000
00000 1 0 1 0000
11
, I
1 00000 0000 1 0 2 2
0 1 0000 000 1 0 1
00 1 000 0000 1 0
x x
S
   
   
   
   
= =
   
   
   
   
   
   
(2.26)

27

0 00 1 0 0 0 1 00 00
0 00 0 1 0 1 0 1 0 00
0 00 0 0 1 0 1 00 00
11
,I
1 0 0000 0 0 0 0 1 0 2 2
0 1 0000 0 0 0 1 0 1
0 0 1 000 0 0 0 0 1 0
y y
S
i i
  
  
−
  
  
−
= =
  
−
  
  
− −
  
  
−
  
(2.27)

1 0 00 0 0 1 000 00
0 1 00 0 0 0 000 00
00 1 0 0 0 00 1 00 0
1
,I
000 1 0 0 00 0 1 0 0 2
000 0 1 0 00 0 00 0
000 0 0 1 00 0 00 1
zz
S
  
   
   
   
−
= =
   
−
   
   
−
  
   
−−
   
. (2.28)

Therefore, the Hamiltonian operator of the system can be rewritten as

2
0 0 () 0
22 2 4
22
0 0 ( ) ()
2 4 24
2
0 0 0 ()
22 4 2
2
() 0 0 0
2 4 22
22
() ( ) 0 0
4 24 2
2
0 ()
4
xx y y
zz z
xy
xx y y
zz
x y xy
xx y y
zz z
x y
xx y y
zz z
x y
xx y y
zz
xy x y
xx y y
xy
B g B gi
B g A
A Ai
B g B gi
B g
A A i A Ai
B g B gi
B g A
A A i
H
B g B gi
B g A
A A i
B g B gi
B g
A Ai A A i
B g B gi
A Ai
−
+
−
−
−
=
+
−−
+
−
+
−
+−
+
+
−+
− 00
2 22
zz z
B g A
−+



















(2.29)
As shown in Eq. (2.29), the Hamiltonian includes the off-diagonal terms. In a low
magnetic field, because of those off-diagonal terms, the eigenstates are a mixture of the
bases. On the other hand, the off-diagonal terms become negligible in a high magnetic
field. For example, we show the calculation for P1 centers (most common impurities in
diamond) at the magnetic field of 8.206 Tesla (applied along z-direction) ( 1/ 2 S = , 1 I = ,
2.0024
x yz
gg g = = = , 82
xy
AA = = MHz, and 114
z
A = MHz). With those parameters, the
eigenvalues are given by,
.
28

115.057 0 0 0 0 0
0 -115.000 0 0 0 0
0 0 -114.943 0 0 0
0 0 0 114.9430 0 0
0 0 0 0 115.000 0
0 0 0 0 0 115.057
E
−
=
 
 
 
 
 
 
 
 
 
GHz, (2.30)

and the eigenfunctions are given by,
As shown in the calculation, at 230 GHz, the Zeeman interaction dominates over the
hyperfine coupling, hence the nontrivial off diagonal terms are negligible, and there is little
to no mixing of basis states. The energies of the six states as a function of the applied
magnetic field are shown in Figure 2.5(a). The position of resonance field can be
computed using the resonance condition ( hE ν = ∆ ).
The second step of the simulation is to apply the perturbation caused by the
microwave radiation which will determine the intensity of the EPR signal. This is done by
calculating the transition probability using Eq.(2.21).
The third step of the simulation is to consider the EPR lineshape. The EPR spectra is
governed by mainly two types of broadening: homogeneous and inhomogeneous
broadenings. In the homogeneous broadening (e.g. broadening due to T1 and T2 spin
relaxations), all spins experience the same fluctuations and have same spin parameters,
thus same lineshape. EPR lineshape with the homogeneous broadening is expected to
be the first derivative of the Lorentzian function,

1
2
3
4
5
6
115.057 4
115.000 5
114.943 6
114.943 3
115.000 2
115.057 1
E
E
E
E
E
E
= −=
= −=
= −=
= =
= =
= =
,
,
,
,
,
.

(2.31)
29

( ) ( )
1
2
2
0 0 0
2
) , (
−













 Γ
+ − − − ∝ B B B B B B L , (2.32)

where Γ is the distance between inflection point (or peak-to-peak distance). On the other
hand, the inhomogeneous broadening of EPR spectrum is due to a superposition of EPR
packets from individual spins, where those individual spins have slightly different Larmor
frequencies due to inhomogeneous local magnetic environments (electron and nuclear
spins).
73
EPR lineshape of the inhomogeneous broadening is often described by the
Gaussian distribution given by,

( )
( )






Γ
−
− − − ∝
2
2
0
0 0
2
exp ) , (
B B
B B B B L ,
(2.33)

where Γ is the peak-to-peak linewidth.
Many experimental EPR lineshape are well-described by either a Lorentzian function,
a Gaussian function, or a combination of the two. In Figure 2.5(a), the spectrum shows
a 230 GHz CW EPR result of P1 center at single orientation.
Furthermore, EPR spectrum of ND powder samples is a combination of EPR signals
from randomly oriented NDs, therefore, we need to take into account the effect of the
random orientation in the simulation. This is done by summing EPR spectra from all
possible orientations. Figure 2.5(b) shows the first derivative EPR simulation of P1
centers. The inset of Figure 2.5(b) shows the absorption spectra of the P1 centers along
with individual component each having specific orientation. The different orientations of
the P1 centers causes a shift in the side peaks resulting in the broadening of the overall
spectrum.

30

Figure 2.5. CW EPR simulation of P1 centers. (a) represent the energy levels of P1
centers as a function of magnetic field. The arrows represent the allowed transitions when
the frequency of the applied microwave is 230 GHz. The simulation spectrum shows one
possible orientation of P1. (b) the simulation of P1 center considering all possible
orientations. The inset represents the absorption spectra (blue) obtained by the addition
of all orientations (red).
31

2.3.2 Slow motion regime
Here we consider CW EPR of nitroxide radicals (the most common spin label used for
labeling biological molecules). Nitroxide radical containing a stable unpaired electron
consists of five or six membered rings containing a nitroxyl group surrounded by four
methyl groups that are attached to adjacent carbons. The unpaired electron in nitroxide
radical occupies a π -orbital that is delocalized over the nitrogen and the oxygen atoms,
thus making its EPR g-values and hyperfine-coupling constants highly anisotropic.
74
EPR
analysis of nitroxide radicals is a powerful tool to probe the dynamics of the molecules
and their local environments because the EPR spectrum representing their g-values and
hyperfine couplings drastically changes with the degree of the molecular dynamics,
characterized by the rotation correlation time (
c
τ ).
When the free radical is placed in a solution with low viscosity, the molecule will
undergo very fast rotations due to thermal motions which results in the averaging out of
the anisotropies of g - and A -tensors, a phenomena called motional narrowing.
Therefore, the EPR spectrum with strong motional narrowing effect can be understood
based on the Hamiltonian with the averaged g and A-values. However, if the nitroxide is
placed in a viscous solution or if it is tethered to a larger molecule such as
biomacromolecules and nanoparticles, there is irregularity in the Brownian motion and
the rotational motion of the molecule. As a results, the anisotropies are only partially
averaged and the spectrum is governed by both motions and spin Hamiltonian. Those
effects in CW EPR has been described by Freed et al.,
67-68, 71, 75
where spin dynamics in
the slow motion regime is expressed by,
32

( , )
ˆ
[ ( ), ( , )] ( ) ( , ) ( ) ( , ) ( ) ( , )
t
i H t t iL t t
t
ρ
ρ ρ ρρ
∂Ω
= − Ω Ω −Γ Ω Ω = − Ω ∂ Ω −Γ Ω Ω
∂
, (2.34)

where Ω is a complete set of random variables which represent Euler angles and
therefore describes the orientation of the molecule.
ˆ
() H Ω is the orientation dependent
total spin Hamiltonian. () L Ω is the Liouville operator associated with the total spin
Hamiltonian. ( , ) t ρ Ω is the density matrix associated with a particular value of Ω . () ΓΩ
is the rotational diffusion operator and depend on the motion model.
The total spin Hamiltonian has two components

ο1
() () H HH Ω= + Ω . (2.35)

o
H is independent of orientation and describes the zero-order energy levels and
transitions, and given by the isotropic Hamiltonian.

B
οο z k z kz
k
H gB S a S I
h
µ
= +
∑
. (2.36)

1
() H Ω is the orientation-dependent Hamiltonian expressed by the product of two
irreducible tensors of rank L

'( , ) ( , )
1 ,, ,
, ,,
() ( 1) ()
K L K L L M
i K M i
i L M K
H FD A
µµ
µ
−
Ω= − Ω
∑∑
,
(2.37)

where
( , )
,
LK
i
F
µ
−
is a spatial function in the molecular frame, and
( , )
,
LK
i
A
µ
are spin operators
quantized in the laboratory frame.
,
()
L
K M
D Ω in the Wigner rotation matrix that includes
transformation from the molecular frame to the laboratory frame.
EPR lineshape associated with the motion derived from Eq. (2.33) is given by,

1
1
( ) Re [( ) ] I v IL v ωω
π
−
∆= ∆−+Γ , (2.38)

33

where v is called the starting vector and includes both the transitions due to spin
operators and the probability distribution function for the orientation of the molecule at
equilibrium. By numerically solving Eq. (2.37), we can extract information about the
molecular motion of the system under study (in the form of
c
τ ) especially in the slow
motion regime in which the spectrum is sensitive to the diffusion operator.
Figure 2.6 shows the simulation of a nitroxide radical obtained with various
c
τ values
(0.03 ns – 100 ns). When nitroxide radicals are free in solution, a fast rotational tumbling
of the molecules (
c
0.03 τ = ns) averages out the g - and A -anisotropies, and the EPR
spectrum is dominated by isotropic hyperfine coupling. Consequently, the EPR spectrum
appears as three signals of comparable intensities as shown in Figure 2.6 (black). When
c
τ of nitroxide radicals increases due to slower rotational motions, it causes different
degrees of averaging which will translate into spectral broadening and uneven changes
of the EPR signal intensities (Figure 2.6).

34

Figure 2.6. Simulation of nitroxide radicals using slow motion regime with different
correlation times. We see that as the correlation time increases, uneven broadening of
the EPR peak occurs. For very slow correlation time, new features appear.

35

2.4 Characterization of paramagnetic impurities in ND using high-frequency CW
EPR
To study the paramagnetic impurities inside the ND, a home-built CW/pulsed 230/115
GHz EPR spectrometer was employed. The 230 GHz/115 GHz EPR system employs a
high-power solid-state source consisting of 8–10 GHz synthesizer, PIN switch, microwave
amplifiers, and frequency multipliers. The output power of the source system is 100 mW
at 230 GHz and 700 mW at 115 GHz. The 230 GHz/115 GHz excitation is propagated
using quasioptical bridge and a corrugated waveguide, and couples to a sample located
at the center of a 12.1 T cryogenic-free superconducting magnet. EPR signals are
isolated from the excitation using an induction mode operation. For EPR detection, the
system employs a superheterodyne detection system in which 230 GHz or 115 GHz is
down-converted into 3 GHz of intermediate frequency (IF), which is then down-converted
again to in-phase and quadrature components of DC signals. (see Ref. [76-77] for details
of the system). The microwave power and magnetic field modulation strength were
adjusted to maximize the intensity of EPR signals without distorting the lineshape
(typically, the microwave power is 2 mW and the modulation amplitude is 0.1 mT with the
modulation frequency of 20 kHz).
The obtained 230 GHz EPR spectrum shows two kinds of EPR signals which are well-
understood by considering P1 centers and the 1/ 2 S = surface impurity in diamond
(Figure 2.7).
78-79
The simulation of the EPR spectrum of P1 centers (magenta) was
performed using 1/ 2 S = ,
,
2.0024
xy
g = , 2.0024
z
g = , and hyperfine coupling to
14
N
nuclear spin ( 1 I = ) Ax = Ay = 82 MHz and 114
z
A = MHz.
80
By fitting the 230 GHz
36

spectrum, we extracted the
g
-factor of the surface impurities (
,
2.0029 0.0001 2.0027 0.0001
xy z
gg =±=± and )

Figure 2.7. Spectral analysis of 100 nm-ND EPR signals. 230 GHz EPR of the NDs is
shown by the solid blue line. The spectrum of the P1 centers was simulated using
, 1 I = , gx,y = 2.0024, gz = 2.0024, and the
14
N hyperfine coupling 82
x y
A A = = MHz,
114
z
A = (magenta). 1/2 S = spins were simulated with 1/2 S = ,
,
2.0029 0.0001
xy
g= ±
and 2.0027 0.0001
z
g= ± (violet). The green dotted line is the sum of the N and 1/ 2 S =
EPR spectra. The simulations were performed using Easyspin.
81

1/2 S =
37

Chapter 3
Dynamics of molecules undergoing Brownian motion

All matters in solution, whether it is a small molecule, a macromolecule or
nanoparticle, undergo random movement, so-called Brownian motion. There exist two
types of Brownian motions, translational and rotational. The Brownian motion is employed
in biological systems to describe many processes, such as diffusion-influenced
biochemical reactions and diffusion of macromolecules in vivo through a crowded
environment.
82-86
For ND applications, it is critical to understand motions of molecules
that are tethered to the surface of ND. EPR analysis allows us to probe the rotational
motion of molecules. In this chapter, the method to model Brownian dynamics will be
described. This allows us to understand the motion effects observed in EPR results.
3.1 Langevin equation
The Langevin dynamic model describes the influence of the fluid molecules on
particles undergoing Brownian motion. Langevin considered that there are two main
forces acting on a particle, namely, the drag viscous force and a fluctuating force due to
random collision of the fluid molecules with the particles. In the present case, the
fluctuating force is considered to be stochastic. Then, by simply taking the viscous force
as a frictional force, the Langevin equation where the inertial force of a particle is
represented by the sum of the frictional force and the stochastic force ( ) (t L )) is given by,
38

2
d d
()
dd
rr
m f Lt
tt
× = −× + , (3.1)

Where m , and r are the mass, and the position vector of the particle respectively, and
f is the friction coefficient. In the case of high friction (
2
dd
dd
rr
fm
tt
− × >> × ), the inertial term
can be ignored and Eq. (3.1) is simplified by

d
()
d
r
f Lt
t
× = . (3.2)
3.2 Free Brownian motion
In this section, we consider a free rotational Brownian motion of a spherical object.
Starting from the Eq. (3.2), because the distribution of () Lt is assumed to be Gaussian-
like having a zero mean and a variance ( ) ( ') 2 ( ') Lt Lt D t t δ = − , therefore () 2 L t DW = .
W represents a white noise process because the spectral density, which is the Fourier
transform, of ( ) ( ') Lt Lt is a constant. D is the diffusion coefficient. f is related to D by

8
B B
r
kT kT
D
fR πη
= =
, (3.3)

where η the viscosity coefficient of the solvent, and R is the radius of the sphere.
If the particle was at position ' r at time 0, the position at time t is given by
The probability distribution of r can be written as
Thus, the analytical solution of Eq. (3.5) can be written as

0
1
' ( ') '
t
r
r r L t dt
f
= +
∫
. (3.4)

2
1/2
()
( , ) (2 ) exp
2
rA
rt B
B
ψπ
−
  −
= −
 
 
,
(3.5)
where
() A rt = and
2
() B rt A = − .

39

3.3 Rotational Correlation function and correlation time
Rotational motion leads to changes in molecular orientation which can be probed
using magnetic resonance (MR). In addition to the NMR and EPR linewidths, the
rotational correlation is considered in the study of the dielectric relaxation and FL
depolarization. This section will present the derivation of the correlation time as was
described by Debye. The object of physical interest in such experiments is the rotational
correlation functions of functions of orientation. These functions are generally the
spherical harmonics (
l
Y ). Hence the correlation function can be written as follow

rot
4
() (cos( ) ( ') ()
5
l ll
t
C t P Yt Yt
π
γ = = ,
(3.7)

where (cos( ))
l
P γ is the Legendre polynomial of order l of the cosine of the angle between
the two orientations. For an isotropic motion, the diffusion process for a distribution of
spins can be described by the Smoluchowski equation given by

where
r
D is the rotational diffusion constant and u represents different orientations. The
solution of such equation is

2
1/2
( ')
( , ) (2 ) exp
4
rr
r t Dt
Dt
ψπ
−
 −
= −


.
(3.6)

2
( , )
( , )
ru
ut
D ut
t
δψ
ψ
δ
= −∇ , (3.8)

2
( , ) exp( ) ( ,0)
ru
u t tD u ψψ = − ∇ , (3.9)
40

At equilibrium,
0
() u ψ should not evolve with time which is true for the condition
0
() 0
r
Du ψ = . Using the conditional probability, which is the probability of being at
orientation u at time t after being at orientation ' u at time 0 ( ( , | ',0) W ut u ), we can define
the correlation function by

*
0
,'
( ) ( ') ( ) ( , | ',0) ( ') '
rot l l
uu
C t u Y u W u t u Y u dudu ψ =
∫∫
.
(3.10)
where
2
( , | ',0) exp( )
u
W u t u tDr = −∇ .

The
0
( ') u ψ is included in Eq. (3.10) to ensure that the initial orientations are weighed
by their equilibrium value. By integrating over ' u , we get
which upon integration becomes
where l is the order of the spherical harmonics.
In slow motion EPR, the g - and A - tensors contributing to the spin Hamiltonian are
by definition the sum of a zero rank tensor and the components of a second rank tensor.
Therefore, l is considered to be 2 . Eq. (3.12) becomes
Furthermore, the rotational correlation time can be extracted from the ()
rot
Ct , as
follows
If ()
rot
Ct is considered to be exponential, then

*2
0
( ) ( ') ( )exp( ) ( ')
rot l u l
u
C t u Y u tDr Y u du ψ= −∇
∫
,
(3.11)

( ) exp( ( 1))
rot r
C t tD l l = − + , (3.12)

( ) exp( 6 )
rot r
C t Dt = − . (3.13)

c
0
()
rot
Ct τ
∞
=
∫
. (3.14)

41

In this case, the characteristic decay τ represents
c
τ . By comparing Eq. (3.13) and (3.15),
we get
c
1/ 6
r
D τ = .
3.4 Simulation procedure
For computational simulation, starting with the set of Langevin equations for three-
dimensional rotational motion,

dx(t)
2
d
x
DW
t
= ,
(3.16)

dy(y)
2
d
y
DW
t
= ,

dz(y)
2
d
DWz
t
= .
To solve Eq. (3.16), we consider discrete time steps ( t ∆ ) by substituting
dx( )
d
t
t
with
1 ii
xx
t
−
−
∆
,
dy( )
d
t
t
with
1 ii
y y
t
−
−
∆
,
d ()
d
zt
t
with
1 ii
zz
t
−
−
∆
. W
θ
and W
ϕ
are
,i
w
t
θ
∆
and
,i
w
t
ϕ
∆
,
respectively.
87
w
θ
and w
ϕ
represent Gaussian distributions with the mean of 0 and the
standard deviation of 1. Therefore, Eq.(2.16) can be rewritten as follow:

1,
2
i i x t
x x D tw
−
= +∆ ,
(3.17)
1 ,
2
i i yt
y y D tw
−
= +∆ ,

1,
2
i i zt
z z D tw
−
= +∆ .

The simulation of such equations is done by repetitive application of Eq. (3.17), taking the
final position of the last step as the initial position of the new one. To calculate the
correlation function from the simulated data we use

rot 2
( ) (cos( )
t
C t P γ = ,
(3.18)
where
2
2
( ) 3 / 2 1/ 2 Px x = − ,
( ) exp( / )
rot
Ct t τ = − , (3.15)

42

1 11
cos ( ) ( ') cos( )cos( ) sin( )sin( )cos( )
i i i i ii
t tt γ θ θ θ θ ϕϕ
− −−
= += + − uu g ,

and θ and ϕ are the polar angle obtained from the x , y , and z coordinates.
Figure 3.1 shows the results of the simulation. The Cartesian coordinates were
simulated using Eq. (3.17) for spherical particle with radius of 25 nm and a radius of 50
nm shown in Figure 3.1 (a) and (b) respectively. The time interval used was 0.01 µs for
a total of 10,000 points. As expected the 25 nm particle diffuses faster than the 5 nm
particle. The trajectory was used to calculate the polar angles which were used to obtain
the correlation functions as described by Eq. (3.8) (Figure 3.1 (c), circles for 25 nm and
dots for 50 nm). The results of the correlation function were then fitted with an exponential
decay to extract the correlation time. As expected the 50 nm particle showed a rotational
correlation time of 22 µs while the 25 nm showed a correlation time of 10 µs. This theory
was used to simulate the Brwonian motion of tethered nitroxide on ND surface (Sec. 5.3
).

43

Figure 3.1. Correlation function simulation of free rotational Brownian motion. (a) and (b)
are the trajectories of particles of radius 25 and 50 nm respectively. (b) shows the results
of the correlation function as a function of time along with the exponential fit.

44

Chapter 4
Surface Chemistry of Diamond to Introduce Functional
Groups

Materials presented in this chapter can also be found in the article titled “Grafting
nitroxide radicals on the surface of nanodiamonds using click chemistry” by Ekaterina
Romanova, Rana Akiel, Franklin H. Cho, and Susumu Takahashi in Journal of
physical Chemistry A 117, 11933-11939 (2013) (Reprinted with permission from Ref.
[88]. Copyright [2013], ACS LLC).

In this chapter, we describe a method to covalently graft nitroxide radicals to the
surface of NDs using copper click chemistry. First, the ND was homogenized with azide
groups using silanization technique. Then, the azide functional NDs were reacted with
alkyne containing nitroxide through copper click chemistry.
4.1 Silanization technique to introduce azide groups to ND surface
The silanization is a reaction between a silane and a hydroxyl group. The
functionalization follows Scheme 4.1. First, all oxygen surface groups of ND where
reduced to hydroxyl groups resulting in hydroxylated NDs (OH-ND). Then, the hydroxyl
groups are reacted with silanes containing a bromide terminated alkyl to give Br-ND.
Finally, the bromide was substituted with azide to give azide functionalized ND (N3-ND).
45

Scheme 4.1. General scheme of functionalization of ND with azide.

4.1.1 Preparation of ND surface for silanization ̶ acid cleaning and borane
reduction
To obtain the starting OH-ND, ND surface was hydroxylated by performing a
combination of a strong acid treatment borane reduction following common procedures.
4,
34, 89-90
Two different sizes of HPHT ND were used: 100 nm (Engis Illinois, USA) and 25
nm ND (Van Moppes, Geneva, Switzerland). The acid cleaning consisted of refluxing
NDs in a 9:1 mixture of concentrated H2SO4 and HNO3 at 75 °C for 72 hours followed by
0.1 M NaOH solution at 90 °C for 2 hours, then by 0.1 M HCl solution at 90 °C for 2 hours.
After the acid treatment, the obtained NDs are repeatedly rinsed with de-ionized (DI)
water, separated by centrifugation (10,000-15,000 rpm for 15 min using Sorvall RC-5C
Plus) and dried under vacuum.
Subsequently, large excess of 1.0 M borane tetrahydrofuran complex solution (Sigma-
Aldrich, Milwaukee, WI, #176192) was added dropwise to a stirring suspension of acid
treated NDs in dry tetrahydrofuran. Then, the mixture was refluxed at 60 °C for 24 hours
and hydrolyzed with 2 M HCl after cooling down to room temperature. Resulting NDs were
washed with DI water (four times) and acetone (once) in consecutive
46

washing/centrifugation cycles to remove the boronic acid produced during the reaction
and dried in vacuum to yield OH-ND.
Scheme 4.2 shows the functionalization of ND with hydroxyl groups. The acid
cleaning is performed to remove any residual metals remaining from the fabrication
process. In addition, a mixture of strong H2SO4 and HNO3 yield the formation of NO2
+

which is a strong oxidizing agent that converts the hydroxyl groups on ND surface to
carboxyl groups. Furthermore, the presence of strong acids hydrolyzes the esters and
anhydrides to carboxylic acids, and the lactone to corresponding carboxylic acid and
alcohol. At the end of this process, the only remaining groups on the ND surface are
carboxyl, hydroxyl and ketones.
The borane reduction, widely used on NDs, is a well-known reaction that is able to
reduce all carbonyl groups to primary alcohols. Therefore, at the completion of the
reaction the ketone and carboxylic groups present at the surface of NDs after acid
cleaning are reduced to hydroxyl groups. This step results in ND surface homogenized
with hydroxyl groups.

Scheme 4.2. Hydroxylation of ND surface.
47

4.1.2 Functionalization with azide through silanization
Functionalization of ND with azide group was done through silanization following a
procedure employed by Ranjan et al.
55

4.1.2.1 Silanization with 3-bromopropyltrichlorosilane
Reaction
Scheme 4.3(a) shows the silanization reaction. In a typical reaction, OH-ND was
suspended in anhydrous toluene under nitrogen atmosphere (10 mg/mL typical
concentration). Because the silane is moisture-sensitive, where it readily polymerizes in
aqueous media, anhydrous toluene is used as solvent to prevent to contamination of the
ND sample with silica particle and to ensure the formation of monolayer around the ND
particles. The flask was sonicated for 30 minutes to disperse the ND, then heated to 80
°C. An excess of 3-bromopropyltrichlorosilane (Sigma-Aldrich, Milwaukee, WI, #437808)
was added dropwise and the resulting solution was heated at 80 °C for 24 hours. To
separate the product of bromide-functional NDs (Br-ND) from unreacted 3-
bromopropyltrichlorosilane, the reaction mixture was re-dispersed in toluene and
centrifuged. This cycle was repeated four times to obtain pure Br-ND.
Mechanism
As shown in Scheme 4.3(b), the silanization reaction follows nucleophilic substitution
mechanism where the hydroxyl groups on the surface of ND act as nucleophiles and
compete with the chloride group attached to the silicone atom in the silane. First, the
oxygen from the hydroxyl group on ND surface attacks the silicon atom and displaces the
chloride, then, deprotonation of the intermediate results in the Br-ND.

48

Scheme 4.3. Silanization.

4.1.2.2 Substitution of bromide with azide
Reaction
Scheme 4.4(a) shows the details of the reaction. To obtain N3-ND, a saturated
solution of sodium azide (NaN3, Sigma-Aldrich, Milwaukee, WI, #438456) in anhydrous
dimethylformamide (DMF) was freshly prepared and added to Br-ND under nitrogen
atmosphere. The mixture was sonicated for 30 minutes, then stirred at 80 °C for 24 hours.
The reaction mixture was re-dispersed in DI water and centrifuged to remove excess of
NaN3. This cycle was repeated four times to yield pure N3-ND.
Mechanism
As depicted in Scheme 4.4(b), this is a substitution reaction were the azide group
from NaN3 attacks the carbon adjacent to the bromide group and forces the bromide
group to leave.

49

Scheme 4.4. Azide substitution of Br in Br-ND

4.2 Copper click reaction applied to NDs
The general scheme of the reaction is shown in Scheme 4.5. In parallel to azide
functionalization of ND, nitroxide radicals were functionalized with an alkyne group. Then
N3-ND was clicked to alkyne-nitroxide in the presence of Cu(I).

Scheme 4.5. General scheme for copper click reaction

50

4.2.1 Synthesis of alkyne functional nitroxide
Reaction
Since we have functionalized the NDs with an azide group as described above, the
copper click chemistry requires an alkyne-containing nitroxide radicals. In this reaction
2,2,6,6-tetramethylpiperidine 1-oxyl radicals (TEMPO) was used. To obtain TEMPO-
alkyne, the precursor 4-hydroxy-TEMPO (TEMPO-OH) (Sigma-Aldrich, Milwaukee, WI;
#176141) was reacted with propagyl bromide (Sigma-Aldrich, Milwaukee, WI; #P51001)
adopting previously established procedure (Scheme 4.6(a)).
91
Typically, a sodium
hydride (NaH, 1.1 eq.) was dissolved in dry DMF and stirred at room temperature for 30
min. Then, TEMPO-OH (1.0 eq in DMF) was added to the stirring suspension of NaH at
0 °C. The solution was stirred at room temperature for 30 minutes, then propagyl bromide
(1.3 eq. in DMF) was added dropwise at 0 °C followed by stirring for 3 hours at room
temperature. The reaction mixture was purified by column chromatography using silica
gel column, and 10 % ethyl acetate in hexane as eluting solvent. The obtained product
was dried under vacuum.
Mechanism
This reaction proceeds in two steps (Scheme 4.6(b)). In the first step, the strong base
NaH causes the deprotonation of the hydroxyl group of the TEMPO-OH. Then, the
resulting alkoxide ion acts as a good nucleophile which attacks the alpha carbon of the
propagyl bromide and displace Br to form the TEMPO-alkyne.

51

Scheme 4.6 TEMPO-alkyne synthesis. (a) Reaction of 4-hydroxy-TEMPO with propagyl
bromide to form TEMPO-alkyne. (b) Mechanism of the reaction.

4.2.2 Copper click reaction of N3-ND with TEMPO-alkyne
Reaction
In this reaction the azide functional ND was reacted with alkyne bearing TEMPO to
give ND grafted TEMPO (TEMPO-ND) following previously reported procedure.
60
First
the N3-ND (~6 mg/mL) were dispersed in anhydrous acetonitrile and sonicated for 30
minutes. The click-reaction was performed by adding an excess of TEMPO-alkyne (1 eq.),
Cu(I) (1 eq.) and TEA (20 eq.) to the suspended N3-ND. The reaction was stirred for 48
hours at room temperature followed by centrifugation and the pellet was re-suspended in
acetonitrile. This procedure was repeated 10–15 times to remove non-reacted nitroxide
52

radicals or until no TEMPO signal was observed in the supernatant. The resulting solid
was dried in vacuum to yield TEMPO-ND.
Mechanism
The widely accepted reaction mechanism is shown in Scheme 4.7.
92
First the
deprotonated alkyne group forms a σ-bond with a copper atom to form copper acetylide
(1). Second, 1 recruits a second copper atom to form the catalytically reactive
intermediate σ-bound acetylide bearing a π-bound enriched copper (2). Third,
nucleophilic β-carbon of the acetilyde attacks the N of the azide to form the first C-N bond
(3). Fourth, the second C-N bond forms displacing one of the coppers and results in a
triazolyl-copper derivative (4). Finally, protonolysis delivers the end product which is the
1,4-disustituted [1,2,3]-triazole ring (5). R1 and R2 represents TEMPO-alkyne and N3-ND
respectively.

Scheme 4.7. The reaction mechanism of the copper click chemistry.

53

4.3 FTIR characterization
Infrared (IR) spectra of functionalized NDs were measured using Bruker Vertex 80
FTIR spectrometer. In the IR measurement, dried ND powder (0.5 - 1 mg) was mixed with
KBr powder (100 mg) in an agate mortar. The mixture was pressed into a pellet using 10
tons load. In our FTIR measurement, FTIR signals from a pure KBr pellet was subtracted
as background signals. All FTIR samples in subsequent chapters are prepared following
the same procedure.
4.3.1 Characterization of the azide functional NDs
During the process of introducing azide groups to the ND surface, the FTIR spectrum
was measured after each step of the reaction. Figure 4.1 shows the FTIR spectra of 100
nm NDs after the acid treatment and after obtaining OH–ND, Br–ND, and N3–ND. The
FTIR spectra of the acid-treated NDs shows characteristic carbonyl (C=O) stretching
vibration modes at ~1100 cm
-1
and ~1760 cm
-1
(the latter corresponding to carboxyl
group) and peaks of vibrational modes of hydroxyl groups (O–H) at ~1630 cm
-1
and in
3000 – 3600 cm
-1
(bend and stretch respectively). The reaction with tetrahydrofuran
complex reduces carbonyl groups to hydroxyl groups typically leading to the decrease in
the C=O IR signals and increase in the O–H and C–H IR signals. As shown in OH–ND in
Figure 4.1, an increase of the C–H stretching vibrational signal (near 2992 cm
-1
) was
visible indicating the presence of hydroxymethyl groups on ND surface. Changes of the
O–H signals were not well pronounced due to the adsorption of water molecules on ND
surface. In addition, the remaining C=O signal in OH–ND suggested that the reduction
step resulted in ND surface partially covered with hydroxyl groups. This is due to the fact
that acid treated NDs form strongly bounded agglomerates which are hard to separate by
54

ultrasonication because of interparticle covalent bonding.
5
Therefore, the reducing agent
will have limited access to ND surface resulting in an inhomogeneous surface
functionalization.
To obtain Product Br–ND, the hydroxyl groups of OH–ND were used to covalently
attach 3-bromopropyltrichlorosilane to ND surface. After the reaction, the binding of silane
was confirmed by the characteristic FTIR signal of the C–Si–O bond at ~1119 cm
-1
shown
in Figure 4.1. Then, Br–ND were reacted with sodium azide to obtain azide-functional
NDs. The success of the reaction was confirmed by the appearance of azide signal at
2100 cm
-1
in the FTIR spectra of N3-ND.
In addition to the reactions characterization, FTIR was used to study the stability of
the silane linkage between the ND and the azide group (C-O-Si bonds) in aqueous
solution specifically in neutral and slightly acidic media. In this investigation, N3-ND was
suspended either in water or in PBS with pH 6.8 and 6.5. The suspension was ultra-
sonicated overnight. Then, the solution was removed and the sample dried and prepared
for FTIR measurements. Figure 4.2 shows the FTIR results of the stability studies
corresponding to OH, C=O, and N3 peaks. The ratio of N3 to C=O peaks was quantified
following the procedure in Sec. A.1. It was determined to be ~3 in all samples namely dry,
water, and PBS pH 6.8 and 6.5. These results indicate that no cleavage of the C-O-Si
bond was observed.
55

Figure 4.1. FTIR spectra of NDs. FTIR signals of 100 nm NDs after acid treated, borane
reduction, silanization with bromide, and azide functionalized ND.

56

Figure 4.2. Results of the stability studies. blue represents the experimental data. The
red represents the multi-peak Lorentzian fits. The green traces represent the Lorenztian
fit of each peak separately which addition results in the red trace. The ratio between N3
and C=O was determined using the green traces corresponding to each group.

57

4.3.2 Characterization of copper click reaction
Using the click-reaction, alkyne-contained nitroxide radicals were covalently attached
to N3-ND through the azide-functional groups to give TEMPO-ND. Figure 4.3(a) shows
the FTIR spectra of N3-ND and TEMPO-ND. Both spectra were normalized by ND mass
considering the intensity of the carboxyl C=O peaks which is not expected to change upon
reaction (see Sect. A.1 for details of normalization). The reaction was confirmed by the
significant reduction of the azide peak at 2100 cm
-1
in the FTIR spectra of TEMPO-ND.
Figure 4.3(b) shows the FTIR peak of the azide vibrational mode at 2100 cm
-1
of N3-ND
and TEMPO-ND. Comparing the areas under the peaks, the reaction efficiency was
estimated to be 98%.

Figure 4.3. FTIR results of copper click chemistry between N3-ND and TEMPO-alkyne.
(a) FTIR spectra of N3-ND and TMEPO-ND. (b) FTIR spectra of azide moieties at 2100
cm
-1
.

58

4.4 Characterization using EPR
For HF EPR measurement of solution samples, we designed a sample holder suitable
for the application. While HF EPR is advantageous for high spectral resolution, a large
amount of water absorption of HF microwave often challenges the implementation of HF
EPR experiments at physiological conditions, i.e. room temperature aqueous samples. In
particular, the absorption is significant at 0.1 – 1 THz.
93
For example, 1 mm thickness of
water absorbs 10 % of microwave power at 10 GHz while at 115 GHz it absorbs 99.98
%. As demonstrated in Ref. [94], one way to overcome the water absorption of HF
microwave is to employ a thin layer of the aqueous sample where the thickness of the
layer (h) is much smaller than the microwave wavelength ( λ ). As shown in Figure 4.4(a),
the magnetic component of the microwave (
1
B ) is maximum at a conducting surface (i.e.
aluminum tape) while the electric component of the microwave (
1
E ) is minimum. This
allows to “mask” thin aqueous samples from the electric field, resulting in reduction of the
microwave absorption.
To implement this idea for our HF EPR system, we designed a sample holder as
shown in Figure 4.4(b). The sample holder made of Teflon is designed to hold an
aqueous sample in a cylindrical well. In the present design, the sample holder is used
with no microwave cavity and aqueous sample solution is placed on a conducting surface
which is the node of the microwave electric component as well as the antinode of the
magnetic component (Figure 4.4(a)). To minimize the microwave absorptions and to
maximize the sample volume, the well height (h) was chosen to be ~100 𝜇𝜇 m and the well
diameter (D) is ~5 mm where D is similar to the aperture of the corrugated waveguide.
The sample well is sealed by an aluminum tape. Moreover, the sample well is located at
59

a distance (d) from the corrugated waveguide that satisfies the condition λ 2
W T
= + hn dn
where
T
n – refraction index of Teflon ( 1.44
T
n = ),
W
n – refraction index of water ( 1.33
T
n = )
and λ – microwave wavelength at 115 GHz.
X-band CW EPR spectroscopy was performed using the Bruker EMX system (Bruker
Biospin) equipped with a high-sensitivity cavity (ER 4119HS, Bruker Biospin). For each
measurement, samples were placed in a quartz capillary (inner diameter: 0.86 mm), with
the typical sample volume being 1–5 µL. CW EPR spectra were obtained by optimizing
the microwave power and magnetic field modulation strength to maximize the amplitude
of EPR signals without distorting the lineshape, with a typical parameter set being:
microwave power of 2 mW; modulation amplitude of 0.03 mT; and modulation frequency
of 100 kHz.

Figure 4.4. Aqueous sample holder. (a) Electric and magnetic field components of
microwave in the sample holder. The sample is positioned on the aluminum tape. (b)
Schematics for aqueous sample design. The top and bottom caps are made of Teflon.
Doted lines indicate threads and volume for screws that are used to tighten top and
bottom caps.
60

4.4.1 Characterization of N3-ND
Here we discuss EPR analysis of azide functionalized ND surface. First, Figure 4.5(a)
shows 115 GHz EPR spectrum of the 100 nm N3-ND sample. The spectrum shows a
signal from 4.098 Tesla to 4.108 Tesla. To confirm that the observed signal corresponds
to the paramagnetic impurities in the diamond, we simulated EPR spectrum using the
same parameters described in Sect. 2.4. As shown in Figure 4.5(a), the simulated result
of ND EPR spectrum agreed reasonably well with the experimental data (Figure 4.5(a),
red), which confirms that the azide functionalization does not change the EPR of NDs, as
we expect. Moreover, the N3-ND sample was measured by X-band EPR spectrometer.
As shown in Figure 4.5(b), the two EPR signals largely overlapped because of the small
difference in their g-values. The obtained EPR data showed a prominent signal at ~0.3328
Tesla and two other signals at ~0.3300 Tesla and ~0.3357 Tesla (Figure 4.5(b), blue).
Using the same parameters and following the same procedure as the HF EPR analysis,
we demonstrated that the simulated X-band spectrum of N3-ND (Figure 4.5(b), green)
agreed with the experimental results too.
61

Figure 4.5. EPR analyses of the N3-ND sample. (a) 115 GHz EPR of N3-ND and (b) X-
band EPR of N3-ND are shown by the solid blue lines. (a) and (b) also show the
contributions of P1 centers (magenta) and the 1/ 2 S = spins (purple) obtained by
simulating the experimental EPR spectra. The dotted green lines overlaid with the
experimental data shows the sum of the N and 1/ 2 S = contributions. The simulations
were performed using Easyspin.
81

62

4.4.2 Characterization of TEMPO-ND
After functionalizing the ND with nitroxide radicals, HF EPR and X-band EPR were
measured for the product (TEMPO-ND). As shown in Figure 4.6(a), HF EPR spectrum of
the TEMPO-ND sample in solution was different from ND EPR spectrum and showed a
distinct signal at ~4.095 Tesla in addition to the ND signals. As a control, HF EPR
spectrum of free TEMPO (mixture of TEMPO and ND (ND/TEMPO) without covalent
binding) was recorded. The EPR spectra of ND/TEMPO showed, in addition to the ND
characteristic, features that are typical of free nitroxide radicals. As seen in Figure 4.6(a)
(bottom trace) the position of the free TEMPO coincides with the position of the extra
feature observed in the case of TEMPO-ND, which proves that this latter is due to
nitroxide radicals. It is clear that the spectrum of tethered TEMPO is different than the
free TEMPO. This is due to slower motion of the tethered TEMPO (see Sec.5.3 for
detailed analysis). Furthermore, we performed X-band EPR measurement of the TEMPO-
ND sample. As shown in Figure 4.6(b), the TEMPO-ND spectrum is represented by a
broad peak at ~0.3325 Tesla resulting from the overlap of nitroxide and surface impurities
on ND.

63

Figure 4.6. CW EPR results on TEMPO-ND. (a) 115 GHZ EPR results of TEMPO-ND.
(b) X-band EPR of TEMPO-ND.
64

Chapter 5
Nitroxide-Functionalization of ND Surface Using
Copper-Free Click Chemistry

Materials presented in this chapter can also be found in an article titled “Investigating
functional DNA grafted on nanodiamond surface using site-directed spin labeling
and electron paramagnetic resonance spectroscopy” by Rana D. Akiel, Xiaojun
Zhang, Chathuranga Abeywardana, Viktor Stepanov, Peter Z. Qin and Susumu
Takahashi (Reprinted with permission from Ref. [95]. Copyright [2016], American
Chemical Society), also in the article titled “High frequency electron paramagnetic
resonance spectroscopy of nitroxide-functionalized nanodiamonds in aqueous
solution” by Rana Akiel, Viktor Stepanov, and Susumu Takahashi in Cell
Biochemistry and Biophysics (2016) (Reprinted with permission from Ref. [96].
Copyright [2016], Springer).

In Chapter 4, we demonstrated the ability to graft nitroxide molecules on azide
functionalized ND with copper catalysed click reaction.

However, the same procedure
was unsucceful in tethering biological molecules, as the amount of Cu(I) catalyst required
resulted in significant degradation. Therefore, we had to adopt the biocompatible copper
free click chemistry.
This chapter demonstrates the ability to use copper free click reaction to attach target
molecules on ND surface. The optimization of the reaction conditions was done by
65

reacting N3-ND with a cyclooctyne and using FTIR to monitor the reaction progress. In
addition, TEMPO was attached to the surface of ND through copper free click reaction
and HF EPR spectroscopy was used to characterize the nitroxide-functionalized NDs in
aqueous solution. Moreover, through EPR spectral analysis, dynamics of nitroxide
radicals on the ND surface were investigated. The latter demonstration sheds light on the
use of HF EPR spectroscopy to investigate biological molecule-functionalized
nanoparticles.
5.1 Copper free click chemistry applied to ND
The copper-free click reaction has been developed to bypass the toxicity of metal
catalyst in biological systems by employing the ring-strain of cyclooctyne to promote the
clicking with an azide group.
97-98
Typically, in the case of linear alkyne, 1,3-cycloaddition
reaction requires an intermediate state with distorted alkyne linearity which needs high
activation energy to proceed, hence the Cu(I) catalyst. However, in the case of
cyclooctynes, the alkyne is already distorted toward the transition state geometry.
Therefore, the energy required to reach the transition state is much smaller, and the
reaction proceeds spontaneously.
99
Therefore the copper-free click chemistry concept
currently is one of the most versatile methods for grafting biological molecules on
nanoparticle surface and has been successfully applied to various nanoparticles, such as
Au,
100
CdSe,
101
Fe2O3,
102
SiO2,
103
and liposome.
104
To characterize the applicability of the
copper free click reaction to ND, the reaction was tested using Dibenzocyclooctyne
(DBCO).
66

5.1.1 Optimization of reaction conditions
5.1.1.1 Method
Reaction
The reaction follows Scheme 5.4(a). A desired amount of N3-ND (ranging from 0.1 –
1.0 mg) was suspended in a mixture of 100 µL 80%/20% (v/v) acetonitrile/water and ultra-
sonicated for 30 min to obtain a homogeneous suspension. Dibenzocyclooctyne-N-
hydroxysuccinimidyl (DBCO-NHS; Click Chemistry Tool, Inc., Scottsdale, AZ) was then
added to the N3-ND suspension at the desired concentration (ranging from 10-320 µM).
The mixture was incubated at room temperature under constant ultra-sonication for 22-
24 hours. Temperature was not controlled during the reaction, and with ultra-sonication
the temperature increased to ~45
o
C. Upon the conclusion of incubation, the reaction
mixture was subjected to centrifugation to recover the NDs as a pellet. The pallet was
then dried under a stream of nitrogen, the sample was weighed and FTIR pallet was
prepared.
Mechanism
The copper free click reaction proceeds as a standard 1,3 cycloaddition following a
concerted peryclic mechanism in which the molecules is in cyclic geometry during the
transition state and all bond breaking and bond forming occurs at the same time (Scheme
5.4(b)).
105
R’ represents ND and R represents NHS.
67

Scheme 5.1 Copper free click between DBCO and ND.
5.1.1.2 Results
FTIR was used to characterize the reactions. In the absence of DBCO (Figure 5.1(a),
magenta trace), a pronounced peak can be observed at 2100 cm
-1
, which corresponds to
N3 functional groups at the ND surface. Upon incubation with DBCO, the N3 signal clearly
reduced (Figure 5.1(a), green trace). The yield of click reactions was determined by
analyzing the reduction in FTIR peak intensity at 2100 cm
-1
, which represents the N3
signal (see Sect. A.2 for details of analysis).
By monitoring changes of the 2100 cm
-1
peak intensity, we found that the reaction is
complete within 24 hours, and the efficiency (i.e., the fraction of N3 reacted calculated
from Eq. (A. 2) depends on the DBCO concentration and the amount of NDs (which is
proportional to the amount of N3) (Figure A. 2, blue symbols). These studies indicated
that for reactions with up to 0.3 mg of N3-ND in a volume of 100 µL solution, an efficiency
of 90±10% can be achieved when the DBCO concentration is ≥ 170 µM (Figure 5.1(b),
blue symbols).

68

Figure 5.1. FTIR results for copper free click chemistry on N3-ND. (a) FTIR spectra of N3-
ND and DBCO-ND. The spectra were normalized by the sample weight. (b) Attachment
efficiency determined based on intensity changes in the N3 FTIR peaks. To allow proper
comparison, each data point was scaled by normalizing the amount of ND to 0.3 mg and
the reaction volume to 100 µL.

69

5.2 Copper free click of N3-ND and TEMPO
5.2.1 Synthesis of TEMPO-DBCO
The reaction follows Scheme 5.2 (a). In this reaction 4-amino-TEMPO (TEMPO-NH2)
(Sigma-Aldrich, Milwaukee, # 163945) was reacted with DBCO-NHS. The 1 eq. of DBCO-
NHS (170 µM, which is the optimized concentration determined during optimization of the
reaction) was reacted with 1.5 eq. of amino-TEMPO (340 µM) in the presence of 1.5 eq.
of triethylamine (340 µM) in 100 µL DMF solution. After incubation overnight, the reaction
solution was evaporated using a speedvac, then the mixture (TEMPO-DBCO, TEMPO,
DBCO-NHS, TEA) was suspended in 100 µL 80%:20% v/v acetonitrile/water. The TEA is
required to keep the amino group deprotonate since it plays the role of nucleophile in this
nucleophilic substitution reaction. (Scheme 5.2(b)).
106

Scheme 5.2. Synthesis of TEMPO-BDCO.

70

5.2.2 Reaction between TEMPO-DBCO and N3-ND
Without further purification, the mixture obtained in the synthesis of TEMPO-DBCO
was added to N3-ND for 22 hours. Then excess reactants were washed away using
multiple washing/centrifugation cycles in acetonitrile until no EPR signal of TEMPO was
observed in the supernatant. The reaction was characterized using HF EPR and X-band
EPR.
5.3 EPR results and analysis
HF EPR and X-band EPR were measured for the product (TEMPO-ND) in aqueous
solution. As shown in Figure 5.2(a), HF EPR spectrum of the TEMPO-ND sample was
different from ND EPR spectrum (Figure 4.5) and showed a distinct signal at ~4.095
Tesla in addition to the ND signals (Figure 5.2(a), blue). In addition, we also performed
X-band EPR measurement of the TEMPO-ND sample. As shown in Figure 5.2(b), the
TEMPO-ND spectrum is represented by a broad peak at ~0.3325 Tesla.
To verify the EPR signals of TEMPO grafted on the ND surface, we performed HF
EPR spectrum analysis of the TEMPO-ND sample. In the analysis, we took into account
the simulated result of ND (Figure 5.2(a), inset green) and simulated EPR spectrum of
TEMPO radicals ( 1/ 2 S = , 1 I = , 2.0086
x
g = , 2.0056
y
g = , 2.0033
z
g = , 6.5
x
A = ,
5.6
y
A = , and 37
z
A = MHz) (Figure 5.2(a), inset magenta). By combining those EPR
signals (Figure 5.2(a) red), we found a good agreement with experimental data.
Moreover, X-band EPR of the TEMPO-ND sample was also performed. Although X-band
EPR spectrum of the TEMPO-ND sample showed that EPR signal of TEMPO at ~0.3308
Tesla largely overlaps with the ND spectrum (Figure 5.2(b), blue), we found a good
71

agreement between the observed X-band spectrum and the simulation using the
parameters obtained from the HF EPR analysis (Figure 5.2(b), red). In addition, control
experiment, where alkyne free TEMPO was mixed with N3-ND, showed no adsorption of
TEMPO on ND (see Sec. A.3 for details). Therefore, the result confirms the observation
of EPR signals of grafted TEMPO.
As shown in Figure 2.6, EPR lineshape of nitroxide radicals in solution depends
greatly on the degree of molecular motions with spectral broadening and uneven changes
of the EPR signal intensities for slower motion. Figure 5.3, bottom trace, shows the
simulated EPR of nitroxide that was added to the simulated ND to obtain a good
agreement with experimental results of TEMPO-ND at X-band (Figure 5.3, red). Clearly,
the nitroxide spectrum of tethered TEMPO is much broader than that of the free TEMPO
(Figure 5.3, top trace). As a matter of fact,
c
τ of simulated tethered TEMPO was found to
be ~4.1 ns which is much slower than the free TEMPO (0.03 ns). In addition, the EPR
analysis shows a much wider line width of the nitroxide component in the TEMPO-ND
spectrum, indicating possible line broadening due to dipolar interaction.

72

Figure 5.2. EPR analyses of the TEMPO-ND sample. (a) 115 GHz EPR data. The
experimental EPR data of nitroxide radicals is magnified by 4 times. The solid blue line is
experimental data, and the green and magenta solid lines in inset are simulated EPR
spectra of ND and nitroxide radical. The red dotted line shows the sum of the simulated
spectra. (b) X-band EPR data are shown by the solid blue lines. The red dotted lines
represent the simulation using same parameters as (a). Samples (a) and (b) were
suspended in phosphate-buffered saline pH 7 at a concentration of 0.1 mg/µL and the
spectra were recorded at room temperature.

73

Figure 5.3. EPR simulation of free TEMPO and of TEMPO tethered to ND.

74

5.4 Simulation of restricted Brownian motion
When TEMPO is tethered to ND surface, two modes of motions contribute to the
dynamics of the nitroxide radical. The first one is the rotational tumbling of the ND particle
itself (Figure 5.4(i)). The second one is the rotation of the TEMPO molecule that is
allowed due to four torsional rotational motion about the bonds that links the nitroxide to
the nanoparticle, called internal motion (Figure 5.4(ii)).
The rotational tumbling of ND can be modeled by the free rotational diffusion of a
spherical particle described in Chapter 3. As shown in Figure 3.1,
c
τ for a sphere of 100
nm diameter is ~22 µs. The influence of the rotational tumbling of ND on the EPR
spectrum is expected to be not significant.
Furthermore, the slower correlation time of TEMPO-ND as compared to free radicals
suggests that the internal motion of the nitroxide molecule is greatly slowed down due to
the grafting on the ND surface. To model such motions, we consider a sphere of radius
R that is attached to a fixed substrate through a rigid tether of length L . We assume that
the Brownian motion is only allowed within a cone model with semi-angle
ο
θ (Figure 5.5).
Following the same procedure as Sec. 3.4, The trajectory of the sphere was simulated.
In the simulation, we consider l LR = + to be constant, namely,

2 2 2
z l xz = −− .
(2.19)

R was set to be 0.1 nm, which is close to the dimension of nitroxide. The length of the
tether (L) was taken to be 1.8 nm for a total 2 l = nm.
Figure 5.6(a) shows simulated trajectory of tethered motion using two different cone
semi-angles 30
o
. In addition, using the simulated trajectory, the correlation function was
calculated by following Eq. (3.7). The correlation time obtained from the exponential fit of
75

the correlation functions was found to be 0.4 ns (Figure 5.6(b)). These
c
τ are slower than
the simulated
c
τ values of a free rotating sphere of radius 0.1 nm which was found to be
0.08 ns (Figure 5.6(c)). Therefore, the simulation results support that slower correlation
time of the nitroxide radicals tethered on the ND surface (TEMPO-ND) and agree with the
experimental observation.

76

Figure 5.4. Motions of grafted nitroxide. i) Global rotational tumbling of nitroxide. This
motion is dictated by the rotational tumbling of ND. It is the same for TEMPO-ND and the
nitroxide-S2-S1-ND. ii) internal motion in the case of TEMPO-ND.

Figure 5.5. Cone model used for the simulation of tethered nitroxide.
77

Figure 5.6. Results of the simulation of the tethered motion. (a) Simulation with cone
semi-angle of 30
o
. (b) result of the correlation time obtained from (a) respectively. (c) The
results of correlation function simulation of free molecules of radius 0.1 nm.

78

Chapter 6
DNA-Functionalization of ND Surface

Materials presented in this chapter can also be found in an article titled “Investigating
functional DNA grafted on nanodiamond surface using site-directed spin labeling
and electron paramagnetic resonance spectroscopy” by Rana D. Akiel, Xiaojun
Zhang, Chathuranga Abeywardana, Viktor Stepanov, Peter Z. Qin and Susumu
Takahashi (Reprinted with permission from Ref. [95]. Copyright [2016], American
Chemical Society).

Many approaches have been developed to deposit the target molecules on the ND
surface either covalently or non-covalently.
1, 4, 8, 107-113
But so far no one has introduced
a way to use the same diamond sample, more specifically same NV, to study different
molecules of interest, as such is very advantageous especially that each NV center is
sensitive to its local environment causing it to have different optical and quantum
properties.
This chapter describes the development of a multipurpose reusable sample stage to
be employed in single molecule imaging of target molecules. This sample stage consists
of covalent functionalization of the ND surface with a single strand DNA (ssDNA). The
ND serves as the magnetic sensor, and the ssDNA will be the platform for attachment of
the molecules through hybridization with a complimentary strand bearing the target
(Scheme 6.1). Since the double stranded DNA can be denatured without affecting the
79

anchoring of ssDNA on the ND surface, the complimentary strand can be removed and
another complimentary strand with a different molecule can be attached. In addition to
reusability, this sample stage allows the imaging of biological molecules under their
physiological environment. Therefore, it will give a better understanding of the
macromolecules dynamics, interactions with other macromolecules, and structure.

Scheme 6.1. Reusable sample stage.

This chapter consists of two main sections. In the first section, the employment of
copper free click chemistry is demonstrated on DBCO tagged oligonucleotide (S1-DBCO)
by functionalizing it with a TEMPO bearing an azide. In the second section, the copper
free click reaction between N3-ND and S1-DBCO is described. Furthermore, using site-
directed spin labeling (SDSL) and EPR, repeated hybridization of the grafted S1 strand
with its complimentary strand (S2) is demonstrated.
80

6.1 Copper free click reaction applied to DNA
6.1.1 Synthesis of DBCO tagged DNA
Reaction
The reaction follows Scheme 6.2(a) and employs previously reported method which
makes use of amine-succinimide chemistry.
114
A 5’ amine modified DNA (S1) (sequence:
5’/amino-hexyl/CAA CAT GTT GGG ACA TGT TC; Integrated DNA Technology, Inc.,
Coralville, IA) was reacted with DBCO-NHS precursor (Click Chemistry Tool, Inc.,
Scottsdale, AZ). A typical reaction mixture included 1 eq. of crude S1, 40 eq. of DBCO in
50%:50% (v:v) 500 mM sodium bicarbonate buffer (NaHCO3) (pH 8.8) to acetonitrile. The
solution was incubated for 24 hours at room temperature with constant mixing.
Upon completion of the DBCO labeling reaction, the product was purified using anion-
exchange high performance liquid chromatography (AX HPLC) followed by reverse-
phase HPLC (RP HPLC). AX HPLC was carried out on an ÄKTA basic system (GE
Healthcare, Inc.) using a DNApac PA-100 column (4 × 250 mm, Dionex Inc., Sunnyvale,
CA), with a solvent gradient formed by buffer A: 20 mM Tris–HCl (pH 6.8), 1 mM NaClO4;
and buffer B: 20 mM Tris–HCl (pH 6.8), 400 mM NaClO4. RP HPLC was carried out using
a ProSphere C18 column (Grace, Inc.), with a solvent gradient formed by buffer A: 5%
acetonitrile, and 0.1 mM Triethylamine acetate (TEAA); and buffer B: 100% acetonitrile.
The purified oligonucleotide was lypholyzed, re-suspended in water, and stored at -20
o
C. The concentration of DBCO-labeled S1 was determined based on its absorption at
260 nm measured using a Beckman Coulter DU800 UV-Vis spectrometer. The
calculations used an extinction coefficient of 192,700 M
-1
cm
-1
, which does not include the
very small contribution of DBCO.
81

Mechanism
This reaction is a nucleophilic substitution were the amine on the DNA acts as a
nucleophile and attack the carbonyl to displace the succinimide (Scheme 6.2(b)). For
amine to act as nucleophile it needs to be deprotonated, hence the pH 8.8.
Scheme 6.2. Synthesis of S1-DBCO.

6.1.2 Click chemistry between S1-DBCO and azide functional TEMPO
To characterize the applicability of the copper-free click reaction, DBCO-S1 was
reacted with a nitroxide precursor, 4-azido-TEMPO (TEMPO-N3, kindly provided by Dr.
Kálmán Hideg, University of Pécs, Hungary) (Scheme 6.3). Typical reactions were
carried out in a mixture containing 1 eq. of S1-DBCO, 2.5 eq. of TEMPO-N3, and
50%/50% (v/v) acetonitrile/water. The solution was incubated for 22 hours at room
temperature with constant mixing. The resulting product was purified with HPLC to obtain
S1-TEMPO.
Scheme 6.3. Copper free click chemistry between S1-DBCO and TEMPO-N3.
82

6.1.3 Results
After the S1 reaction with DBCO-NHS the product was purified by AX HPLC. Figure
6.1 shows the HPLC traces of the DBCO-NHS, S1 precursors, and the S1-DBCO product.
The S1-DBCO clearly elutes later than either the DNA or the DBCO as expected since
DBCO renders the DNA more hydrophobic, indicating successful coupling.
After AX HPLC the reaction was desalted using RP HPLC. The pure product S1-
DBCO obtained was then reacted with TEMPO-N3. After HPLC purification EPR of the
product S1-TEMPO was measured. EPR lineshape of nitroxide radicals in solution
depends greatly on the degree of molecular motions. EPR spectra showed the
characteristic three-peak pattern from
14
N nitroxide radicals as determined through EPR
analysis (Figure 6.2). By fitting the data,
c
τ of nitroxide was determined to be 0.57±0.04
ns. The obtained correlation time is slower than free nitroxide radicals, and similar to the
reported value for spin-labeled single-stranded DNAs.
115
Therefore, the result confirmed
successful copper-free click reaction with S1-DBCO.

83

Figure 6.1. Anion-exchange HPLC of DNA-DBCO. The traces show precursors (top:
DBCO-NHS; middle: DNA S1-NH2) and DBCO-reacted products (bottom).

84

Figure 6.2. EPR spectrum of S1-TEMPO. 𝜏𝜏 c
was determined to be 0.57±0.04 ns based
on EPR lineshape simulation (solid blue line). The inset shows EPR data of a free
nitroxide, which gives 𝜏𝜏 𝑐𝑐 = 0.03±0.01 ns under the same experimental conditions.
Lineshape simulations were performed using Easyspin with the isotropic motion model
and the TEMPO spin Hamiltonian ( 1/ 2 S = , 2.0085
x
g = , 2.0059
y
g = , 2.0021
z
g = ,
6.5
x
A = MHz, 5.6
y
A = MHz, 37
z
A = MHz).

85

6.2 Attachment of S1-DBCO to N3-ND and multiple hybridization of different
complimentary strand
6.2.1 Methods
6.2.1.1 Attachment of S1-DBCO to N3-ND
The reaction follows Scheme 6.4. Using procedures developed from the DBCO/ND
studies (Sec. 5.1), typical reaction mixture (100 µL) included 0.3 mg N3-ND, 100 µM S1-
DBCO, and 80%/20% (v/v) acetonitrile/water. Typically, N3-ND was suspended in 50 µL
of 80%/20% (v/v) acetonitrile/water, and the mixture was subjected to ultra-sonication for
30 min to obtain a homogeneous suspension. Then, the proper amount of S1-DBCO was
added to the N3-ND, and the volume was adjusted to 100 µL (as smaller volumes caused
the NDs to aggregate in the presence of DNA). The mixture was incubated for one day at
room temperature while subjected to constant ultra-sonication. Upon conclusion of the
incubation, the reaction mixture was subjected to centrifugation to recover the ND
particles as the pellet. To remove unreacted S1-DBCO that are non-covalently adsorbed
at the ND surfaces, the recovered ND particles were re-suspended in 20 µL solution of
80%/20% (v/v) acetonitrile/water. The mixture was homogenized using ultra-sonication,
then the ND particles were recovered by centrifugation. The washing cycle was repeated
until the UV-Vis absorption spectrum (220–320 nm) of the supernatant showed no
detectable signal for DNA. The DNA-tagged ND product was then dried under a stream
of nitrogen and characterized using FITR and EPR.

86

Scheme 6.4 Copper free click between S1-DBCO and N3-ND.

6.2.1.2 Hybridization of the tethered DNA with the complimentary strand
The S2 strand, which is complementary to the S1 strand, has a sequence of 5’-GAA
CAT GTC CCA ACA TGT TG-3’. The work reported in this study used S2 strands with
either R5 or R5a nitroxides (see Scheme 6.5(a) and (b) for structure) attached at the
backbone of either the 4
th
and 17
th
nucleotide (Scheme 6.5(c)). Following previous
established procedures, the S2 strands were synthesized with the desired modification
using solid-phase chemical synthesis (Integrated DNA Technology, Inc. San Diego, CA),
and labeled with the corresponding nitroxide radical.
116-118

In each hybridization reaction, ~100 µM labeled S2 was mixed with ~0.15 mg of S1-
ND in 10 µL of 100 mM PBS (pH 7). The concentration of S2 to be added was estimated
according to the amount of DNA on ND that was indirectly calculated by determining the
amount of DNA in all the washes and subtracting it from the initial amount that was added.
The reaction was incubated overnight with constant mixing. The excess S2 strand was
removed by multiple cycles of washing/centrifugation in PBS until the supernatant shows
no nitroxide signal as detected by X-band EPR spectroscopy.

87

Scheme 6.5. Nitroxide structures and DNA sequence. (a) R5 structure. (b) R5a structure.
(c) S2-S1 duplex tethered to ND. Arrows indicate the backbone phosphate sites at the S2
strand at which spin labels have been attached.

6.2.1.3 Denaturing
For the denaturing process, the S2-S1-ND sample was incubated three times in large
volume of 50%:50%% acetonitrile:water for one hour. Because the ND is stable in
acetonitrile, and because water is needed to dissolve the salt, 50%:50% acetonitrile:water
was chosen. After denaturing the sample was washed 5 times to remove the free spin
labelled complimentary strand. Finally, the X-band EPR spectrum of the ND was
recorded.

6.2.2 Results
6.2.2.1 Characterization of copper free click chemistry between S1-DBCO and N3-
ND using FTIR
We performed the copper-free click reaction to covalently attach the DBCO-
functionalized S1 DNA strand onto NDs. Upon extensive wash with 80%/20% (v/v)
acetonitrile/water to remove unreacted S1 (see Sect. A.4 for details on why
acetonitrile/water was chosen as the wash solution), FTIR spectrum was recorded. The
88

spectra of S1-ND showed characteristic PO
2-
symmetric and asymmetric stretching
vibration (~1065 cm
-1
and ~1223 cm
-1
respectively) corresponding the oligomer
backbone, and peaks characteristic of C-N and C=N stretch (~1408 cm
-1
and ~1585 cm
-
1
respectively) corresponding to DNA basis (Figure 6.3(a)). Furthermore, due to the
presence of methyl groups in DNA, an increase in the CH stretch near 2992 cm
-1
was
observed. FTIR analyses revealed significant reduction in the N3 peak of NDs in samples
reacted with S1-DBCO ( Figure 6.3(b)), with the yield reaching as high as 90±10% based
on changes of FTIR signals. Additionally, in a control experiment using the S1 strand
without DBCO attached, we did not observe any change in the FTIR signal at 2100 cm
-1

(Figure 6.4(b)), demonstrating that changes observed at 2100 cm
-1
indeed reports
reaction of N3–ND with S1-DBCO.

Figure 6.3. FTIR results of S1-ND. (a) The spectra were normalized by the sample weight
as describe in Sect. A.2. (b) ND-weight-normalized FTIR signal at the 2100 cm
-1
region.

89

6.2.2.2 Characterization of Hybridization using EPR
In an attempt to demonstrate that the grafted S1 could play the role of a platform to
attach the target molecule, we examined the ability of S1-ND to hybridize with its
complementary strand (designated as S2). To characterize the hybridization different
standard techniques were test. In the UV-Vis spectroscopy, DNA signal was hard to
characterize because of the scattering due to the presence of ND particles. Furthermore,
the attempt to use gel electrophoresis was also unsuccessful because of the inability of
the ND particles to run through the gel. Therefore, we employed EPR spectroscopy to
study the hybridization. EPR spectroscopy is widely used to investigate dynamical
behaviors and microenvironments of various molecules including free radicals and
biological molecules.
63, 119-120
In particular, for investigation of biological molecules, site-
directed spin labeling (SDSL) has been developed to introduce a spin probe at a specific
site within macromolecule.
121-122
Investigation using SDSL and EPR spectroscopy has
been performed in many biological systems including proteins (T4-lysozyme,
64, 123

rhodopsin,
124
bacterirhodopsin,
64, 125
proteorhodopsin,
126
etc) DNAs and RNAs.
117

In our studies, two different nitroxide labels were attached to S2 strand: one denoted
R5, and the other one denoted R5a. Both R5 and R5a are nucleotide-independent
nitroxide labels that are attached to the phosphate backbone of the DNA.
First, a S2 DNA strand labeled at p4 with R5 spin label (R5(p4)-S2) was hybridized to
the S1 strand grafted on the ND surface. The X-band CW EPR was measured before
(S1-ND) and after the completion of the reaction (R5(p4)-S2-S1-ND). As shown in Figure
6.4(a), S1-ND sample showed the EPR spectrum of the NDs, ensuring that the tethering
of S1 to the ND surface did not cause any changes to the ND surface. On the other hand,
90

the EPR result of the R5(p4)-S2-S1-ND sample showed three distinct signals at ~0.3307
Tesla, ~0.3324 Tesla, and ~0.3339 Tesla in addition to the ND signal (Figure 6.4(a)).
Importantly, we studied the adsorption of DNA on the ND surface under reaction
conditions (see Sec. A.4 for the details of the study). In the absence of S1 tethering to
NDs, incubation of N3-ND and R5(p4)-S2 (R5(p4)-S2/N3-ND) yielded EPR spectrum
showing only ND signals after extensive washing (Figure A. 4(c)), indicating that non-
specific association of DNA to ND surface would not be sufficient to account for the
observed EPR signal from the hybridized samples. Together, the data strongly indicated
that S1 tethered to ND maintains the ability to hybridize to its complementary strand.
We further tested the ability of ND-tethered DNAs to undergo repeated hybridization.
Upon washing the hybridized R5(p4)-S2 to S1-ND sample repeatedly in an excess
amount of 50%:50% acetonitrile:water without added salt, the observed EPR spectrum
was found to show only ND signals (Figure 6.4(b), middle spectra), with the residual
nitroxide signal being < 3%. This indicates successful denaturing of the S1-S2 duplex
which resulted in removal of the nitroxide signal associated with the spin-labelled S2.
Significantly, when the same S1-ND sample was hybridized with the same spin labelled
S2 (R5(p4)-S2) following the protocol described above, identical features of the EPR
spectra were observed (Figure 6.4(b), bottom spectrum). In addition, when the ratio
between the intensity of nitroxide and ND was compared between the first and the second
hybridization, it was found that > 90% of the nitroxide EPR signal was recovered
considering the first hybridization as 100%. This clearly indicates successful re-
hybridization onto DNAs tethered on NDs.
91

Additionally, two different S2 strands were used in subsequent re-hybridization; one
labelled with R5 at the seventeenth phosphate (R5(p17)-S2) instead of the fourth, and
the second one labelled at the fourth phosphate with a R5a spin label (R5a(p4)-S2). The
EPR spectra of R5(p17)-S2-S1-ND and R5a(p4)-S2-S1-ND (Figure 6.4(c)) showed two
different components one corresponding to ND and the other one corresponding to
nitroxide, similar to the results of R5(p4)-S2-S1-ND. Together these results indicate that
S1 on ND is able to hybridize to the complimentary strand despite the nature of the
nitroxide label and the position of labeling.

92

Figure 6.4. Repeated hybridization with DNA grafted on ND surface. (a) EPR spectrum
of DNA-tethered ND sample (S1-ND) before (top) and after (center) hybridization with
R5(p4)-labeled complementary strand (R5(p4)-S2-S1-ND) and EPR results measured
after adsorption studies (bottom). The microwave frequency was ~9.33 GHz. (b) The
results of the 1st hybridization of R5(p4)-S2 with S1-ND, EPR spectrum of S1-ND after
denaturing, and the 2nd hybridization of the R5(p4)-S2 with the S1-ND obtained after
denaturing. (c) EPR spectrum of different S2 strand hybridized to S1-ND. The top
spectrum is the hybridization of R5(p4), the middle spectrum is the hybridization of
R5(p17)-S2 and the bottom spectrum is the R5a(p4)-S2. The R5a(p4)-S2 is the result of
a third hybridization on the same S1-ND from (b).
93

6.2.3 EPR analysis
Starting with R5(p4)-S2-S1-ND where R5 spin label was position at the p4 site of S2
(~6.9 nm away from the ND surface), to confirm that the features observed are due to
nitroxide, we simulated the spectra of R5 label (Figure 6.5(a), magenta) ( 1/ 2 S = , 1 I = ,
2.0085
x
g = , 2.0059
y
g = , 2.0021
z
g = , 6.5
x
A = , 5.6
y
A = MHz and 37
z
A = MHz)
127-128

and we added it to simulated ND (Figure 6.5(a), green), then we compared the resulting
spectrum (red) to the recorded spectrum (blue). As shown, we found a good agreement
between the experiment and the simulation. Furthermore, the simulated nitroxide (Figure
6.5(a), magenta) showed three prominent signals with uneven intensities. From the EPR
analysis, we obtained
c
τ of 1.0 ns.
Second, we will discuss the EPR analysis of the R5(p17)-S2-S1-ND sample where R5
spin label was position at the p17 site of S2 (~2.9 nm away from the ND surface). As seen
in Figure 6.5(b), the X-band EPR results were similar to that at p4 site. Using the same
EPR parameters for the R5(p4)-S2-S1-ND sample, we successfully simulated the
observed EPR spectrum. Based on the analysis, we obtained
c
τ of R5 to be 1.2 ns.
Next, we discuss the case where a R5a spin label is positioned at the p4 site of the
S2 sample (R5a(p4)-S2-S1-ND). Difference between R5a and R5 labels is that R5a has
bromine in the C4 site of the pyrroline ring while R5 has hydrogen. Figure 6.5(c) shows
the EPR data and the result of the analysis. The obtained EPR spectrum of the R5a spin
label shows splitting at 0.3305 Tesla. To explain the splitting at low field, the nitroxide
simulation was carried out using the microscopic ordered macroscopic disordered
(MOMD) model, with,
7.95 -1
10 s
x
R = ,
8.42 -1
10 s
y
R = ,
7.8 1
10 s
z
R
−
= , 2.01º D α = , 60.62º D γ = ,
94

and the orienting potential coefficients to be 1.3. The corresponding rotational
c
τ (
ave
( / ) 16
c
R τ = where
( )
ave
/3
x yz
R R R R = ++ ) was found to be 1.2 ns.
The nitroxide spectra obtained from the three hybridization experiments show features
depending on the identity and location of nitroxide labels, which provide information on
behaviors of the ND-tethered DNA duplexes. Specifically, R5(p4)-S2 and R5(p17)-S2
both were comparable to previous studies of R5-labeled duplexes in solution.
116
This is
characteristic of highly mobile nitroxides, and indicates that at either site R5 has little
direct contact with the ND surface. As such, the ND-tethered duplexes are not lying down
on the ND surface, but instead spreading out in solution and undergoing relatively free
motions with respect to the ND. The high mobility of the tethered DNA may stem from the
use of a relatively long and flexible linker to tether the DNA to the ND surface (Scheme
6.5(b)). This conclusion is supported by multiple additional pieces of evidence. First,
c
τ of
R5(p4)-S2-S1-ND was 1.0±0.1 ns, which is comparable to
c
τ of free S1-TEMPO (Figure
6.2) as well as free DNA duplex in solution.
129
Second, R5 motions are slightly slower at
the p17 site (
c
τ ~ 1.2 ns) than that at p4 (
c
τ ~ 1.0 ns), which can be expected as p4 is
further away from the tethering point (Scheme 6.5(b)). Third, when R5 is substituted by
R5a at the p4 site, the resulting spectrum becomes broader, and shows splitting at the
low-field manifold. R5a-S2 results are strongly indicative of a nitroxide undergoing
anisotropic rotation under the restriction of a potential, and arises from the substitution of
–H by –Br at the 4-position of the pyrroline ring (Scheme 6.5(a)), which impedes rotations
about bonds connecting the pyrroline ring to the DNA.
116, 130
This bears strong similarity
to results obtained from R5a-labeled DNA duplexes, including those tethered to a protein
95

(streptavidin).
129, 131
Because bromine atom is much larger than hydrogen atom, we
expect that side-chain motions of the R5a label will be more restricted than the R5 label,
thus the EPR analysis indicates that the obtained correlation time is strongly influenced
by DNA motions.
In the case where nitroxide is tethered to the surface of ND through DNA hybridization,
we have three modes of motions that affect the EPR spectrum. The first is rotational
tumbling of NDs (Figure 6.7(i)). The second is DNA rotational motion caused by torsional
rotations of the ten bonds linking the DNA duplex to the ND surface (Figure 6.7(ii)). The
third is side-chain motions of the R5 spin label caused by the three torsional rotation of
the bonds separating nitroxide from S2 strand (Figure 6.7(iii)). As shown in the case of
the TEMPO sample, the slow rotational tumbling on the ND particle eliminates the
rotational motion of ND and causes the dynamics of the EPR spectrum to be dominated
by the DNA motion and the internal motion of TEMPO. Moreover, the observed correlation
time comparable with that of the TEMPO-ND sample indicates that DNA duplex grafted
on the ND surface have a larger degree of freedom in their motions which is expected
given that the linker used for DNA was relatively longer than the linker for TEMPO.
Finally, in addition to dynamics, EPR analysis allowed us to determine the surface
load of nitroxide labeled S2 on ND surface. We estimated the number of tethered DNA
molecules in the R5(p4)-S2-S1-ND sample by analyzing the intensity of EPR signals in
the observed spectrum. By comparing the observed EPR intensity of the simulated
R5(p4)-S2 in R5(p4)-S2-S1-ND sample (Figure 6.5(a), magenta) with that of known
concentrations of R5(p4)-S2 in a buffer solution (Figure A. 4(a)), the number of R5(p4)-
S2 DNA on the ND surface was estimated to be 0.27 nmol, corresponding to 1.6 × 10
14

96

R5(p4)-S2 molecules on surfaces of 0.1 mg 100-nm-ND. Using 3.51 g/cm
3
for the density
of diamond and ~5.2 × 10
-16
cm
3
for the volume of one 100-nm diameter spherical ND,
the number of ND particles in the measured sample was estimated to be ~5.5×10
10
. Thus,
each 100 nm ND has ~2900 of R5(p4)-S2 molecules on the surface, which corresponds
to the average separation between the R5(p4)-S2 molecules of ~3.3 nm.

97

Figure 6.5. EPR results along with the simulations of (a) R5(p4)-S2-S1-ND, (b) R5(p17)-
S2-S1-ND, and (c) R5a(p4)-S2-S1-ND. In all images the green spectra correspond to the
ND simulation, the magenta spectra correspond to nitroxide simulation, the red spectrum
is the addition of ND and nitroxide, and the blue spectra are the experimental results.
98

Figure 6.6. Motions of nitroxide in S2-S1-ND. i) Rotation tumbling of nitroxide dictated by
the rotational tumbling of ND. ii) DNA internal motion dictated by 10 torsional rotation of
the bonds attaching DNA to ND. ii) internal motion of nitroxide dictated by three torsional
rotation of the bonds attaching nitroxide to DNA.

99

Chapter 7
Conclusion

In summary, we demonstrated efficient functionalization of ND with small molecules
using click chemistry. We used EPR spectroscopy to characterize the click chemistry
reactions and to investigate motions of molecules grafted on the ND surface. In particular,
we developed and employed the HF EPR method to investigate the dynamics of the
molecules on the ND surface with high spectral resolution, which is critical to spectrally
separate EPR signals of NDs (paramagnetic impurities in NDs) and grafted molecules
(i.e. nitroxide radicals). The demonstration sheds light on the use of HF EPR
spectroscopy to investigate dynamics of biological molecules grafted on the surface of
various nanoparticles.
Furthermore, we have successfully extended the click chemistry method to efficiently
graft single-stranded DNAs on the surface of NDs. After the click reaction, using EPR
spectroscopy of the spin-labeled double-strand DNAs, we showed that the tethered DNAs
are capable to hybridize spin-labeled complementary strands repeatedly. Using further
EPR analysis, we also demonstrated that the ND-tethered duplexes are functional and
behaving similarly to those in solution. Thus, this work is a key step to graft functional
target biological molecules on the diamond surface for NV-based magnetic sensing
applications.

100

Appendix
A.1 Normalization of the FTIR spectra in the case of copper click reaction
To quantify the intensity of the azide peak, the C=O of carboxyl at ~1760 peak was
used as a reference because it only depends on the weight of ND and is not expected to
change upon addition of solutions or upon copper click reaction. Since this peak overlaps
with OH bend which changes depending on the water content, to determine the intensity
of carboxylic C=O, both peaks were fitted with a Lorentzian function. In addition, the N3
peak was fitted also with a Lorentzian function. The result of the C=O fits can be used to
normalize the data by mass by simply dividing it by the intensity of the fit. In addition, it
can be used to determine the ratio between the C=O and N3 and compare the results
between different data. Figure A. 1 shows typical results of Lorenzian fits of each of the
three peaks.

101

Figure A. 1. FTIR results showing C=O, OH, and N3 peak. Blue trace represents the
experimental data, the green traces represent the Lorentzian fit of each of the peak, and
the red trace in the addition of the green traces.

102

A.2 Calibration curve of intensity of EPR vs. concentration of TEMPO
FTIR intensity depends on the amount of ND, which is proportional to the amount of
N3 groups. Therefore, we first calibrated the FTIR intensity as a function of the ND weight
(Figure A. 2(a)). A linear dependence can be clearly observed. Each point in the
calibration curve was the average of the FTIR signal measured from five different areas
on the same pallet. The variations at each data points were small, indicating a
homogeneous distribution of the ND samples in the pellets being measured.
This calibration study allowed us to define the normalized FTIR intensity for unreacted
NDs (I0) as:
I0= (1.9±0.2) × (the weight of NDs in mg). (A. 1)

Then, using the measured intensity from the weight-normalized FTIR spectra (Iexp)
and the normalized intensity, the reaction yield, defined as the percentage of azide groups
reacted, is then calculated as:
Yield (%) = 100 × (I0 - Iexp)/I0.
(A. 2)
Figure A. 1(b) shows representative ND-weight-normalized FTIR spectra at 2100 cm
-
1
. The normalization was done using Eq. (A. 1). FTIR signal was clearly reduced for N3-
ND reacted with S1-DBCO, but remained unchanged with S1 (i.e., N3-ND mixed with S1
in the absence of DBCO). As shown in Figure 6.3 and Figure A. 2, the click reaction yield
can be driven to close to 100% if sufficient amount of S1-DBCO can be supplied in the
reaction.
103

Figure A. 2. Normalization of FTIR data. (a) FTIR intensity of the N3 signal (at 2100 cm
-
1
) as a function of ND weight. The solid line is a fit to a linear function. The measurements
were performed five times at each sample. The mean and distribution in the five
measurements are represented by the square point and error bar. (b) ND-weight-
normalized FTIR signal at the 2100 cm
-1
region.

104

A.3 Adsorption Studies of TEMPO on the surface of ND
To eliminate the possibility of non-specific interactions of TEMPO on the surface of
ND, adsorption studies were performed where TEMPO without DBCO was mixed with
N3-ND. Hence, TEMPO is not expected to bond to ND. Typically, ~97 µM of TEMPO and
~0.1 mg of ND were mixed in a total volume of 100 µL of 80%:20% acetonitrile-water.
The mixture was ultrasonicated overnight, followed by centrifugation. 90 µL of the
supernatant was collected, then the sample was washed multiple times using suspension
in 20 µL 80% acetonitrile, followed by centrifugation with 20 µL of supernatant collected
each time. This process was repeated until no EPR signal of TEMPO was observed in
the supernatant. After the sample was cleaned, X-band EPR of the pallet was recorded.
The concentration of TEMPO in each wash was determined by comparison to known
TEMPO concentration. Figure A. 3 (a) shows the concentration of TEMPO in supernatant
after each was. The total mol of TEMPO collected after all washes ~9.6 nmol) was
comparable to the amount of TEMPO added to the ND (9.7 nmol). In addition, the EPR
of the pallet (Figure A. 3 (b)) after the conclusion of the cleaning process shows a typical
ND spectrum. Together these results suggest that the TEMPO does not adsorb on the
ND surface. This experiment provides a control for both copper click reaction and coper
free click reaction. it confirms that the additional signals observed in TEMPO-ND indeed
is due to covalent bonding.
105

Figure A. 3. Results of adsorption of TEMPO on the ND surface. (a) The moles of TEMPO
collected after all washes. (b) x-band EPR spectrum of the pallet. The red dotted trace is
the fit of the data performed with ND parameters.

106

A.4 Verification of the wash procedure for removing DNA strands non-
specifically associated with ND

The wash procedure was verified by measuring cw EPR spectra of the spin-labeled
complementary strand (R5(p4)-S2) in the supernatant (80%/20% (v/v) acetonitrile/water
or PBS solutions) after incubation of N3-ND (i.e., without the S1 strand attached) and
R5(p4)-S2. As shown in the Figure A. 4(a), the intensity of EPR spectrum was first
calibrated with the known concentration, then the EPR intensity was measured as a
function of the number of wash. The amount of DNA was determined by the EPR
spectrum analysis using Easyspin.
81
As shown in the inset of Figure A. 4(b), the EPR
intensity decreased significantly after wash. Figure A. 4(b) shows the amount of DNA
present in the supernatant as a function of the number of wash. Using either solution, the
amount of DNA in the supernatant quickly reduced and became too small to measure
after only a few washes. The data also indicated that the acetonitrile solution is more
effective in removing the non-specifically associated DNA. After the wash process, we
performed EPR spectroscopy. As shown in Figure A. 4 (c), only EPR signals of NDs were
observed in the incubated samples.

107

Figure A. 4. Wash procedure and adsorption studies results. (a) EPR intensity as a
function of the known concentration of R5(p4)-S2 in PBS. For the fit: S = 1/2, gx = 2.0085,
gy = 2.0059, gz = 2.0021, Ax = 6.5 MHz, Ay =5.6 MHz and Az = 37 MHz, 0.01 mT of the
linewidth and 0.4 ns of the rotational correlation time were used. (b) A plot of the central
line intensity measured from cw EPR spectrum of the supernatant vs. the number of wash.
Inset shows the corresponding EPR spectra obtained from washes using PBS solution,
which were measured with identical acquisition parameters and were not subjected to
spectral normalization. The EPR intensity analyses were performed with 0.4 ns and 0.6
ns of the rotational correlation time for the PBS and acetonitrile solutions, respectively.
(c) EPR spectra of the incubated samples after the wash process. The spectra was
simulated using spin parameters of NDs:
88

14
N impurity: S = 1/2, gx,y = 2.0024, gz = 2.0024,
Ax,y = 82 MHz, and Az = 114 MHz; surface impurities: S = 1/2, gx,y = 2.0029 and gz =
2.0027.

108

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Abstract (if available)

Abstract Nanodiamonds (NDs) are a new and attractive class of materials for sensing and delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable in these applications, yet reported approaches for covalent modification with biological macromolecules are still limited, and characterizing behaviors of ND-tethered bio-molecules is difficult. The approach we apply in our group employs click chemistry and electron paramagnetic resonance (EPR) to graft and characterize different target molecules on the ND surface. ❧ This dissertation will focus on describing the techniques used to efficiently graft small molecules and large biological molecules in particular DNA to the ND surface as well as the study of the dynamics of ND-grafted molecules through EPR analysis. In Chapter 1, an introduction to ND describing the methods of production, the properties, and the different functionalization approaches will be given. In Chapter 2, an insight about the EPR principles along with the different simulation approaches used throughout the dissertation is presented in addition to EPR characterization of paramagnetic impurities in NDs. Chapter 3 describes the Brownian motion simulation which will be used to understand the motion of the tethered molecules. In Chapter 4, grafting of nitroxide radicals on the surface of NDs using Cu(I)-catalyzed azide/alkyne. In Chapter 5, the investigation of the dynamics of the nitroxide radicals grafted on ND surface using EPR analysis is presented. Finally, in Chapter 6, the use of copper-free click chemistry to covalently attach DNA strands at ND surfaces and the employment of site-direct spin labeling and EPR spectroscopy to study the ability of the tethered DNA strands to undergo repetitive hybridizations is showed.

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Creator Akiel, Rana Dib (author)

Core Title Functionalization of nanodiamond surface for magnetic sensing application

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry (Chemical Physics)

Publication Date 11/09/2016

Defense Date 10/10/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag click reaction,DNA,electron paramagnetic resonance,FTIR,nanodiamond,OAI-PMH Harvest,spin label

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Takahashi, Susumu (committee chair), Pinaud, Fabien (committee member), Thompson, Mark (committee member)

Creator Email akiel@usc.edu,ranadori@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-319143

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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