surface functionalization of inorganic colloidal...
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SURFACE FUNCTIONALIZATION OF INORGANIC COLLOIDAL NANOPARTICLES FOR BIOCHEMICAL APPLICATIONS
By
YUAN LIU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Yuan Liu
To my beloved parents, wife, and daughter
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ACKNOWLEDGMENTS
First of all, I’d like to sincerely thank my research advisor, Dr. Weihong Tan who
offered me such a precious opportunity to study under his guidance at the University of
Florida. His trust and support help me overcome one difficulty after another. His
encouragement and inspiration make me a more confident, independent and critical
scientist. I am a stronger, more proactive individual due to his tutelage.
I am also grateful to my committee members, Dr. Daniel R. Talham, Dr. Jiangeng
Xue, Dr. George Christou, and Dr. Adam S. Veige for their helpful discussion, advice
and assistance. I would like to acknowledge Dr. Daniel L. Purich for collaborating with
me on my experiments involving reaction kinetic of enzyme mimic. Your advice and
willingness to talk helped me through many challenging times and my appreciation can
never be fully shown. I would like to give my particular thanks to Dr. Kathryn R. Williams
for her patient revisions and critical suggestions on my manuscripts.
To all my fellow group mates in Tan group for their assistance and valuable
discussions. Special thanks goes to Dr. Tao Chen for teaching me the basic skills
needed to move forward in all of my research endeavors, as well as Dr. Cuichen Wu,
Dr. Mingxu Liu, Dr. Guizhi Zhu, Cheng Cui, Dr. Liqin Zhang, Sena Cansiz. I am also
thankful to Dr. Weijun Chen, Dr. Ying Jiang, Dr. Tao Zhang, Dr. Liping Qiu, Dr. Juan Li,
Dr. Xiaohong Fang, Dr. Rong Hu, Dr. Zhenbao Liu, Dr. Carole Champanhac, Weijia
Hou, Yanyue Wang, I-Ting Teng, Xigao Chen, Shuo Wan, Kimberly Stewart, Ren Cai,
Yian Guo, Xiaowei Li, Xiaoshu Pan, Long Li and others for their love and friendship.
I would like to thank all of my friends at UF. I might not have been able to
complete my studies at UF if it were not for you all’s support.
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Finally, I would like to acknowledge my parents and sister for their support and
unfaded love which are the driving source for me to overcome difficulties, move forward,
and be successful. I am so grateful to my wife, Xiaoman Zhai, who is a fantastic lover,
friend and mom. I appreciate her for her support, accompany, tolerance and everything
she has dedicated to our family.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF FIGURES ........................................................................................................ 10
ABSTRACT ................................................................................................................... 13
CHAPTER
1 INTRODUCTION .................................................................................................... 15
Inorganic Colloidal Nanoparticles Synthesis ........................................................... 15
Inorganic Colloidal Nanoparticles Biochemical Applications ................................... 17 Magnetic Resonance Imaging .......................................................................... 17
Magnetic Hyperthermia .................................................................................... 17 Quantum Dots Fluorescence Imaging .............................................................. 18 Upconversion Nanoparticles Photodynamic Therapy ....................................... 18
Targeted Drug Delivery .................................................................................... 19 Nanozyme ........................................................................................................ 20
Inorganic Colloidal Nanoparticles Surface Functionalization .................................. 21 Hydrophobic Colloidal Nanoparticles Phase Transfer ...................................... 21
Colloidal nanoparticles Ligand Exchange .................................................. 21
Hydrophobic Colloidal Nanoparticles Ligand Encapsulation ...................... 23 Colloidal Nanoparticles Surface Functionalization ............................................ 23
Oligonucleotide sequence synthesis ................................................................ 25 DNA Aptamer Selection Using the Cell-SELEX Strategy ................................. 25
2 FACILE AND EFFICIENT SURFACE FUNCTIONALIZATION OF HYDROPHOBIC MAGNETIC NANOPARTICLES .................................................. 35
Background ............................................................................................................. 35
Experimental Section .............................................................................................. 37 Synthesis of Hydrophobic Magnetic Nanoparticles .......................................... 37 Aqueous Phase Transfer of Hydrophobic Magnetic Nanoparticles .................. 38
Various pH Solubility Tests of MNPS ............................................................... 38
MNP Surface Function with Fluoresceinamine ................................................. 38 MNP Surface Functionalization with DNA Aptamer .......................................... 39 Target Binding Test .......................................................................................... 39 MNP Surface Functionalization with Enzyme and Catalytic Activity Test ......... 40
Results and Discussion........................................................................................... 40
Conclusions ............................................................................................................ 43
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3 IONIC FUNCTIONALIZATION OF HYDROPHOBIC COLLOIDAL NANOPARTICLES TO FORM IONIC NANOPARTICLS WITH ENZYMELIKE PROPERTIES ......................................................................................................... 52
Background ............................................................................................................. 52 Experimental Section .............................................................................................. 54
Synthesis of Inorganic Colloidal Nanoparticles ................................................. 54 Ionization of Colloidal Nanoparticles ................................................................ 56 Calculation of Concentration of Nanozymes ..................................................... 59
Instrumentation ................................................................................................. 62 Results and Discussion........................................................................................... 63
Ionization and Characterization of Colloidal Nanoparticles .............................. 63 Peroxidase-Like Activities of Ionic Colloidal Nanoparticles............................... 64
Generalization of Colloidal Nanoparticles Ionization ........................................ 67 Conclusions ............................................................................................................ 68
4 THIOL-ENE CLICK REACTION-BASED BIOCONJUGATION OF COLLOIDAL NANOPARTICLES ................................................................................................. 82
Background ............................................................................................................. 82 Experimental Section .............................................................................................. 83
Synthesis of N-(2-[3,4-Dihydroxyphenyl] Ethyl) Acrylamide ............................. 83
Synthesis of Inorganic Colloidal Nanoparticles ................................................. 84 Acrylation of Hydrophobic Nanoparticles .......................................................... 85
Peglation of Acrylated Nanoparticles ................................................................ 86
UCNP Conjugation with HS-DNA ..................................................................... 86
UCNP Conjugation with Horseradish Peroxidase (HRP) Enzyme .................... 86 Target Binding Test with Flow Cytometry ......................................................... 87 Agarose Gel Electrophoresis ............................................................................ 87
SDS-Page Gel Electrophoresis ........................................................................ 87 Instrumentation ................................................................................................. 88
Results and Discussion........................................................................................... 89 Ligand Exchange of Inorganic Colloidal Nanoparticles and Characterization... 89 Bioconjugation of UCNP ................................................................................... 91
Conclusions ............................................................................................................ 92
5 CONCLUSIONS ................................................................................................... 101
Surface Functionalization of Inorganic Colloidal Nanoparticles for Biochemical Applications ....................................................................................................... 101
Facile and Efficient Surface Functionalization of Hydrophobic Magnetic Nanoparticles ..................................................................................................... 101
Ionic Functionalization of Hydrophobic Colloidal Nanoparticles to Form Ionic Nanoparticles with Enzymelike Properties ......................................................... 102
Thiol-ene Click Reaction-based Bioconjugation of Hydrophobic Colloidal Nanoparticles ..................................................................................................... 103
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LIST OF REFERENCES ............................................................................................. 105
BIOGRAPHICAL SKETCH .......................................................................................... 116
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LIST OF TABLES
Table page 4-1 Detailed sequence information of HS-DNA and HS-Aptamer. ............................ 94
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LIST OF FIGURES
Figure page 1-1 Nanoscale size effect of WSIO nanocrystals on magnetism and induced
magnetic resonance signals. .............................................................................. 28
1-2 Size-dependent optical properties of CdSe nanocrystals in solution. ................. 28
1-3 Traditional ELISA diagram .................................................................................. 29
1-4 Comparison of nature enzyme based ELISA and artificial nanozyme based NLISA. ................................................................................................................ 30
1-5 EDC/Sulfo-NHS crosslinking reaction scheme. .................................................. 30
1-6 Maleimide reaction scheme for chemical conjugation to sulfhydryl. ................... 31
1-8 Schematic representation of DNA aptamer selection using the cell-SELEX strategy. .............................................................................................................. 32
1-9 Modified nucleic acid CPG/Phosphoramidite for chemical synthesis of DNA sequences. ......................................................................................................... 33
1-10 Examples of functional CPG and phosphoramidite. ........................................... 34
2-1 Detailed aptamer sequence information of DNA aptamer and library. ................ 45
2-2 Ligand exchange using tetrahydrofuran, DHCA and NaOH. .............................. 45
2-3 TEM images of magnetic nanoparticles before and after ligand exchange ........ 46
2-4 TEM images of MNPs in water at different pH values. ....................................... 47
2-5 Dynamic light scattering of MNP before and after ligand exchange ................... 48
2-6 Zeta potential of MNP in water at different pH values. ........................................ 48
2-7 The as-transferred MNPs in water and PBS at different pH values .................... 49
2-8 Normalized fluorescence of MNPs before and after incubation with FLAM ........ 49
2-9 Catalysis comparison of MNP and AP. ............................................................... 50
2-10 Catalytic performance of MNA-AP after 5 rounds of washing ............................. 50
2-11 A 10-round catalytic activity test of MNP-AP. ..................................................... 51
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2-12 Schematic illustration of pH controlled reversible aggregation and dissociation of MNPs. ......................................................................................... 51
3-1 Zeta-potentials of different ionic nanoparticles. .................................................. 69
3-2 Comparison of Michaelis-Menten parameters for different ionic nanoparticles with different ligands.. ......................................................................................... 69
3-3 IR spectra of dopamine and FePt before and after ligand exchange. ................. 70
3-4 IR spectra of DHCA and FePt before and after ligand exchange ....................... 70
3-5 IR spectra of 4-ATP and CdSe before and after ligand exchange. ..................... 71
3-6 IR spectra of 4-MCBA and CdSe before and after ligand exchange ................... 71
3-7 Ionization of hydrophobic colloidal nanoparticles................................................ 72
3-8 Zeta-potential measurement of FePt-Dopamine and FePt-DHCA . .................... 73
3-9 Zeta-potential measurement of Pd-4-ATP and Pd-4-MCBA. .............................. 73
3-10 pH dependent relative peroxidase activity of ionic nanoparticles........................ 74
3-11 Michaelis-Menten kinetics for the oxidation of TMB catalyzed by different nanoparticles. ..................................................................................................... 75
3-12 Plot of maximal velocity versus total nanozyme concentration. .......................... 76
3-13 Peroxidase activity of amino terminal FePt with oxygen and argon saturated TMB solution. ...................................................................................................... 76
3-14 UV/Vis absorbance of TMB and oxidized TMB. .................................................. 77
3-15 Peroxidase activity of CdSe-4-ATP and CdSe-4-MCBA.. ................................... 77
3-16 Peroxidase activity of CdSe-4-ATP at different conditions ................................. 78
3-17 A control experiment was conducted under different conditions for CdSe-4-MCBA. ................................................................................................................ 78
3-18 NMR results of ligands released from Fe3O4 before and after ligand exchange. ........................................................................................................... 79
3-19 Optical absorption spectra of CdSe stabilized with different ligands in different solvents. ............................................................................................... 80
3-20 TEM and photoluminescence spectra of NaYF4 before and after ligand exchange and ionization. .................................................................................... 81
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4-1 Hydrophobic colloidal nanoparticles ligand exchange and thiol-ene bioconjugation. ................................................................................................... 95
4-2 H-NMR of dopamine acrylate. ............................................................................ 95
4-3 C-NMR of dopamine acrylate. ............................................................................ 96
4-4 IR spectroscopy of UCNP before and after ligand exchange ............................. 96
4-5 Photographs of the UCNP in chloroform ............................................................ 97
4-6 Photoluminescence of UCNP before ligand exchange and after thiol-ene crosslinking with HS-PEG and HS-DNA. ............................................................ 98
4-7 Zeta-potential of UCNP after thiol-ene crosslinking with HS-PEG and HS-DNA. ................................................................................................................... 98
4-8 The fluorescence of UCNP-S-PEG and UCNP-S-PEG-FITC. ............................ 99
4-9 Agarose gel and flow cytometry histograms of CEM and Ramos cells ............... 99
4-10 SDS-PAGE gel and enzymatic activity of UCNP-S-HRP via thiol-ene crosslinking. ...................................................................................................... 100
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SURFACE FUNCTIONALIZATION OF INORGANIC COLLOIDAL NANOPARTICLES
FOR BIOCHEMICAL APPLICATIONS
By
Yuan Liu
December 2016
Chair: Weihong Tan Major: Chemistry
Inorganic colloidal nanoparticles synthesized in nonpolar solvents by high-
temperature thermolysis show attractive chemical and physical properties due to
nanoscale size. Through rational engineering, colloidal nanoparticles have been
demonstrated broad application in biodetection, targeted drug delivery, and cancer
therapy. Therefore, the objective of this research is to develop facile surface
functionalization methods of colloidal nanoparticles for biochemical applications.
First, we developed a facile, high efficiency, single-phase and low-cost method to
convert hydrophobic magnetic nanoparticles (MNPs) to an aqueous phase using
tetrahydrofuran, NaOH and 3, 4-dihydroxyhydrocinnamic acid without any complicated
organic synthesis. The as-transferred hydrophilic MNPs are water soluble over a wide
pH range (pH=3 to 12), and the solubility is pH-controllable. The as-transferred MNPs
with carboxylate can be readily adapted with further surface functionalization, varying
from small molecule dyes to oligonucleotides and even enzymes.
Second, we report a simple model to ionize various types of hydrophobic
colloidal NPs including FePt, cubic Fe3O4, Pd, CdSe, and NaYF4 (Yb 30%, Er 2%, Nd
1%) NPs, to multicharged (positive and negative) NPs via ligand exchange. Surfaces of
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neutral hydrophobic NPs were converted to multicharged ions, thus making them
soluble in water. Furthermore, intrinsic peroxidase activity was observed for ionic FePt,
Fe3O4, Pd, and CdSe NPs, of which FePt and CdSe catalyzed the oxidation of colorless
substrate 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) to blue in the absence of H2O2, while
Pd and Fe3O4 catalyzed the oxidization of TMB in the presence of H2O2.
Finally, bioconjugation based on crosslinking primary amines to carboxylic acid
groups has found broad applications in protein modification, drug development, and
nanomaterial functionalization. However, amino acid-rich protein typically gives
nonselective bioconjugation when using primary amine based crosslinking. In order to
control protein orientation and activity after conjugation, site-specific bioconjugation is
desirable. In response, we developed an efficient and highly selective thiol-ene click
reaction-based bioconjugation using colloidal nanoparticles. The resulting aptamer and
enzyme nanoconjugates demonstrated excellent target binding ability and enzymatic
activity, respectively.
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CHAPTER 1 INTRODUCTION
Inorganic colloidal nanoparticles are crystalline materials that have sizes from 1
to 100 nm in dimension1. At this size regime, inorganic colloidal nanoparticles display
unique chemical and physical properties2, 3. Synthesis of inorganic colloidal
nanoparticles can be conducted either in aqueous or organic phase4, 5. However,
hydrophobic inorganic colloidal nanoparticles synthesized in nonpolar solvents by high-
temperature thermolysis show some advantages, such as tunable size with narrow size
distribution and low crystalline defect6, 7, 8, 9. The applications of inorganic colloidal
nanoparticles have been studied for decades. Colloidal nanoparticles based
nanotechnology have potential advantages in bioanalytical10, 11, 12, bioimaging13,14,
targeted drug delivery15, 16, 17, 18, and cancer therapy19, 20, 21, compared with traditional
methods.
Inorganic Colloidal Nanoparticles Synthesis
Over the past decades, various inorganic colloidal nanoparticles, including iron
oxide22, 23, semiconductor quantum dots 24, 25, 26, noble metal27, 28, 29, alloy metal30, 31, 32,
and upconversion nanoparticles33, 34 et al. have been synthesized by chemists.
Inorganic colloidal nanoparticles synthesis can be categorized to organic phase
synthesis and aqueous phase synthesis from the point of solvents. As representative,
Paul Alivisatos and Xiaogang Peng developed the synthesis of semiconductor quantum
dots with various structures. Taeghwan Hyeon developed an ultra large scale synthesis
method of monodisperse nanocrystals including iron oxide and manganese monoxide35.
Younan Xia synthesized various noble metal and alloy metal nanoparticles, such as Ag,
Au, and Pd-Pt36, 37. Chunhua Yan38 and Xiaogang Liu developed the synthesis of
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lanthanide doped upconversion nanocrystals. In aqueous phase synthesis, Yadong Li
developed a general method to synthesize various colloidal nanoparticles, including
noble metals, semiconductors, metal oxide et al39.
Compared with aqueous phase synthesis, inorganic colloidal nanoparticles
synthesis in organic phase shows some advantages, such as narrow size distribution,
highly crystalline structure, and controllable size tuning35. In a typical synthesis of iron
oxide, iron oleate was synthesized first using iron (III) chloride and sodium oleate. As a
precursor, iron oleate was added to the solvent 1-octadecene (ODE). Oleic acid, this
organic molecule was mixed with iron oleate in ODE as capping ligand which is used to
stable the nanocrystals as a colloidal status in organic solvent. As the reaction
temperature increases, the iron oleate decomposed thermally and led to the formation
of iron oxide nanocrystals. After nucleation, nanocrystals grow bigger and bigger and
finally reach equilibrium and monodisperse spherical particles. Both fast-heating and
slow-heating can produce uniform monodisperse nanoparticles6. However, the size of
nanoparticles can be controlled by setting different reaction time and different reaction
temperature. Typically, long-reaction time and high reaction temperature can result
large size of iron oxide nanoparticles (5-30 nm). The shape of iron oxide also can be
controlled, for example, from spherical to cubic40. This shape control can be realized by
controlling the adding of surfactant sodium oleate.
Other metal oxide and alloy metals nanoparticles can also be synthesized
through this hyperthermal reaction, such as MnO41, 42, and FePt nanoparticles43. In the
last decade, much research has been devoted to the shape control of semiconductor
quantum dots, gold nanoparticles, and upconversion nanoparticles. Semiconductor
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nanorods (CdS)44, 45, nanowire (CdS)46, nanosheets (CdS, CdSe)47, 48, core/shell
quantum dots (CdS/ZnS)49, 50, 51, doped quantum dots and their optical properties have
been reported. Gold nanorods52, 53 with different size (length: 20-200 nm) have also
been reported. Upconversion nanocrystals, including nanorods54(length: 50 to 200 nm),
spherical nanoparticles55 (8 to 50 nm) and hexagonal nanoplates56 (50 to 250 nm) with
lanthanide dopant, and their optical properties have been studied as well.
Inorganic Colloidal Nanoparticles Biochemical Applications
Magnetic Resonance Imaging
The application of inorganic colloidal nanoparticles has been widely studied.
Magnetic resonance imaging is a popular medical technique using magnetic field and
pulses of radio wave energy to make pictures of organs inside the body. Magnetic
nanoparticles which have the capability to increase the magnetic resonance signal now
is an emerging contrast agent57, 58, 59. Signal of magnetic resonance imaging is
correlated with the size of magnetic nanoparticles. As the size of iron oxide
nanoparticles increased from 4 nm to 12 nm, the T2-weighted MR signal intensity
decreased, thus gave a darker magnetic resonance imaging60 (Figure 1-1). With this
enhanced magnetic resonance signal, magnetic nanoparticles can be used as cancer
diagnosis.
Magnetic Hyperthermia
Due to nanoscale size effect, colloidal iron oxide nanomagnets which can
generat heat when an alternating magnet field was applied have been used for
magnetic hyperthermia61, 62. In 1993, Jordan et al. reported the application of magnetic
nanoparticles for hyperthermia therapy63. Tumor cells which are more sensitive to
temperature increase than healthy cells can be killed by hyperthermia therapy. The
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heating power of iron oxide is determined by the mechanism of magnetic energy
dissipation for single-domain particles which is strongly depends on the size of
nanocrystal. Specific loss power, the generated heat absorbed by surrounding
environment, increased when the size of iron oxide increased from 5 to 16 nm. Shape of
iron oxide is also a factor that affect the Magnetic hyperthermia efficiency. A bigger area
of hysteresis loop from cubic particles than that of spherical was observed. Cubic iron
oxide exhibit higher anisotropy energy values than that of spheres was also confirmed64.
Quantum Dots Fluorescence Imaging
Quantum dots, also known as fluorescent semiconductor nanocrystals, have
been studied from electronic materials science to biological application over the past
three decades. Quantum dots are single crystals with a few nanometers in diameter.
Their size and shape can be precisely controlled by reaction time and temperature.
Quantum dots are excellent probes and can be observed and tracked over an extended
period of time with confocal microscopy because they are characterized by a very large
absorption cross section. Compared with organic fluorophores, quantum dots have
unique optical properties, such as size-dependent absorption and emission from visible
to infrared wavelengths65, 66 (Figure 1-2). So bioconjugated quantum dots are suitable
probes for in vivo targeting and imaging of cancer cells.
Upconversion Nanoparticles Photodynamic Therapy
Conventional photodynamic therapy is limited because of the short penetration
depth of visible light. Upconversion nanoparticles which can convert long wavelength
near infrared radiation into short wavelength visible radiation overcome the limitation of
conventional biolabels such as organic dyes and quantum dots67, 68. Typically,
upconversion nanoparticles are composed of inorganic host lattice and lanthanide ions
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doped in the host lattice. Upon irradiation with infrared light, the host lattice can alter the
upconversion processes by exerting a crystal field around the dopant ion through lattice
vibration. As representative, NaYF4 has been regarded as one of the most efficient
upconversion hosts for lanthanide dopant69.
In the regard of photodynamic therapy, photosensitizers such as Ce6 and
mecrocyanine 540, were usually conjugated or loaded to upconversion nanoparticles70.
High-energy photons excited by irradiating low-energy near infrared light to
upconversion nanoparticles can active the photosensitizers to generate the singlet
oxygens which can kill the cancer cells. Mesoporous silica coated NaYF4 with Yb and Er
dopant has two main peaks, green (~540 nm) and red (~660 nm). Two photosensitizers,
mecrocyanine 540 and zinc (II) phthalocyanine who have the absorption that matched
with the emission of NaYF4: Yb, Er were loaded into the mesoporous silica layer. By
encapsulating a combination of two photosensitizers as agents, an enhanced
photodynamic therapy efficiency was experimentally confirmed71.
Targeted Drug Delivery
Targeted drug delivery is a technique using nanomaterial as drug carrier, DNA
aptamer as targeting tool to deliver drug to tumor target in order to achieve a high
efficient drug delivery and cancer therapy72. Porous nanomaterials, such as silica
nanoparticles and iron oxide nanoparticles can provide pores in nanostructure for drug
loading to increase the circulation time73, 74. Molecules, such as folic acid and DNA
aptamer can lead a target binding after conjugated on the nanoparticles surface75, 76.
Hybridized double strand DNA with groves can also be used to load drugs such as
doxorubicin77.
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Nanozyme
Nanozyme, nanomaterials based artificial enzyme, as an emerging application in
biochemical detection and immunoassays, have been established as high stable and
low cost alternatives to natural enzymes during the past decades78, 79, 80. Enzyme mimic,
including oxidase mimic and peroxidase mimic, have been found among various
materials, such as noble metals, metal oxide, porphyrin, dendrimers, alloy metals, and
quantum dots81, 82. Significant progress of nanozymes have been achieved in
biosensing, immunoassay, cancer diagnostics and therapy83.
Different nanomaterials can mimic different enzymes. As a first report to mimic
nature enzyme, iron oxide nanoparticles exhibit an intrinsic hydrogen peroxide activity to
catalyze the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) to give a colored product.
The catalytic activity of iron oxide nanoparticles is size dependent. The smaller size, the
better catalytic activity. The performance of iron oxide nanoparticles also varies with pH
and temperature. For example, iron oxide nanoparticles display high catalytic activity at
lower pH and room temperature, rather than high pH and high temperature81. Noble
metals or alloy metals received considerable attention as nanozyme due to their stability
under harsh environment.
Dual-enzyme mimic of nanoceria have been studied for oxidase mimic and
peroxidase mimic84, 84, 85, 86, 87. Oxidase is a natural enzyme to convert molecular oxygen
into either water or hydrogen peroxidase. Nanoceria can efficiently imitate the catalytic
activity of oxidase due to the oxidation reduction reaction84 of Ce3+/Ce4+. Besides
oxidase mimic, nanoceria can also mimic the hydrogen peroxidase to catalyze the
decomposition of hydrogen peroxide into molecular oxygen and water, during which
TMB can be oxidase to oxidize TMB with blue color. Based on this color change of
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TMB, it has been widely used in colorimetric analysis to determine the concentration of
a chemical element or chemical compound in a solution88.
Enzyme-Linked ImmunoSorbent Assay (ELISA) is a plate-based assay technique
to detect and quantify substances such as peptides, proteins, antibodies and hormones.
ELISA has been widely used in biosensing and immunoassay (Figure 1-3). Developing
a Nanozyme-Linked ImmunoSorbent Assay is a promising strategy to overcome the
limitations ELISA due to the susceptibility of enzymes to denaturation or
decomposition89 (Figure 1-4). Nanozyme is a good candidate to replace the natural
enzyme because of its excellent enzymatic activity. By modifying the nanozyme with
antibody or aptamer, enzyme can be replaced by nanozyme to develop a Nanozyme-
Linked ImmunoSorbent Assay.
Inorganic Colloidal Nanoparticles Surface Functionalization
Hydrophobic Colloidal Nanoparticles Phase Transfer
Inorganic colloidal nanoparticles have demonstrated wide application in
biodetection, bioimaging, drug delivery, cancer diagnoses and therapy90, 91, 92. However,
organic phase synthesized colloidal nanoparticles are typically soluble in organic phase
which is hydrophobic. Thus, the bioapplications of inorganic colloidal nanoparticles have
been limited because of their poor solubility in aqueous phase, so a phase transfer from
the hydrophobic phase to hydrophilic phase is very necessary to overcome the limitation
of hydrophobic inorganic nanoparticles81.
Colloidal nanoparticles Ligand Exchange
Various methods have been developed to overcome the poor solubility of
colloidal nanoparticles in the aqueous phase. There are two popular methods to transfer
the hydrophobic colloidal nanoparticles to aqueous phase. The first one is ligand
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exchange93, 94. Typically, a hydrophilic ligand either soluble in water or modified with
polyethylene glycol was used to replace the original hydrophobic ligand such as oleic
acid. In this phase transfer process, colloidal nanoparticles were dispersed in organic
solvent, such as toluene or chloroform, while replacement ligands were dissolved in
aqueous phase, such as water. The resulting mixture of colloidal nanoparticles and
replacement ligands were stirred for 24 hours to complete the ligand exchange process.
After ligand exchange, the hydrophobic colloidal nanoparticles were transferred to
aqueous phased because of the replacement ligands has a stronger binding affinity to
the colloidal nanoparticles. As the representatives, dihydrolipoic acid, dopamine, 3,4-
dihydroxyhydrocinnamic acid, 4-mercaptobenzoic acid, and polyethylene glycol
modified dihydrolipoic acid or dopamine have been widely used to transfer the
semiconductor quantum dots, magnetic nanoparticles, gold nanoparticles, silver
nanoparticles, upconversion nanoparticles to the aqueous phase95, 96, 97.
Regarding the replacement ligands, there are several important requirements.
First, they are soluble in aqueous phase thus the colloidal nanoparticles with
replacement ligands can be maintained as colloidal states in aqueous phase. For
example, polyethylene glycol modification to the replacement ligand is a popular method
to improve the solubility of replacement ligand. Second, the replacement ligand should
have binding groups which has stronger binding ability than the original ligands thus a
fulfill ligand exchange can be completed81. For example, thiol group in dihydrolipoic acid
or 4-mercaptobenzoic aicd has stronger binding ability to noble metal nanoparticles
such as gold and silver nanoparticles. The two phenolic hydroxyl groups can form a five
member chelate structure with iron ions on the iron oxide nanoparticles surface which is
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more stable that the chelate binding formed by carboxylic acid98. So a stronger binding
group to nanoparticles is the driving force for the ligand exchange.
Hydrophobic Colloidal Nanoparticles Ligand Encapsulation
The second method to transfer the hydrophobic colloidal nanoparticles is ligand
encapsulation96, 97, 98, which involves formation of a hydrophilic shell on the surface of
hydrophobic colloidal nanoparticles. To conduct the ligand encapsulation, an
amphiphilic ligand with lipid on one side and hydrophilic tail on the other side was
dispersed in aqueous phase and mixed with colloidal nanoparticles which were
dispersed in organic phase, such as chloroform or toluene. After thoroughly stirring,
hydrophobic colloidal nanoparticles were transferred into aqueous phase. Both
amphiphilic polymer and amphiphilic DNA lipid have been successfully used to transfer
the hydrophobic colloidal nanoparticles to aqueous phase99, 100.
Colloidal Nanoparticles Surface Functionalization
Colloidal nanoparticles phase transfer from hydrophobic phase to hydrophilic
phase, whether through ligand exchange or amphiphilic ligand encapsulation is just the
first step for biochemical application. The second step and the most significant step is
the surface functionalization of as transferred aqueous colloidal nanoparticles101, 102.
The conjugation of nanoscaled materials and small biomolecules enable unpredictable
new properties or new functions, such as targeted bioimaging and targeted drug
delivery76. Colloidal nanoparticles surface functionalization determines their further
applications. However, the replacement ligand or encapsulation ligand determine what
kind of surface functionalization method should be used. Various surface
functionalization strategies have been studied for different applications, such as
bioimaging, enzyme conjugation, drug conjugation and DNA aptamer conjugation72, 75.
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As one of the most popular methods for nanoparticles bioconjugation, EDC/NHS
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide • HCl/N-hydroxysuccinimide or sulfo-
NHS) coupling has been wide used103 (Figure 1-5). In a typical reaction, carboxylic acid
was firstly active by EDC to form an unstable intermediate which can further either react
with primary amine to form a stable conjugate or react with NHS/sulfo-NHS to form an
amine-reactive NHS ester and finally react with primary amine to form a stable
conjugate. There are many free amine and carboxylic acid groups in protein. So
EDC/NHS coupling is the primary choice for antibody nanomaterials conjugation when
nanomaterials surface was modified with amine or carboxylic acid groups. Replacement
ligands with amine or carboxylic acid group such as dopamine, 4-mercaptobenzic acid
and 3,4-dihydroxyhydrocinnamic acid are primary candidate for ligand exchange81, 104.
On the one hand, hydrophobic colloidal nanoparticles can be transferred to the aqueous
phase via replacement ligand. On the other hand, the carboxylic acid or amine group of
the replacement ligand on the nanoparticles surface can be used for further surface
functionalization such as protein conjugation and dye conjugation104. The most efficient
environment for EDC/NHS coupling is acidic conditions such as pH=4.5. Extraneous
carboxyls and amines should be devoid in the reaction buffers.
Maleimide reaction is another popular crosslink method for bioconjugation105
(Figure 1-6). The maleimide group reacts specifically with sulfhydryl groups to form a
stable thioether linkage at neutral conditions when pH is 6.5-7.5. In basic environment
when pH is higher than 8.5. the hydrolysis of the maleimide group will give a non-
reactive maleamic acid. When maleimide reaction was used for protein conjugation, the
disulfide bonds in protein structures have to be reduced to free thiols by dithiothreitol
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(DTT) or Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) before react with
maleimide group.
Oligonucleotide sequence synthesis
DNA aptamers are short sequences of oligonucleotides which can specifically
bind to their targets106, 107. DNA aptamers are generated from Systematic Evolution of
Ligands by Exponential enrichment (SELEX)108, 109. DNA aptamer can bind to their
targets from small molecules, proteins to cells specifically because of their secondary or
tertiary structures110, 111. DNA aptamer can be synthesized through a chemical synthesis
method developed by Caruthers group in 1983112. In this chemical synthesis, controlled
pore glass beads were used as the solid support, protected phosphoramidites were
used as monomers, and tetrazole was used as catalytic reagent.
In a typical chemical synthesis of DNA sequences112 (Figure 1-7), four steps:
deprotection, coupling, capping, and stabilization were included. In the first step-
deprotection, trichloroacetic acid was used to remove the trityl group which attached to
the 5’ carbon of the recipient nucleotide’s sugar. In the second step-coupling, hydroxyl
group after first deprotection reacts with the tetrazolyl phosphoramidite intermediate
formed from tetrazole and phosphoramidite nucleoside. This coupling step leads the
formation of the 5 prime to 3 prime linkage. In the capping step, coupling failures are
removed before adding the next base by reacting with acetic anhydride and N-
methylimidazole. The last step is to stabilize the phosphate linkage. By repeating these
four steps, a desired DNA sequence will be obtained.
DNA Aptamer Selection Using the Cell-SELEX Strategy
With the advantages of chemical synthesis of DNA sequence, any desired DNA
sequence will be able to synthesized efficiently. Over the last decade, SELEX have
26
been developed in the Tan lab113 (Figure 1-8). Many specific aptamers for different
diseased and cancerous cells have been generated from SELEX, such as lymphocytic
leukemia114, myeloid leukemia115, liver cancer116, lung cancer117, 118, colorectal
cancer119, and others. In a typical cell-based SELEX, a library with random sequences
(30-40 bases) and 1013-1016 single stranded oligonucleotides were obtained through
chemical synthesis and incubated with target cells at 4 °C, which can effectively inhibit
undesired oligonucleotide internalization. In this step, some sequences will bind with
target cells with high affinity. Others may bind with target cells weakly or not at all. So,
the next step is to separate the bound sequences and other sequences such as non-
bound, weakly bound and physically absorbed, and collect those bind to target cells with
high affinity. The third step is to incubate the control cells with the eluted sequences and
then collect the sequences that do not bind with the control cells but bind with target
cells. Finally, the eluted DNA sequences are used as the library for next round after
polymerase chain reaction amplification. This cycle has to be repeated for 15 to 25
times to get a desired level and then the enriched poo is cloned and sequenced to
identify a panel of potential aptamers. The advantages of the aptamers from the SELEX
are easy preparation, good stability, facile modification, and minimal immune response.
Recently, various phosphoramidite with different functional groups have been
developed to meet the application of DNA oligonucleotide sequences (Figure 1-9). With
functional group such as biotin, amine, thiol et al., DNA oligonucleotide sequence can
be conjugated nanomaterials, antibody or proteins, and small molecules through
crosslinking with streptavidin120, carboxylic acid and maleimide. Dye labeled
phosphoramidite, such as FITC-phosphoramidite and Tamra-CPG, have been
27
developed in order to modify the DNA sequence for further bioimaging applications121,
122, 123 (Figure 1-10). In addition, DNA labeling have enabled nanomaterials with wide
applications not only in biochemical detection124, 125, bioimaging126, 127, drug delivery128,
129 and cancer therapy130, 131, but also in nanocrystals synthesis132, 133, 134, nanostructure
controlling135, 136.
28
Figure1-1. Nanoscale size effect of WSIO nanocrystals on magnetism and induced magnetic resonance signals. Reprinted with permission from Jun, Y. et al. J. Am. Chem. So. 130, 5732-5733 (2005).
Figure 1-2. Size-dependent optical properties of CdSe nanocrystals in solution.
Reprinted with permission from Murray, C. & Bawendi, N. J. Am. Chem. Soc. 115, 8708-8715 (1993).
29
Figure 1-3. Traditional ELISA diagram. Reprinted with permission from https://en.wikipedia.org/wiki/ELISA
30
Figure 1-4. Comparison of nature enzyme based ELISA and artificial nanozyme based NLISA. Reprinted with permission from Wan, Y., Qi, P., Zhang, D., Wu, J. & Wang, Y. Biosens. Bioelection. 33, 69-74 (2012).
Figure 1-5. EDC/Sulfo-NHS crosslinking reaction scheme. Reprinted with permission
from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/carbodiimide-crosslinker-chemistry.html
31
Figure 1-6. Maleimide reaction scheme for chemical conjugation to sulfhydryl. Reprinted
with the permission from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/sulfhydryl-reactive-crosslinker-chemistry.html
Figure 1-7. Chemical synthesis scheme of nucleic acids. Reprinted with the permission from Chen, T., Ph.D., Functional nucleic acid-incorporated nanomaterials for bioanalytical and biomedical applications. University of Florida, 2013.
32
Figure 1-8. Schematic representation of DNA aptamer selection using the cell-SELEX strategy. Reprinted with permission from Sefah, K., Shangguan, D., Xiong, X., O’Donoghue, M. B. & Tan, W. Nat. Protocols 5, 1169-1185 (2010).
33
Figure 1-9. Modified nucleic acid CPG/Phosphoramidite for chemical synthesis of DNA sequences. Reprinted with permission from http://www.glenresearch.com/Catalog/index1.php
34
Figure 1-10. Examples of functional TAMRA CPG, amino-modifier C6 phosphoramidite, biotin phosphoramidite, thiol-modifier C6 S-S phosphoramidite, cyanine 5.5 phosphoramidite, and dithiol serinol phosphoramidite. Reprinted with permission from http://www.glenresearch.com/Catalog/index1.php
35
CHAPTER 2 FACILE AND EFFICIENT SURFACE FUNCTIONALIZATION OF HYDROPHOBIC
MAGNETIC NANOPARTICLES
Background
Hydrophobic nanocrystals synthesized in nonpolar solvents by high-temperature
thermolysis show attractive properties, such as tunable size with narrow size
distribution, and low crystalline defect.6, 10 Among these materials, magnetic
nanoparticles (MNPs), because of their unique nanoscale physical and chemical
properties, have demonstrated prominent potential in biomedical applications, including
magnetic resonance imaging (MRI),72, 137, 138 targeted drug delivery15, 139 and
hyperthermia for cancer treatment.140-143 Despite their success in biomedical science,
the preparation of biocompatible MNPs is made difficult by the presence of hydrophobic
surfactant stabilizer on their surface.
Many strategies have been developed to transfer the hydrophobic MNPs to an
aqueous phase. One popular method is ligand exchange, in which small molecules are
used to replace the hydrophobic ligand. The other is amphiphilic ligand encapsulation,
which involves formation of a hydrophilic shell on the surface of MNPs. Unfortunately,
the ligand exchange method is typically performed in two phases: MNPs in the
hydrophobic phase and small molecules in the hydrophilic phase138,144,145. For example,
dopamine hydrochloride has been used for ligand exchange on MNPs98, 146, 147.
However, because of the lack of solubility in nonpolar solvents, a two-phase system is
needed. Furthermore, MNPs tend to aggregate when mixed with polar solvents. Thus,
the exchange efficiency is typically low because of inefficient interaction between the
MNPs and small molecules, and further aggregation may occur from the low exchange
36
ratio. In addition, dopamine transferred MNPs have poor biocompatibility as a result of
poor solubility in biological buffers.
Although scientists have tried various modifications, such as the addition of PEG,
to the small molecules to improve the stability of MNPs, extensive organic synthesis are
needed, which are time-consuming and labor-intensive, thus making the ligand
exchange method more complicated. Amphiphilic ligand encapsulation is simple, but it
may not result in stable MNPs by the noncovalent hydrophobic interactions, and
additional organic syntheses are necessary as well.
To address these obstacles, we have developed a facile, high efficiency, single-
phase and low-cost method to transfer hydrophobic MNPs to an aqueous phase over a
wide pH range (pH=3 to 12) using 3, 4-dihydroxyhydrocinnamic acid (DHCA) in
tetrahydrofuran (THF). As shown in Figure 2-2, the surfactant stabilizer oleic acid was
replaced by DHCA, which forms a robust anchor on the surface of magnetic
nanoparticles via a five-membered metallocyclic chelate. The MNPs were then
neutralized with NaOH to precipitate the sodium salt, which is not soluble in THF. The
ionic form was then dispersed in aqueous solution at moderate concentrations. The
resulting water-soluble MNPs were very stable over a wide pH range from 3 to 12
(Figure 2-4 and Figure 2-7). In addition, MNPs coated with DHCA can be robustly
functionalized via the carboxyl group to form a peptide linkage with other amine-
containing molecules, varying from small molecule dyes to oligonucleotides and even
enzymes. Typically, DHCA without modification was dissolved in THF in a three-necked
flask. Hydrophobic MNPs (28±2 nm) were added dropwise at 50 °C and kept for 3 hours
at this temperature. Upon cooling the reaction mixture, NaOH (0.5 M) was added to
37
precipitate the MNPs, which were collected by centrifugation and redispersed in water.
The as-transferred MNPs were spherical and fairly monodisperse without aggregation,
as shown by transmission electron microscopy (TEM) images (Figure 2-3) and dynamic
light scattering (DLS) (Figure 2-5).
Experimental Section
Synthesis of Hydrophobic Magnetic Nanoparticles
Iron oleate was synthesized using a modified literature method.6 Typically, 10.8 g
iron chloride (FeCl3∙6H20, 40 mmol) and 36.5 g sodium oleate (120 mmol) were
dissolved in a mixed solvent composed of 80 mL ethanol, 60 mL distilled water and 140
mL hexane. The resulting solution was heated to 60 °C and refluxed for 4 hours. When
the reaction was finished and cooled to room temperature, the upper organic layer
containing the iron oleate complex was washed three times with distilled water using a
separatory funnel. After removal of hexane, the resulting iron oleate complex was in a
waxy solid form.
Hydrophobic magnetic nanoparticles were synthesized using a modified
protocol.2 Iron oleate 0.9 g (1 mmol) and oleic acid 0.156g (0.55 mmol) were added to a
three-neck flask (25 mL) with a solvent mixture of 1-octadecene (ODE)/n-tetracosane
(TCA) (3.5g/1.5g). The reaction mixture was heated to 320 °C at a heating rate of ~18
°C/min. After 1 hour, the reaction solution was quickly cooled to room temperature by
blowing air across the reaction flask. The resulting iron oxide magnetic nanoparticles
were purified with acetone/hexane (precipitation/redispersion) for three rounds. After
purification, the product was dispersed in chloroform or tetrahydrofuran (THF) for further
use.
38
Aqueous Phase Transfer of Hydrophobic Magnetic Nanoparticles
Fifty mg of 3, 4-dihydroxyhydrocinnamic acid (DHCA) were dissolved in 6 mL of
THF in a three-neck flask (25 mL). The resulting solution was heated to 50 °C under
argon flow. Then, 20 mg of hydrophobic magnetic nanoparticles dispersed in 1 mL of
THF were added dropwise. After 3 hours, the reaction was cooled to room temperature,
and 500 µL NaOH (0.5 M) were introduced to the solution to precipitate the magnetic
nanoparticles. The precipitate was collected by centrifugation (3000 rpm/min) and
redispersed in 2 mL water for further use.
Various pH Solubility Tests of MNPS
Twenty µL of the as-transferred MNPs were dissolved in 500 µL of water with pH
= 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. MNPs aggregated when pH was 2
or lower. NaOH (0.5 M) was used to increase the pH to 7 or higher. The MNPs then
became soluble and the solution cleared again. HCl (0.1 M) was used to decrease the
pH to 2 or lower, and the MNPs reaggregated.
MNP Surface Function with Fluoresceinamine
A 30 µL aliquot of as-transferred MNPs was dispersed in 300 µL of phosphate
buffered saline (PBS). Ten µL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC) (20 mM, freshly prepared) were added to the above solution and
incubated for 15 min with shaking. Then, 10 µL of N-hydroxysuccinimide (NHS) (25 mM,
freshly prepared) and 10 µL of fluoresceinamine (0.25 mM) were added, and the
mixture was incubated for 2 hours. Finally, MNPs were collected by centrifugation or
with a strong bar magnet and washed three times with PBS buffer. The resultant
product was redispersed in PBS buffer.
39
MNP Surface Functionalization with DNA Aptamer
The TAMRA labeled Sgc8 aptamer/library with amino group (detailed sequence,
Figure 2-1) was synthesized using an ABI3400 DNA/RNA synthesizer (Applied
Biosystems, Foster City, CA, USA). The TAMRA-labeled aptamer/library was
deprotected in 3 mL of deprotection solution (methanol:tert-butylamine:water=1:1:2) for
4 h at 65 °C. Then, 250 µL 3 M NaCl and 6 mL cold ethanol were used to precipitate the
deprotected sequences. The precipitated aptamers were collected by centrifugation and
dissolved in 400 µL of triethylammonium acetate (TEAA) for further purification by
reversed-phase HPLC (ProStar, Varian, Walnut Creek, CA, USA) using a C18 column
and acetonitrile-TEAA solvent. The purified aptamer/library was quantified by UV-vis.
Typically, 30 µL of as-transferred MNPs were dispersed in 300 µL of PBS. Ten
µL of EDC (20 mM, freshly prepared) were added to the above solution and incubated
for 15 min while shaking. Then, 10 µL of NHS (25 mM, freshly prepared) and 10 µL of
TAMRA labeled Sgc8 aptamer/library with amino group (500 µM) were added and the
mixture was incubated for 2 hours. Finally, MNPs were collected by centrifugation or
with a strong bar magnet and washed three times with PBS buffer. The resultant
product was redispersed in PBS buffer.
Target Binding Test
To demonstrate the specific target binding ability of MNP-ASAT to different cell
lines, fluorescence measurements were obtained on a FACSAriaTM IIu cytometer
(Becton Dickinson, San Jose, CA, USA) using a 488 nm laser as excitation source.
Samples containing CEM/Ramos cells with a concentration of 106 cells/mL were
incubated with the desired concentration of cells + ASAT, cells + ALT, cells + MNP-
ASAT and cells + MNP-ALT in a 200 µL volume of binding buffer at 4 °C for 30 min. The
40
resulting cells were washed 3 times with washing buffer and redispersed in binding
buffer for flow cytometry analysis by counting 10,000 events.
MNP Surface Functionalization with Enzyme and Catalytic Activity Test
Thirty µL of as-transferred MNPs were dispersed in 300 µL of PBS. Ten µL of
EDC (20 mM, freshly prepared) were added, and the mixture was incubated for 15 min
while shaking. Then, 10 µL of NHS (25 mM, freshly prepared) and 10 µL of alkaline
phosphatase (25 µM) were added, and the mixture was incubated for 2 hours. Finally,
MNPs were collected by centrifugation or with a strong bar magnet and washed three
times with PBS buffer. The resultant product was redispersed in PBS buffer.
MNP-AP were collected by centrifugation or with a strong bar magnet and
washed 5 times. For each washing step, the supernatant, which may have contained
free AP enzyme, was collected. The catalytic activities of the supernatant and MNP-AP
from each precipitation were tested with the substrate p-nitrophenyl phosphate (pNPP).
After washing 3 times, the supernatant did not catalyze the pNPP hydrolysis at all,
indicating that the free AP enzyme had been removed. However, the MNP-AP could still
catalyze the pNPP reaction, indicating that the AP enzyme was immobilized on the
surface of MNPs. To test the reusability of MNP-AP, after 5 washes, MNP-AP catalysis
was repeated 10 times.
Results and Discussion
After ligand exchange, water with pH values ranging from 1 to 12 was used to
test the solubility and stability of the as-transferred MNPs. MNPs aggregated when the
pH was 2 or lower (Figure 2-12). When NaOH (0.5 M) was introduced to the aggregated
MNPs (pH=2) to raise the pH to 7, the aggregated MNPs dissociated and redissolved,
and the soluble MNPs aggregated when HCl (0.1 M) was added to decrease the pH to 2
41
or lower (2). Thus, the aggregation and dispersion of MNPs is reversible by controlling
the pH.
Isoelectric point precipitation (IPP) was introduced to explain the reversibility of
MNP aggregation. IPP is the pH at which the net primary charges of a protein become
zero, leading to aggregation from reduced electrostatic repulsions. The IPP value for
MNPs was experimentally determined to be 2 according to the solubility test above. To
prove this hypothesis, the zeta-potentials of as-transferred MNPs at different pH values
were measured (Figure 2-6). Consistent with the solubility test, the results showed that
the zeta-potential of an MNP is -1.4 mV at pH 2. Addition of NaOH makes the MNP
surfaces more negative, thus increasing the electrostatic repulsion between the MNPs
and allowing them to be dispersed in water (2 (B)). Both TEM images and DLS results
in water with different pH values indicate that the MNPs were monodisperse and stable
for a period of several months. In addition, to test the stability of as-transferred MNPs,
either phosphate buffered saline (PBS) or cell culture medium was used as solvent and
no obvious aggregation was observed over a period of 3 months.
The DHCA anions not only offer MNPs excellent water solubility but also provide
a platform for further surface functionalization via the carboxyl group to form a peptide
linkage with other amine-containing molecules. Therefore, we systematically
investigated surface functionalization with fluoresceinamine (FLAM), a DNA aptamer,
and an enzyme. FLAM was chosen as a model small-molecule probe to test MNP
surface functionalization because FLAM has an amine group which can form a peptide
bond with the carboxyl group by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC)/N-hydroxysuccinimide (NHS) coupling to give emission at 519 nm.
42
In order to rule out the possibility of physical adsorption, a control experiment was
conducted with and without EDC/NHS coupling. Fluorescence measurements (Figure 2-
8) showed that MNP and MNP/FLAM without EDC/NHS coupling do not fluoresce, while
MNP/FLAM with the EDC/NHS crosslinking gives a significant fluorescent signal at 519
nm, indicating that FLAM was covalently linked onto the surface of MNPs, and that the
carboxylated MNP surface could serve as a platform for robust biomolecule
functionalization.
Having determined that the as-transferred MNPs are stable in biological systems,
we next established their utility in biomedical applications, amine modified Sgc8
aptamer (ASAT) (see Figure 2-1 for all DNA sequences114) labeled with
carboxytetramethylrhodamine (TAMRA) was used to modify MNPs based on EDC/NHS
coupling and to test their binding ability to target cancer cells. In this study, we designed
an amine-modified random oligonucleotide library labeled with TAMRA (ALT) as control
to ASAT (Figure 2-1). Selected from a large library by SELEX (Systematic Evolution of
Ligands by Exponential Enrichment), aptamers are single-stranded oligonucleotides and
could specifically bind to their targets (CEM cells for Sgc8) by folding into distinct
secondary or tertiary structures.113 Ramos cells, which are not recognized by Sgc8,
were chosen as control cells. According to the flow cytometry histogram (3), a large shift
was observed for CEM cells, while only a negligible shift was observed for Ramos cells
when treated with MNP-ASAT. For MNP-ALT, no obvious shift was observed for either
CEM or Ramos cells. For target CEM cells, a slightly larger shift was observed for MNP-
ASAT compared to free ASAT. This can be attributed to the multivalent effect of multiple
aptamers on the surface of MNP-ASAT, thus resulting in enhanced binding affinity to
43
the target cancer cells. Thus, the ASAT-modified MNPs have excellent binding ability on
target cancer cells and can be used as specific fluorescence imaging agents.
Protein enzymes, while being widely used because of their high biocatalytic
activity, are limited from the low stability and low recycling capability. Nanobiocatalysis,
whereby an enzyme is immobilized on a nanoparticle surface while retaining its
biocatalytic activity, is of significant importance for industrial reuse.148, 149 Therefore, we
conducted a facile and robust covalent enzyme immobilization on the MNP surface
using alkaline phosphatase (AP) as a model enzyme. The hydrolysis of p-nitrophenyl
phosphate can be catalyzed efficiently by AP when pH=9.8. The assay results (Figure
2-10) indicated that MNP-AP possesses excellent catalytic activity. A 10-round catalytic
recycle of MNP-AP demonstrated that MNP-AP retains its catalytic activity after many
uses, indicating that the as-transferred MNP is an excellent nano-supporter for enzyme
immobilization (Figure 2-11).
Conclusions
In summary, we have developed a facile, high efficiency, single-phase and low
cost method for aqueous phase transfer of hydrophobic MNPs using DHCA and THF.
Without any complicated organic synthesis, MNPs neutralized with NaOH show
excellent water solubility and stability in biological environments, demonstrating that the
approach is more cost-effective and labor-efficient than the traditional two-phase ligand
exchange method. Moreover, we report the first hydrophilic nanoparticles with wide pH-
range solubility and pH-controllable aggregation. The as-transferred MNPs with
carboxylate can be readily adapted by further surface functionalization which is simple
and robust. Based on these superior features, we believe that this method can be
applied to other ligand exchange methods for nanocrystals, such as quantum dots and
44
nanorods, and that the as-transferred nanoparticles will find widespread application in
nanobiotechnology.
45
Figure 2-1. Detailed aptamer sequence information of DNA aptamer and library. Underscore indicates the full sequence of Sgc8 aptamer.
Figure 2-2. Ligand exchange using tetrahydrofuran 3, 4-dihydroxyhydrocinnamic acid (DHAC) and NaOH.
46
Figure 2-3. TEM images of magnetic nanoparticles before ligand exchange in chloroform (A) and after ligand exchange in water (B). Scale bar is 200 nm. Solvent dispersity of MNPs (C) before and (D) after ligand exchange. Photo courtesy of author.
47
Figure 2-4. TEM images of MNPs in water at different pH values (scale bar for pH=1 and 2 is 500 nm; others are 200 nm).
48
Figure 2-5. Dynamic light scattering of MNP before (37 nm) and after (30 nm) ligand exchange in THF and water.
0 1 2 3 4 5 6 7 8 9 10 11 12 13
-60
-50
-40
-30
-20
-10
0
10
20
Ze
ta P
ote
ntia
l (m
V)
pH
Figure 2-6. Zeta potential of MNP in water at different pH values.
49
Figure 2-7. The as-transferred MNPs in water and PBS at different pH values from 1 to 12. Photo courtesy of author.
Figure 2-8. Normalized fluorescence of MNPs before and after incubation with FLAM with and without EDC/NHS.
50
Figure 2-9. Catalysis comparison of MNP and AP: (A) 5 µl (1 mg/mL) of MNP in 200 µL of pNPP; (B) 1 µL (2 µM) of AP in 200 µL pNPP; (C) 200 µL of pNPP. Photo courtesy of author.
Figure 2-10. Catalytic performance of MNA-AP after 5 rounds of washing. S: catalysis of supernatant; P: catalysis of precipitate (MNP-AP). Photo courtesy of author.
51
Figure 2-11. A 10-round catalytic activity test of MNP-AP. Photo courtesy of author.
Figure 2-12. Schematic illustration of pH controlled reversible aggregation and dissociation of MNPs. Photo courtesy of author.
52
CHAPTER 3 IONIC FUNCTIONALIZATION OF HYDROPHOBIC COLLOIDAL NANOPARTICLES
TO FORM IONIC NANOPARTICLS WITH ENZYMELIKE PROPERTIES
Background
Colloidal NPs with excellent physical and chemical properties have been
employed in applications ranging from solar cells150 to light-emitting diodes151 and
photocatalysis152. As biosensors, colloidal nanoparticles, including quantum dots11, gold
nanoparticles153, upconversion nanoparticles154 and alloyed plasmonic nanoparticles155,
have attracted intense interest because of their unique optical properties. However,
despite success in the synthesis of hydrophobic colloidal NPs1, 35, 54, 156, the
development of reproducible high-quality nanocrystal biosensors remains
challenging157. To stabilize the colloidal NPs in organic media at high temperature, a
layer of hydrophobic surfactant is often needed158, thus resulting poor solubility in
aqueous phase.
To improve the application of hydrophobic colloidal NPs in bioanalytical research,
most current methods use ligand exchange158 or amphiphilic ligand encapsulation159 to
transfer the hydrophobic colloidal NPs to an aqueous phase. Typically, ligand exchange
has been conducted in a two-phase system with hydrophobic colloidal NPs in a non-
polar phase and the reacting ligands in a polar phase, but this leads to serious
aggregation and poor solubility. Amphiphilic ligand encapsulation has been performed
at high temperature and requires extra washing steps, thus leading to low transfer
efficiency and poor long-term stability. Although molecular metal chalcogenide
complexes provide stable hydrophilic NPs160, their biocompatibility and toxicity are
concerns in biosensor applications.
53
To solve these solubility and cytotoxicity problems, we have designed a simple
model system to transfer hydrophobic colloidal NPs to an aqueous phase via ligand
exchange and ionization. The process is conducted in a single phase (THF solvent) with
gentle heating. Four different compounds, including 4-aminothiophenol (4-ATP), 4-
mercaptobenzoic acid (4-MCBA), dopamine, and 3, 4-dihydroxyhydrocinnamic acid (3,
4-DHCA), were all tested in separate experiments to replace the long hydrocarbon
chain ligands on colloidal NP surfaces, as shown in Figure 3-7. After ligand exchange,
aqueous HCl was added to the ligand exchange solutions containing either 4-ATP or
dopamine to protonate the –NH2 groups. Likewise, aqueous NaOH was added to ligand
exchange solutions containing either 4-MCBA or 3, 4-DHCA to deprotonate the –COOH
groups. Thus, the neutral hydrophobic NP surfaces were converted to multicharged
ions, called ionic NPs (INPs), which were collected by centrifugation and redispersed in
water.
Various colloidal NPs, including alloyed metal, metal oxide, noble metal, and
semiconductor nanoparticles, can be transferred to the aqueous phase by this ionization
process. In our model system, dopamine and 3, 4-DHCA were used for metal alloys and
metal oxides, respectively, while 4-ATP and 4-MCBA were used for noble metals and
quantum dots, respectively. In particular, we selected FePt, cubic Fe3O4, Pd, and CdSe
as representative samples of metal alloy, metal oxide, noble metal, and semiconductor
NPs, respectively.
The four replacement ligands fulfill several important requirements. (i) They are
soluble in tetrahydrofuran with hydrophobic colloidal NPs forming a single-phase ligand-
exchange environment. (ii) The nucleophilic thiol groups in 4-ATP and 4-MCBA form
54
stable complexes with the surfaces of Pd and CdSe NPs to facilitate ligand
exchange104. Stable metallocyclic chelates formed between the undercoordinated metal
atoms at the colloidal NPs surface (FePt and Fe3O4), meanwhile, the two phenolic
hydroxyl groups of dopamine and 3, 4-DHCA also promote ligand exchange165. (iii)
They have either acidic (COOH) or basic (NH2) groups which could be neutralized by
NaOH or HCl to form ionic NPs.
Experimental Section
Synthesis of Inorganic Colloidal Nanoparticles
FePt alloy nanoparticles were synthesized similar to previously reported
procedures156. In a typical synthesis of FePt, platinum acetylacetonate (0.5 mmol), 1, 2-
hexadecanediol (1.5 mmol) and dioctylether (20 mL) were mixed and degassed 3 times
before heating to 100 °C. Oleic acid (0.16 mL), oleylamine (0.17 mL) and Fe(CO)5 (0.13
mL) were added. Then the mixture was heated to 300 °C and refluxed for 30 min under
argon flow. After reflux, the reaction system was rapidly cooled to room temperature.
FePt nanoparticles were washed with ethanol and hexane and finally redispersed in
tetrahydrofuran (THF).
Fe3O4 nanocubes. Iron oleate precursor was first synthesized using previously
reported procedures161. Typically, 10.8 g iron chloride (FeCl3∙6H20, 40 mmol) and 36.5 g
sodium oleate (120 mmol) were dissolved in a solvent mixture composed of 80 mL
ethanol, 60 mL distilled water and 140 mL hexane. The resulting solution was heated to
60 °C and refluxed for 4 hours. When the reaction was finished and cooled to room
temperature, the upper organic layer containing the iron oleate complex was washed
three times with distilled water using a separatory funnel. After removal of hexane, the
resulting iron oleate complex was in a waxy solid form. To synthesize Fe3O4 nanocubes,
55
iron oleate 0.9 g (1 mmol) and sodium oleate 0.32 g (1.05 mmol) were added to a three-
neck flask (25 mL) with a solvent of 1-octadecene (5 g). The reaction mixture was
heated to 200 °C and maintained for 1 hour, followed by heating the mixture to 320 °C.
After 40 min, the reaction solution was quickly cool to room temperature by blowing air
across the reaction flask. The resulting Fe3O4 nanocubes were washed with hexane and
acetone/ethanol. After purification the product was dispersed in THF.
Pd nanoparticles were synthesized based on reported procedures162. In a typical
synthesis, Pd(acac)2 (0.1 g) was added to 1 mL of trioctylphosphine to form an orange
solution. Then 10 mL of oleylamine were introduced and degassed for 10 min. The
resulting solution was slowly heated to 250 °C (5 °C/min). After 30 min, the reaction
system was cooled quickly, washed with ethanol and hexane, and finally redispersed in
THF.
CdSe semiconductor nanoparticles were synthesized using a procedure similar
to the previously published method163. Precursor cadmium myristate was first prepared
using the following procedure: to a solution of sodium myristate in methanol (0.025 M,
240 mL), cadmium nitrate in methanol (0.05 M, 40 mL) was added dropwise to form a
white precipitate, which was washed twice with methanol and dried under vacuum
overnight. To synthesize CdSe nanocrystals, selenium powder (0.05 mmol) and
cadmium myristate (0.1 mmol) were added to a flask with 1-octadecene (5.0 g). The
mixture was degassed for 30 min under vacuum at room temperature. Then the solution
was heated to 240 °C and maintained for 20 min. The CdSe nanoparticles were washed
with ethanol and toluene and redispersed in tetrahydrofuran (THF).
56
Upconversion nanoparticles (NaYF4 (Yb 30%, Er 2%, Nd 1%)) were synthesized
similar to previously reported procedures164. Typically, Y(CH3CO2)3 (0.67 mmol),
Yb(CH3CO2)3 (0.3 mmol), Er(CH3CO2)3 (0.02 mmol) and Nd(CH3CO2)3 (0.01 mmol)
were added to a 50-mL flask containing oleic acid (7.5 mL) and 1-octadecene (17.5
mL). The resulting mixture was heated to 150 °C and maintained for 0.5 hours and then
cooled to room temperature. Subsequently, a methanol solution (6 mL) containing NH4F
(4 mmol) and NaOH (2.5 mmol) was added and stirred at 50 °C for 0.5 hours. The
reaction mixture was then heated to 100 °C to remove the methanol from the reaction
mixture. Finally, the reaction solution was heated to 290 °C and maintained for 2 hours
under argon flow. The resulting nanoparticles were washed with hexane and ethanol
and redispersed in THF.
Ionization of Colloidal Nanoparticles
Ionization of FePt with dopamine: dopamine (50 mg) was first dissolved in 300 µL
of deionized water, followed by adding 4.7 mL of THF. The mixture was transferred to a
25mL-flask and heated to 50 °C under argon flow. To the 25mL-three-neck flask, FePt
(15 mg) nanoparticles in THF (2 mL) were added and incubated for 5 hours at 50 °C.
After incubation, 100 µL of HCl (1 M) were added to the mixture to form a precipitate,
which was collected by centrifugation and redispersed in Ultrapure Millipore water (18.2
Ω).
Ionization of FePt with 3, 4-dihydroxyhydrocinnamic acid (3, 4-DHCA): to a
25mL-three-neck flask, 3, 4-dihydroxyhydrocinnamic acid (50 mg) dissolved in 5 mL of
THF was added. The mixture was heated to 50 °C. Then 15 mg of FePt nanoparticles
dissolved in 2 mL of THF was added to the flask and incubated for 5 hours. After
incubation, 200 µL of NaOH (0.5 M) were added to the mixture to form a precipitate,
57
which was collected by centrifugation and redispersed in Ultrapure Millipore water (18.2
Ω).
Ionization of Pd with 4-aminothiophenol (4-ATP): replacement ligand 4-
aminothiophenol (40 mg) was dissolved in 5 ml of THF in a 25mL-three-neck flask and
heated to 50 °C. Then 10 mg of Pd nanoparticles dissolved in 2 ml of THF was added to
the flask and incubated for 5 hours. After incubation, 100 µL of HCl (1 M) were added to
the mixture to form a precipitate, which was collected by centrifugation and redispersed
in Ultrapure Millipore water (18.2 Ω).
Ionization of Pd with 4-mercaptobenzoic acid (4-MCBA): replacement ligand 4-
mercaptobenzoic acid (40 mg) was dissolved in 5 mL of THF in a 25mL-three-neck flask
and heated to 50 °C. Then, 10 mg of Pd nanoparticles dissolved in 2 mL of THF was
added to the flask and incubated for 5 hours. After incubation, 200 µL of NaOH (0.5 M)
were added to the mixture to form a precipitate, which was collected by centrifugation
and redispersed in Ultrapure Millipore water (18.2 Ω).
Ionization of Fe3O4 nanocubes with dopamine: dopamine (50 mg) was first
dissolved in 300 µL of deionized water, followed by adding 4.7 mL of THF. The above
mixture was transferred to a 25mL-three-neck flask and heated to 50 °C under argon
flow. To the 25mL-three-neck flask, Fe3O4 nanocubes (15 mg) dissolved in THF (2 mL)
were added and incubated for 5 hours at 50 °C. After incubation, 100 µL of HCl (1 M)
were added to the mixture to form a precipitate, which was collected by centrifugation
and redispersed in Ultrapure Millipore water (18.2 Ω).
Ionization of Fe3O4 nanocubes with 3, 4-dihydroxyhydrocinnamic acid: to a
25mL-three-neck flask, 3, 4-dihydroxyhydrocinnamic acid (50 mg) dissolved in 5 mL of
58
THF was added. The mixture was heated to 50 °C. Then, 15 mg of Fe3O4 nanocubes
dissolved in 2 mL of THF was added to the flask and incubated for 5 hours. After
incubation, 200 µL of NaOH (0.5 M) were added to the mixture to form a precipitate,
which was collected by centrifugation and redispersed in Ultrapure Millipore water (18.2
Ω).
Ionization of CdSe with 4-aminothiophenol: replacement ligand 4-
aminothiophenol (40 mg) was dissolved in 5 mL of THF in a 25mL-three-neck flask and
heated to 50 °C. Then, 10 mg of CdSe nanoparticles dissolved in 2 mL of THF was
added to the flask and incubated for 5 hours. After incubation, 100 µL of HCl (1 M) were
added to the mixture to form a precipitate, which was collected by centrifugation and
redispersed in Ultrapure Millipore water (18.2 Ω).
Ionization of CdSe with 4-mercaptobenzoic acid. replacement ligand 4-
mercaptobenzoic acid (40 mg) was dissolved in 5 mL of THF in a 25mL-three-neck flask
and heated to 50 °C. Then, 10 mg of CdSe nanoparticles dissolved in 2 mL of THF was
added to the flask and incubated for 5 hours. After incubation, 200 µL of NaOH (0.5 M)
were added to the mixture to form a precipitate, which was collected by centrifugation
and redispersed in Ultrapure Millipore water (18.2 Ω).
Ionization of UCNP (NaYF4 (Yb 30%, Er 2%, Nd 1%)) with dopamine: dopamine
(50 mg) was first dissolved in 300 µL of deionized water, followed by adding 4.7 mL of
THF. The above mixture was transferred to a 25mL-three-neck flask and heated to 50
°C under argon flow. To the 25mL-three-neck flask, UCNP (15 mg) dissolved in THF (2
mL) was added and incubated for 5 hours at 50 °C. After incubation, 100 µL of HCl (1
59
M) were added to the mixture to form a precipitate, which was collected by
centrifugation and redispersed in Ultrapure Millipore water (18.2 Ω).
Ionization of UCNP (NaYF4 (Yb 30%, Er 2%, Nd 1%)) with 3, 4-
dihydroxyhydrocinnamic acid: to a 25mL-three-neck flask, 3, 4-dihydroxyhydrocinnamic
acid (50 mg) dissolved in 5 mL of THF was added. The mixture was heated to 50 °C.
Then 15 mg of UCNP dissolved in 2 mL of THF was added to the flask and incubated
for 5 hours. After incubation, 200 µL of NaOH (0.5 M) were added to the mixture to form
a precipitate, which was collected by centrifugation and redispersed in Ultrapure
Millipore water (18.2 Ω).
To prepare the NMR samples, hydrophobic Fe3O4 NPs (before ligand exchange)
capped with oleic acid and hydrophilic Fe3O4 NPs (after ligand exchange) capped with
3, 4-DHCA were dissolved by HCl (1.2M) via sonication. The mixtures were dried by air
blow and further degassing with schlenk line. Because both oleic acid and 3, 4-DHCA
can be dissolved in methanol, the ligand residue including FeCl3 was dissolved in d-
methanol for NMR tests. To better compare the characterization of ligands released
from Fe3O4 NPs, two parallel samples (Oleic acid with FeCl3 in d-methanol and 3, 4-
DHCA with FeCl3 in d-methanol) were prepared. From the NMR results, a near
quantitative ligand exchange was achieved, indicating a high degree of ligand
exchange.
Calculation of Concentration of Nanozymes
Number of FePt nanoparticles
Average size of FePt dFePt = 3.2 nm
Density of FePt: ρFePt = 14 g/cm3
60
Concentration of aqueous FePt solution: CFePt = 3.5 µg/µL
Mass of one FePt nanoparticle is 𝑚FePt = ρ ∙4
3∙ π ∙ (
d
2)
3
= 2.4 × 10−19g
Concentration of aqueous FePt in number: CNFePt=
CFePt
mFePt= 1.46 × 1013/µL
For each catalytic reaction, 5 µL (V) of FePt nanoparticles were added to a 200
µL volume of TMB solution. Thus the total number of FePt nanoparticles is NFePt =
CNFePt∙ V = 7.3 × 1013, and the concentration of FePt [EFePt] is given by
NA = 6.02 × 1023 mol−1
Vtotal = 5 + 200 = 205 µL
[EFePt] =
NFePt
NAVtotal
⁄= 5.9 × 10−7 mol/L
For Fe3O4, 10 µL of Fe3O4 nanoparticles were added to a 200 µL volume of TMB
solution each time.
Average size of Fe3O4 aFe3O4= 14.9 nm
Density of Fe3O4: ρFe3O4= 5 g/cm3
Concentration of aqueous Fe3O4 solution: CFe3O4= 2.5 µg/µL
Similarly, the concentration of Fe3O4 is [EFe3O4] = 1.2 × 10−8 mol/L
For Pd, 10 µL of Pd nanoparticles were added to a 200 µL volume of TMB
solution each time.
Average size of Pd dPd = 4.2 nm
Density of Pd: ρPd = 11.9 g/cm3
Concentration of aqueous Pd solution: CPd = 2.0 µg/µL
Similarly, the concentration of Pd is [EPd] = 3.4 × 10−7 mol/L
61
For CdSe, 20 µL of CdSe nanoparticles were added to a 300 µL volume of TMB
solution each time.
Average size of CdSe dCdSe = 4.5 nm
Density of CdSe: ρCdSe = 5.8 g/cm3
Concentration of aqueous CdSe solution: CCdSe = 3.5 µg/µL
Similarly, the concentration of CdSe is [ECdSe] = 0.9 × 10−6 mol/L
Calculation of the initial velocity and kinetic parameters of nanozyme.
𝐴 = 𝜀 ∙ 𝑐 ∙ 𝑙
𝑐 = 𝐴𝜀 ∙ 𝑙⁄
The length of cuvette is 0.2 cm.
The extinction coefficient (S7) of oxidized TMB product at 652 nm is
𝜀652 = 3.9 × 104 M−1 cm−1
The initial velocity was determined by
ν𝑖𝑛𝑖𝑡 =𝑑[𝑃]
𝑑𝑡
𝜈𝑖𝑛𝑖𝑡 =𝑐1 − 𝑐2
∆𝑡=
𝐴1𝜀 ∙ 𝑙⁄ −
𝐴2𝜀 ∙ 𝑙⁄
∆𝑡=
1
𝜀 ∙ 𝑙∙
𝐴1 − 𝐴2
∆𝑡= 12820 × 10−8 ∙
𝐴1 − 𝐴2
∆𝑡 𝑀
ν𝑖𝑛𝑖𝑡 =𝑑[𝑃]
𝑑𝑡=
𝑉𝑚𝑎𝑥[𝑆]
𝐾𝑚 + [𝑆]
1
𝜈𝑖𝑛𝑖𝑡=
𝐾𝑚 + [𝑆]
𝑉𝑚𝑎𝑥[𝑆]=
𝐾𝑚
𝑉𝑚𝑎𝑥
1
[𝑆]+
1
𝑉𝑚𝑎𝑥
𝑘𝑠𝑙𝑜𝑝𝑒 =𝐾𝑚
𝑉𝑚𝑎𝑥
𝑑𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 =1
𝑉𝑚𝑎𝑥
𝑉𝑚𝑎𝑥 = 𝑘𝑐𝑎𝑡[𝐸]
62
Where A refers to the absorbance of oxidized TMB product at 652 nm, ε refers to
the extinction coefficient of oxidized TMB product at 652 nm, 𝒍 refers to the optical path
length in the cuvette, c refers to the concentration of oxidized TMB product, νinit refers to
the initial velocity of TMB oxidation, [S] is the TMB substrate concentration, Vmax is the
maximum rate, [E] is the nanozyme concentration, kcat is the turnover number which
means the maximum number of substrate molecules converted to product per enzyme
molecule per second, Km is the Michaelis constant. It is the substrate concentration at
which the reaction rate is 𝑉𝑚𝑎𝑥
2.
Instrumentation
Transmission electron microscopy (TEM). Imaging was carried out using a
Hitachi H-7000 transmission electron microscope at 100kV. Five-µL samples of
hydrophobic nanocrystals in hexane or water were dropped onto a carbon-coated
copper grid (Ted Pella), and then dried for TEM.
Zeta-potential. Zeta-potentials were determined at room temperature using a
Zetasizer Nano-ZS (Malvern).
FT-IR spectra were recorded with a near- and mid-IR spectrometer (a Nicolet
Nexus 670) in KBr pellets.
UV-Vis absorption spectra were recorded using a Shimadzu UV-1800.
Nanocrystals were dissolved in hexane or water for measurement.
A Millenia eV laser (2nd harmonic of Nd:YAG, 532 nm) was used to pump a
Spectra-Physics Tsunami femtosecond Ti:Sapphire laser with a repetition rate of 80
MHz. The output of the Ti:Sapphire laser was tuned to 980 nm, had pulse widths of <
63
100 fs, and had a power of 375 mW. Steady-state emission spectra were collected on a
Fluoromax-3 spectrophotometer.
Results and Discussion
Ionization and Characterization of Colloidal Nanoparticles
Transmission electron microscopy (TEM) revealed that the monodisperse INPs
retain their shape and size in the aqueous phase (Figure 3-7, (c)-(e), (g)-(i), (k)-(m), (o)-
(q)). Infrared (IR) spectra (Figure 3-3, Figure 3-4, Figure 3-5, Figure 3-6), both before
and after ligand exchange, show characteristic C-H stretching peaks from oleic acid
(OA)/oleylamine (OAm), or myristic acid (MA), stabilizers before ligand exchange.
However, these peaks disappear after replacement with dopamine and 3, 4-DHCA, or
4-ATP and 4-MCBA, indicating successful exchange of the original ligands. To further
determine the degree of ligand exchange, we used NMR to characterize the ligands
before and after ligand exchange. Fe3O4 NPs were used as an example, because they
can be dissolved by HCl easily to release the surface ligands. From the NMR results
(Figure 3-18), a near quantitative ligand exchange was achieved, demonstrating a high
degree of ligand exchangeAs support, the photographs in 1 ((f), (j), (n), (r)) show that
FePt, cubic Fe3O4, Pd, and CdSe NPs were transferred to the aqueous phase from the
organic phase after ligand exchange and ionization.
After ligand exchange, ζ-potentials were measured for surface characterization of
INPs in the aqueous phase. The ionic colloidal NPs, whether using carboxylic acid- or
amine- functionalized ligands, were redispersed in neutral (pH=7) Ultrapure Millipore
water (18.2 Ω). Figure 3-1 shows that the INPs modified by amine-functionalized ligands
and neutralized by HCl gave positive ζ-potentials, while the INPs modified by carboxylic
acid-functionalized ligands and neutralized by NaOH gave negative ζ-potentials. When
64
aqueous HCl or NaOH were introduced after ligand exchange, both amine group and
carboxyl groups were neutralized to form salts, either NP-NH3Cl or NP-COONa, which
aggregated in tetrahydrofuran. In water, however, the positive or negative surface
charge prevented aggregation, thus maintaining the colloidal state of INPs.
Peroxidase-Like Activities of Ionic Colloidal Nanoparticles
The availability of our INPs led us to explore their catalytic properties, especially
because their ionic properties would commend their use in aqueous solutions and
because their thermal stability may exceed that of most biological catalysts. Indeed,
some nanozymes, which are nanomaterial-based enzyme mimics, offer high stability,
low cost, and good catalytic efficiency78, 166, making them useful in immunoassays,
biosensing, bioremediation, and cancer diagnostics167, 168. For greatest effectiveness, it
is important to fabricate high-quality nanozymes of uniform size and defined structure to
best fulfill specific tasks169. Given that horseradish peroxidase is a heme-iron protein
that is widely used in bioanalytical chemistry, we examined our FePt, Fe3O4, Pd, and
CdSe nanoparticles for their ability to catalyze oxidation reactions. Of these, ionic FePt
and CdSe NPs were found to catalyze the oxidation of the colorless substrate 3,3’,5,5’-
tetramethylbenzidine (TMB), which is blue in the absence of hydrogen peroxide, while
Pd and Fe3O4 NPs could catalyze the oxidation of TMB in the presence of H2O2.
(Spectra of the colorless substrate and blue product are shown in Figure 3-14)
Mindful that most nanoparticles lack the cardinal features of enzymes (e.g.,
homogeneous composition, structurally defined active sites, substrate specificity, and
high catalytic rate enhancements), we were interested in determining whether INP
catalysis could be modeled phenomenologically by the Michaelis-Menten equation (v =
kcat[Etot}/{1 + (Km/[S]}), where v is the initial velocity, kcat is the turnover number, [Etot] is
65
total catalyst concentration, Km is the Michaelis constant, and [S] is the substrate
concentration. In the absence of detailed information about likely binding sites, we
assumed that there was one catalytic center per nanoparticle, but multiple independent
centers on each particle would only affect the true value of kcat. (Future work using the
Langmuir Equation with unreactive substrates should clarify the actual number of sites
per particle.) Reaction conditions were optimized to assure that initial velocity data were
obtained, insofar as product formation was linear with time and v was found to be
directly proportional to INP concentration. The resulting rate data are presented in
Figure 3-11, and the derived rate parameters are presented in Figure 3-2.
We found that this peroxidase-like activity of the above NPs show different
catalytic properties and pH-dependencies. In the absence of H2O2, for example, FePt
displays better catalytic activity than CdSe, which requires up to several hours (Figure
3-14, Figure 3-15, Figure 3-16, Figure 3-17) to realize an obvious color change with
TMB. In the presence of H2O2, Fe3O4 shows better catalytic activity than Pd
nanoparticles. To study the effect of pH on peroxidase-like activity, we used pH values
ranging from 3 to 12 in TMB solutions. All INPs showed the best catalytic activity at pH
3 (Figure 3-10). Thus, pH 3 and room temperature were adopted as the standard
conditions for the steady-state kinetics assay. Typical Michaelis-Menten curves were
observed for FePt, Fe3O4, and Pd with several replacement ligands (Figure3-11), and
the parameters (Figure 3-2) were determined according to the fitted Michaelis-Menten
model and Lineweaver-Burk plots (2)170. The Km values of amino terminal INPs were
lower than those of the carboxyl terminal INPs, suggesting that INPs with terminal NH3+
have higher affinity for substrate than the INPs with terminal COO⁻. This can be
66
attributed to the surface charges of the INPs and the aromatic amine structure of TMB.
Amino terminal INPs with strong positive charge by R-NH3+ can easily attract and
template the nucleophilic aromatic TMB.
To study the mechanism underlying INP peroxidase-like activity, different
catalytic conditions were considered as mentioned above. FePt and CdSe INPs could
catalyze TMB in the absence of H2O2. However, O2 played a critical role in the catalytic
activity of FePt. Oxygen-saturated TMB solution showed superior catalytic ability when
compared to argon-saturated TMB solution (Figure 3-13). Therefore, the catalysis of
FePt on TMB was considered as a two-substrate mechanism:
1. FePt + O2 ⇄ FePt―O2 2. FePt―O2 + TMB ⇄ TMB―FePt―O2 → FePt + Oxidized―TMB + H2O
Reaction (i) with first substrate-O2 is rapid and reaches equilibrium before
reacting with the second substrate, TMB. In a typical catalytic reaction, O2 is reduced in
a 4e- process171 and converted into H2O (O2+4H++4e-→ 2H2O) by FePt, in which TMB is
used as electron donor. Thus, in the peroxidase-like activity mimic, O2 is first templated
by FePt to form the FePt-O2 complex, followed by templating of TMB by FePt-O2 to form
a TMB-FePt-O2 complex. In the second complex, each TMB donates one electron to
form a 3,5,3’,5’-tetramethylbenzidine semiquinone-imine cation free radical, finally
yielding an oxidation product after deprotonation and charge transfer25 (Equation (ii)).
A control experiment was conducted using ionic CdSe in the absence of H2O2,
both in the dark and in visible light, and no oxidized TMB was observed from ionic CdSe
in the dark. However, the amino terminal ionic CdSe catalyzed TMB slowly under
illumination (Medium Bipin Base Bulb, 32 Watt). The absorption peak of ionic CdSe was
67
626 nm. The UV/vis spectra showed no obvious wavelength shift, either before or after
ligand exchange (Figure 3-19). Under visible light irradiation, the excited electrons in the
valence band were transferred to the conduction band with holes induced in the valence
band. Valence band holes are powerful oxidants and could directly react with the
electron donor TMB to form TMB radical cations and finally yield the oxidized product.
This process indicates that the peroxidase-like activity of ionic CdSe could be attributed
to its photocatalytic property3.
Ionic Fe3O4 nanocubes and Pd NPs do not catalyze TMB oxidation in the
absence of H2O2. Fe3O4 synthesized in aqueous phase displayed peroxidase-like
activity by the presence of rich Fe2+ on the surface22. It has been reported that porous
Pd NP assemblies can be used as horseradish peroxidase substitutes30. Ionic Fe3O4
nanocubes and Pd NPs, which were synthesized in the organic phase, also exhibited
excellent peroxidase-like activity, indicating that the peroxidase-like activity of Fe3O4
and Pd NPs originates from the NPs themselves. Ligand exchange and ionization
transfer hydrophobic colloidal NPs to the aqueous phase, but without affecting their
peroxidase-like activity.
Generalization of Colloidal Nanoparticles Ionization
Ionic functionalization of colloidal NPs via ligand exchange in a single phase
provides a facile method to transfer the highly crystalline NPs synthesized through
pyrolysis to an aqueous phase. Notably, ligand exchange and ionization can be
generalized to other nanoparticle systems, such as upconversion nanoparticles
(UCNPs), which are used extensively in bioimaging and photodynamic therapy. As a
representative, NaYF4 (Yb 30%, Er 2%, Nd 1%) was selected and synthesized20 in
order to study optical properties before and after ligand exchange and ionization. TEM
68
indicated that both shape and size were maintained (Figure 3-20, (a)-(c)), and no
obvious change was observed in the photoluminescence spectra (Figure 3-20, (d)). ζ-
potential measurements (Figure 3-20, (e)) showed that dopamine-modified ionic NaYF4
has a positive surface charge and that the 3, 4-DHCA-modified ionic NaYF4 has a
negative surface charge.
Conclusions
In conclusion, ionic functionalization of hydrophobic colloidal nanoparticles to
form hydrophilic ionic NPs via ligand exchange offers the opportunity to develop
reproducible high-quality nanoparticle biosensors with maximal performance in
physiological conditions and to achieve large-scale application and impact. Advanced
nanoparticle biosensors can be engineered for visualization and detection based on the
ionic NPs. Future work can also benefit from the rational design of nanozymes, allowing
natural horseradish peroxidase to be replaced by nanozymes, which are cheaper and
more stable.
69
Figure 3-1. Zeta-potentials of ionic FePt (dopamine and 3, 4-DHCA), Fe3O4 (dopamine and 3, 4-DHCA), Pd (4-ATP and 4-MCBA), and CdSe (4-ATP and 4-MCBA).
Figure 3-2. Comparison of Michaelis-Menten parameters for ionic FePt, cubic Fe3O4 and Pd with different ligands. Km is the Michaelis constant, Vmax is the maximal reaction velocity, [E] is the ionic nanoparticle concentration, and kcat is the catalytic constant, where 𝑘𝑐𝑎𝑡 = 𝑉𝑚𝑎𝑥/[𝐸].
70
Figure 3-3. IR spectra of dopamine and FePt before (OA/OAm) and after ligand exchange with dopamine.
Figure 3-4. IR spectra of 3, 4-DHCA and FePt before (OA/OAm) and after ligand exchange with 3, 4-DHCA.
71
Figure 3-5. IR spectra of 4-ATP and CdSe before (myristic acid (MA)) and after ligand exchange with 4-ATP.
Figure 3-6. IR spectra of 4-MCBA and CdSe before (MA) and after ligand exchange with 4-MCBA
72
Figure 3-7. Ionization of hydrophobic colloidal nanoparticles. (a), Schematic representation of ionization of alloy metal (FePt) and metal oxide (Fe3O4) NPs with dopamine and 3, 4-DHCA. (b), Schematic representation of ionization of noble metal (Pd) NPs and quantum dots (CdSe) with 4-ATP and 4-MCBA. (c), (g), (k) and (o) are TEM images of ionic FePt, cubic Fe3O4, Pd and CdSe nanoparticles with 3, 4-DHCA and 4-MCBA in water, respectively. (d), (h), (l) and (p) are TEM images of FePt, cubic Fe3O4, Pd and CdSe nanoparticles in hexane. (e), (i), (m) and (q) are TEM images of ionic FePt, cubic Fe3O4, Pd and CdSe nanoparticles with dopamine and 4-ATP in water, respectively. (f), (j), (n) and (r) are corresponding photographic images of FePt, cubic Fe3O4, Pd and CdSe nanoparticles in hexane and water after ligand exchange and ionization. Scale bar: FePt, Pd, and CdSe are 50 nm. Cubic Fe3O4 is 100 nm. Photo courtesy of author.
73
Figure 3-8. ζ-potential measurement for FePt-Dopamine shows positive surface charge, and FePt-3, 4-DHCA shows negative surface charge in water.
Figure 3-9. ζ-potential measurement for Pd-4-ATP shows positive surface charge, and Pd-4-MCBA shows negative surface charge in water.
74
Figure 3-10. pH dependent relative peroxidase activity of ionic nanoparticles. To determine the relative peroxidase activity, FePt-dopamine (5 µL, 3.5 mg/mL), FePt-3, 4-DHCA (5 µL, 3.5 mg/mL), Fe3O4-dopamine (10 µL, 2.5 mg/mL), Fe3O4-3, 4-DHCA (10 µL, 2.5 mg/mL), Pd-4-ATP (10 µL, 2 mg/mL), and Pd-4-MCBA (10 µL, 2 mg/mL) were added to 200 µL of TMB solution (1.5 mM) with different pH values. Absorbance (652 nm) was taken at 15 min and normalized.
75
Figure 3-11. Michaelis-Menten kinetics (ν (initial velocity) versus [substrate]) for the oxidation of TMB catalyzed by ionic FePt, cubic Fe3O4, and Pd nanoparticles. The initial velocities of (a) and (b) were measured by adding 17.5 µg of ionic FePt to 200 µL of standard TMB solution (pH=3) with different concentrations at room temperature. The initial velocities of (c)-(f) were measured by adding 25 µg of ionic Fe3O4 or 20 µg of ionic Pd to 200 µL of standard TMB solution (pH=3) with 400 mM of H2O2. Insets: Lineweaver-Burk plots.
76
Figure 3-12. Plot of maximal velocity versus total nanozyme (FePt) concentration.
Figure 3-13. Peroxidase activity of amino terminal FePt with oxygen and argon saturated TMB solution (absorbance at 652 nm). To monitor the peroxidase activity, 5 µL of FePt (3.5 mg/mL) nanoparticles were added to a 200 µL volume of standard TMB solution (1.5 mM, pH=3).
77
Figure 3-14. UV/Vis absorbance of TMB and oxidized TMB.
Figure 3-15. Peroxidase activity of CdSe-4-ATP and CdSe-4-MCBA. To monitor the peroxidase activity of CdSe, 20 µL of CdSe-4-ATP (3.5 mg/mL) and CdSe-4-MCBA (3.5 mg/mL) were added to separate 300 µL-TMB solutions (1.5 mM, pH=3). Absorbance (652 nm) was measured at different times.
78
Figure 3-16. Peroxidase activity of CdSe-4-ATP at different conditions (no H2O2 added): a is TMB solution only (1.5 mM), b is TMB (1.5 mM, pH=3) with added CdSe-4-ATP (20 µL, 3.5 mg/mL) after 11 hours of illumination, c is TMB (1.5 mM, pH=3) with added CdSe-4-ATP (20 µL, 3.5 mg/mL) in the dark for 11 hours, d is TMB (1.5 mM, pH=7) with added CdSe-4-ATP (20 µL, 3.5 mg/mL) after 11 hours of illumination, e is TMB (1.5 mM, pH=11) with added CdSe-4-ATP (20 µL, 3.5 mg/mL) after 11 hours of illumination. All of the TMB solutions are 200 µL. Photo courtesy of author.
Figure 3-17. A control experiment was conducted under different conditions (no H2O2 added) for CdSe-4-MCBA: a is TMB solution only (1.5 mM), b is TMB (1.5 mM, pH=3) with CdSe-4-MCBA (20 µL, 3.5 mg/mL) after 11 hours of illumination, c is TMB (1.5 mM, pH=3) with added CdSe-4-MCBA (20 µL, 3.5 mg/mL) under dark for 11 hours, d is TMB (1.5 mM, pH=7) with added CdSe-4-MCBA (20 µL, 3.5 mg/mL) after 11 hours of illumination, e is TMB (1.5 mM, pH=11) with added CdSe-4-MCBA (20 µL, 3.5 mg/mL) after 11 hours of illumination. All TMB solutions are 200 µL. Photo courtesy of author.
79
Figure 3-18. NMR results of ligands released from Fe3O4 before and after ligand
exchange. The purple line shows 3, 4-DHCA and FeCl3; the dark green line shows ionized Fe3O4 NPs (after ligand exchange) capped with 3, 4-DHCA dissolved by HCl (1.2 M); the light green line shows Fe3O4 NPs (before ligand exchange) capped with oleic acid dissolved by HCl (1.2 M); the red line shows oleic acid and FeCl3. All of the samples were dissolved in d-methanol for NMR tests.
80
Figure 3-19. Optical absorption spectra of CdSe stabilized with different ligands in
different solvents.
81
Figure 3-20. TEM and photoluminescence spectra of NaYF4 (Yb 30%, Er 2%, Nd 1%)
before and after ligand exchange and ionization. TEM of (a) ionic NaYF4 with 3,4-DHCA in water, (b) NaYF4 before ligand exchange in hexane, and (c) ionic NaYF4 with dopamine in water. (d) Photoluminescence spectra of NaYF4 before and after ligand exchange and ionization. (e) ζ-Potential measurement for NaYF4−dopamine shows positive surface charge, and NaYF4−3,4-DHCA shows negative surface charge in water.
82
CHAPTER 4 THIOL-ENE CLICK REACTION-BASED BIOCONJUGATION OF COLLOIDAL
NANOPARTICLES
Background
With their unique physicochemical properties, hydrophobic colloidal nanoparticles
have wide applications in biochemistry157, such as bioimaging, drug delivery, cancer
therapy, and enzyme mimicry172-177 On the other hand, the lack of biocompatibility has,
to some extent, limited their applications81, 104. To overcome this obstacle, hydrophobic
colloidal nanoparticles must first be transferred to aqueous phase, followed by surface
functionalization through EDC/NHS coupling, or maleimide reaction.104, 179 However,
EDC/NHS coupling usually has low crosslinking efficiency. Although maleimide reaction
is rapid and has been widely used for antibody drug conjugates, the succinimide linkage
of the maleimide addition product is susceptible to hydrolysis180, 181. Therefore, even
though nanomaterial bioconjugates have enjoyed success, the chemistry of
nanoparticle-biomolecular linkage still determines their applications in biochemistry.
“Click” chemistry, or tagging, is a class of biocompatible reactions that join
substrates to biomolecules in a quick, selective, and high-yielding manner182. With its
efficiency and selectivity, click chemistry is a powerful tool in the field of biomolecular
labeling, cell surface modification and drug development183, 184. Many chemical ligations
have been employed to fulfill the demands of bioorthogonal reactions, including azide-
alkyne reactions185, 186. However, there are no azides or alkynes in native biomolecules,
thus making it necessary to specially introduce these groups into proteins or DNA.
Compared to azide-alkyne reaction, we suggest that the thiol functional group of
cysteine containing proteins makes bioconjugation more readily achievable through
thiol-ene click reaction. Indeed, most recently, thiol-ene click reaction has been
83
extensively studied in synthetic methodologies, surface and polymer modification, and
polymerization187, 190.
At the intersection of biology and nanomaterials, bionanotechnology aims to
utililize the unique properties of nanomaterials within a biological context to overcome
the problems associated with systemic administration of drugs and contrast agents191-
193.43-45 We have previously reported a facile ligand exchange method for colloidal
nanoparticle surface functionalization81, 104. Now, with the advantages of click reaction,
we herein report a selective and robust crosslinker and applied it to thiol-ene click
reaction for the bioconjugation of nanomaterials. Specifically, the ene group was
modified on the replacement ligand and then anchored on the hydrophobic colloidal
nanoparticle surface via single-phase ligand exchange. The bioconjugation of colloidal
nanoparticles via thiol-ene crosslinker was tested by HS-PEG, HS-DNA, and cysteine-
containing enzyme (Figure 4-1).
Experimental Section
Synthesis of N-(2-[3,4-Dihydroxyphenyl] Ethyl) Acrylamide
Dopamine hydrochloride (2 g, 10.6 mmol) and triethylamine (1.46 mL, 10.6
mmol) were dissolved in 20 mL anhydrous methanol in a 100 mL flask. The flask was
cool in an ice bath for 30 min. Acryloyl chloride (1.22 mL, 1.26 mmol) in 1 mL of
tetrahydrofuran, and triethylamine (2.20 mL, 15.9 mmol) in 2 mL of methanol were
added to the flask by dropwise in 20 min. Then ice bath was removed and the reaction
mixture was stirred for 2 hours at room temperature. Solvent was removed using
rotavapor. Ethyl acetate (50 mL) was added to dissolve the residue. Hydrochloric acid
(15 mL, 1M) and brine (15 mL) were used to wash the product. The organic layer was
collected and dried over with anhydrous magnesium sulfate, and then filtered and
84
concentrated by evaporation. The product can be recrystallized to give a white solid
compound with 50 % yield.
Synthesis of Inorganic Colloidal Nanoparticles
NaYF4 (Yb 30%, Er 2%) nanoparticles were synthesized using a procedure
similar to the previously published method164. Typically, Y(CH3CO2)3 (0.68 mmol),
Yb(CH3CO2)3 (0.3 mmol), Er(CH3CO2)3 (0.02 mmol) were added to a 100 mL flask
containing oleic acid (7.5 mL) and 1-octadecene (17.5 mL). The resulting mixture was
heated to 150 °C and kept for 0.5 h. Subsequently, a methanol solution (6 mL)
containing NH4F (4 mmol) and NaOH (2.5 mmol) was added after cooled to room
temperature. The mixture solution was stirred for 0.5 h at 50 °C, and then was heated to
100 °C to remove the methanol. Finally, the reaction solution was heated to 290 °C and
maintained for 2 h under argon flow. The resulting nanoparticles were washed with
hexane and ethanol and redispersed in THF.
Iron oleate was synthesized using a modified literature method6. Typically, 10.8 g
iron chloride (FeCl3∙6H20, 40 mmol) and 36.5 g sodium oleate (120 mmol) were
dissolved in a mixed solvent composed of 80 mL ethanol, 60 mL distilled water and 140
mL hexane. The resulting solution was heated to 60 °C and refluxed for 4 hours. When
the reaction was finished and cooled to room temperature, the upper organic layer
containing the iron oleate complex was washed three times with distilled water using a
separatory funnel. After removal of hexane, the resulting iron oleate complex was in a
waxy solid form. Hydrophobic magnetic nanoparticles were synthesized using a
modified protocol. Iron oleate 0.9 g (1 mmol) and oleic acid 0.156 g (0.55 mmol) were
added to a three-neck flask (25 mL) with a solvent of 1-octadecene (5 g). The reaction
mixture was heated to 320 °C at a heating rate of ~18 °C/min. After 1 hour, the reaction
85
solution was quickly cooled to room temperature by blowing air across the reaction
flask. The resulting iron oxide magnetic nanoparticles were purified with
acetone/hexane (precipitation/redispersion) for three rounds. After purification, the
product was dispersed in tetrahydrofuran (THF) for further use.
Manganese oleate was prepared based on a reported method194. In a typical
synthesis, 40 mmol of manganese chloride tetrahydrate and 80 mmol oleic acid were
mixed together in 200 mL of methanol. Then, 80 mmol of sodium hydroxide dissolved in
200 mL of methanol was added to the above solution by dropwise. A red oily
precipitation was obtained from a clear colorless mixture. After two hours stirring, the
precipitation was washed with water, ethanol and acetone, then dissolved in hexane.
The oily residue was dried with anhydrous MgSO4. Finally, a deep red waxy solid was
obtained after evaporating the solvent and dried in vacuum. In a typical synthesis, 1
mmol of manganese oleate were dissolved in 5 g of 1-octadecene and degassed at 80
°C for 1 hour to remove the moisture and oxygen. Then the reaction mixture was heated
to 270 °C and maintained for 1 hour. The as-prepared nanoparticles were washed with
hexane/ethanol, and finally dispersed in tetrahydrofuran for future ligand exchange.
Acrylation of Hydrophobic Nanoparticles
Dopamine acrylate (50 mg) was dissolved in 5 mL of THF in a 25 mL three-
necked flask and heated to 40 °C. Then 10 mg of hydrophobic nanoparticles (UCNP,
iron oxide, manganese oxide) in 2 mL of THF were added to the flask and the mixture
was incubated for 3 hours. After incubation, the acrylated nanoparticles were washed
with ethanol and THF, and finally redispersed in THF.
86
Peglation of Acrylated Nanoparticles
Acrylated nanoparticles (UCNP, iron oxide, manganese oxide) in THF (1 mL, 0.5
mg/mL) were mixed with HS-PEG1000 (HS-PEG3400-FITC) in water (200 µL, 20 mg/mL).
Then, 20 µL of trimethylamine (TEA) in THF (5 mg/mL) was added as catalyst to
promote the thiol-ene click reaction. The reaction mixture was incubated for 2.5 hours at
room temperature. After incubation, nanoparticles were washed 3 times with water and
finally redispersed in water for characterization.
UCNP Conjugation with HS-DNA
Thiol modified DNA was first reduced with dithiothreitol (DTT) and then purified
by desalting column to get HS-DNA. Acrylated nanoparticles (UCNP, iron oxide,
manganese oxide) in THF (800 µL, 0.5 mg/mL) mixed with HS-DNA (200 µL, 50 µM).
Then 20 µL of TEA (5 mg/mL) was added to promote the thiol-ene click reaction. The
reaction mixture was incubated for 2.5 hours at room temperature. After incubation,
UCNP-S-DNA was washed three times with water, and finally redispersed in water.
UCNP Conjugation with Horseradish Peroxidase (HRP) Enzyme
Cysteine is a very important residue in HRP. There are 8 cysteines and 4
disulfide bonds were formed in HRP. To reduce the disulfide bond, HRP (250 µL, 1
mg/mL) was mixed with DTT (100 mM) for 15 mins. Then the reduced HRP was purified
by desalting column. To conjugate HRP with UCNP, acrylated UCNP in THF (800 µL,
0.5 mg/mL) was mixed with reduced HRP (100 µL, 0.5 mg/mL). Then 20 µL of TEA (5
mg/mL) was added to promote the thiol-ene click reaction. The reaction mixture was
incubated for 2.5 hours at room temperature. After incubation, UCNP-S-HRP was
washed four times with water until no catalytic activity was observed in supernatant.
Finally, UCNP-S-HRP was redispersed in water.
87
Target Binding Test with Flow Cytometry
To demonstrate the specific target binding ability of UCNP-S-Aptamer to different
cell lines, fluorescence measurements were obtained from a FACS AriaTM IIu cytometer
(Becton Dickinson, San Jose, CA, USA) using a 555 nm laser as excitation source.
Samples containing CEM/Ramos cells with a concentration of 106 cells/mL were
incubated with the desired concentration of UCNP-Sgc8 and Sgc8 aptamer in a 200 µL
of binding buffer at 4 °C for 30 min. The resulting cells were washed 3 times with
washing buffer and redispersed in binding buffer for flow cytometry analysis by counting
10,000 events.
Agarose Gel Electrophoresis
To analyze the thiol-ene crosslinking stability of UCNP and HS-DNA (UCNP-S-
DNA), agarose gel (3%) was prepared using agarose (0.9 g), TBE (30 mL, 1X), and EB
(5 µL). Agarose gel electrophoresis was conducted at 100 V, for 30 min. HS-DNA and
UCNP-DNA without thiol-ene crosslinking were used as control study. Then wash the
gel with deionized water before taking pictures.
SDS-Page Gel Electrophoresis
To analyze the thiol-ene crosslinking stability of UCNP and HRP (UCNP-S-HRP),
SDS-page gel (NuPage 4-12% Bis-Tris Gel) electrophoresis was conducted at 200 V,
for 45 min. UCNP only, HRP only and UCNP-HRP without thiol-ene crosslinking were
used as control study. After SDS-page gel electrophoresis, the gel was washed twice
with deionized water, then stained with GelCodeTM Blue Safe Protein Stain for 30 min.
After staining, the gel was washed three times with deionized water before taking
pictures.
88
Instrumentation
Transmission Electron Microscopy (TEM) imaging was carried out using a Hitachi
H-7000 transmission electron microscope at 100 kV. Five microliter samples of colloidal
nanocrystals in water were dropped onto a carbon-coated copper grid (Ted Pella) and
then dried for TEM.
ζ-Potentials were determined at room temperature using a Zetasizer Nano-ZS
(Malvern).
FT-IR Spectra. Spectra were recorded with a near- and mid-IR spectrometer (a
Nicolet Nexus 670) in KBr pellets.
Optical Absorption Spectroscopy. UV−vis absorption spectra were recorded
using a Shimadzu UV-1800. Nanocrystals were dissolved in hexane or water for
measurement.
Synthesis of thiol modified Sgc8 aptamer. The TAMRA labeled Sgc8 aptamer
with disulfide group (detailed sequence, Table 1) was synthesized using an ABI3400
DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA). The disulfide
modified TAMRA-labeled aptamer was deprotected in 3 mL of deprotection solution
(ammonium hydroxide, methylamine 50:50) for 30 min at 65 °C. Then, 250 µL 3 M NaCl
and 6 mL cold ethanol were used to precipitate the deprotected sequences. The
precipitated aptamers were collected by centrifugation and dissolved in 400 µL of
triethylammonium acetate (TEAA) for further purification by reversed-phase HPLC
(ProStar, Varian, Walnut Creek, CA, USA) using a C18 column and acetonitrile-TEAA
solvent. The purified aptamer was quantified by UV-vis.
89
Results and Discussion
Ligand Exchange of Inorganic Colloidal Nanoparticles and Characterization
To increase thiol-ene click reaction efficiency, we selected acryloyl chloride,
instead of methacryloyl chloride, as the ene group. The ene group of acryloyl chloride is
a better electrophile than that of methacryloyl chloride. Therefore, as shown in Figure 4-
1, when reacted with dopamine, the product, dopamine acrylamide, as the replacement
ligand, could replace the surfactant stabilizer oleic acid to form robust anchor on the
surface of colloidal nanoparticles via a five-membered metallocyclic chelate146. Using
this method, hydrophobic lanthanide-doped upconversion nanoparticles (UCNP), iron
oxide nanoparticles, and manganese oxide nanoparticles were all synthesized to test
the thiol-ene-based crosslinker. As an example, NaYF4 (Yb 30%, Er 2%) was selected
to study optical properties and biocompatibility after bioconjugation with HS-PEG, HS-
DNA, and cysteine-containing Horseradish Peroxidase.
C-/H-NMR demonstrated that dopamine acrylamide was successfully
synthesized (Figure 4-3). Both colloidal nanoparticles and replacement ligand could be
dissolved in Tetrahydrofuran (THF). Thus, ligand exchange was conducted in THF, as a
single phase system, at 40 °C for 3 hours. Infrared (IR) spectra of UCNP showed that
the characteristic peaks of amide C=O and phenol C-O appeared after ligand exchange,
indicating successful ligand exchange (Figure 4-4). After ligand exchange, HS-PEG1000
or HS-DNA, following reduction by 1, 4-dithiolthreitol (DTT) and purification through
desalting column dissolved in water, was mixed with dopamine acrylamide functional
UCNP in THF for 3 hours. Triethylamine was used as a catalyst to promote the thiol-ene
click reaction. Transmission electron microscopy (TEM) revealed that the
monodispersed UCNP, iron oxide, and manganese oxide nanoparticles retained their
90
shape and size in aqueous phase after conjugation with HS-PEG1000 and HS-DNA
(Figure 4-5). No obvious change was observed from the photoluminescence of UCNP
after conjugation with HS-PEG1000 and HS-DNA upon excitation at λ=980 nm (Figure 4-
6). Zeta-potential of UCNP after conjugation with HS-PEG showed almost neutral
surface charge (-3.2 mV). Because DNA oligonucleotide is negatively charged, UCNP-
S-DNA showed negative surface charge (-25.9 mV), as shown in S5 (Figure 4-7).
UCNPs gave significant fluorescence signal at 525 nm after linking with FITC-modified
HS-PEG3400, indicating a successful covalent thiol-ene crosslinking (Figure 4-8).
Stability of thiol-ene crosslinker is a significant factor contributing to its
application in bioconjugate chemistry. Therefore, we used agarose gel electrophoresis
to study the stability of thiol-ene crosslinker with UCNP (Figure 4-9). Four parallel
samples, including UCNP-S-DNA with thiol-ene crosslinker, UCNP-DNA without thiol-
ene crosslinker, UCNP alone, and DNA alone, were prepared for agarose gel
electrophoresis. Based on agarose gel imaging (Figure 4-9, left), only UCNP alone had
no band. UCNP-DNA without thiol-ene crosslinking had a very strong band at the free
DNA position based on the release of physically absorbed DNA on the nanoparticle
surface under electrophoresis. A less intense band was observed at the free DNA
position for UCNP-S-DNA with thiol-ene crosslinking, as a likely result of some physical
absorption. However, there was a band in line 2 which was very strong and did not
move. This immobility could be explained by the covalent linkage of DNA on the
nanoparticles surface via thiol-ene crosslinker and the large size of UCNPs (28 nm)
under electrophoresis. Thus, it was concluded that thiol-ene click reaction could provide
a stable and robust crosslinker.
91
Bioconjugation of UCNP
The biomedical applications of UCNP were further studied using HS-DNA-
aptamer. Thiol- modified Sgc8 aptamer labeled with carboxytetramethylrhodamine
(TAMRA) was conjugated on the UCNP surface via thiol-ene click reaction to test its
binding ability to target cancer cells (Table 4-1). Aptamer Sgc8 can bind with the
membrane protein PTK7 which is highly expressed on CEM cells114, 195. Ramos cells
without PTK7 were used as a negative control. As shown by flow cytometry histograms
in Figure 4-9 (right), an obvious shift was observed from CEM cells, while only a
negligible shift was observed for Ramos cells.
Protein bioconjugation with nanomaterials is a powerful reagent in biochemistry
and medicine196. However, the control of protein orientation on the nanoparticles, which
is essential in catalysis, delivery, and therapy, necessitates selective bioconjugation197-
199. Cysteine is a significant residue for the chemical modification of proteins due to the
unique reactivity of the thiol group and low abundance of cysteine residues in nature
proteins. Therefore, cysteine-selective conjugation for bionanoconjugates is desired179,
180. In order to study nanoparticle-protein conjugation, Horseradish Peroxidase (HRP)
was selected for conjugation with UCNP. HRP has 8 cysteines which form 4 disulfide
bonds. The red bond in 3a indicates the disulfide bond. Before thiol-ene crosslinking,
the disulfide bond was reduced by treatment with DTT. The reduced HRP was purified
by desalting column before crosslinking with dopamine acrylamide functional UCNP.
Triethylamine (TEA) was added as a catalyst to promote the thiol-ene click reaction.
The resultant UCNP-S-HRP conjugates were then analyzed by SDS-PAGE (sodium
dodecyl sulfate polyacrylamide electrophoresis). As shown in Figure 4-10 (c), HRP
without reduction or TEA catalysis was physically absorbed on the surface of UCNPs
92
and showed a HRP band and a long tail band under electrophoresis. In contrast, the
UCNP-S-HRP conjugated via thiol-ene crosslinking gave a single band, but did not
move owing to the large size of UCNPs, indicating that a stable and robust thiol-ene
linkage had occurred between UCNPs and HRP.
As a result of structural disturbance, enzymatic activity can be affected by
conjugation with nanomaterials. However, enzymatic activity is dependent on active
sites with access to the environment196. HRP is an enzyme which can catalyze the
oxidation of 3,3’5,5’-Tetramethylbenzidine (TMB) in the presence of hydrogen peroxide.
Thus, we further studied the catalytic activity of HRP after conjugation with UCNP to
form UCNP-S-HRP via thiol-ene click reaction. As shown in Figure 4-10 (d), UCNP
alone shows no catalytic activity. On the other hand, UCNP-S-HRP was washed 4 times
after conjugation until all free HRP enzyme was washed away and no catalytic activity
was observed from the supernatant. Under these conditions, we observed excellent
catalytic activity from UCNP-S-HRP in the presence of TMB. For HRP enzyme to be
active, its substrate TMB must react with the active sites and finally release the product.
Therefore, the cysteine-selective conjugation with dopamine acrylamide functional
UCNPs via thiol-ene click reaction had no effect on the active sites of HRP enzyme after
conjugation.
Conclusions
In conclusion, we have developed a thiol-ene click reaction-based bioconjugation
of colloidal nanoparticles, including iron oxide and manganese oxide, as well as
UCNPs, and tested the crosslinker by HS-PEG peglation, HS-aptamer labeling, and
enzyme immobilization. Gel electrophoresis demonstrated that the thiol-ene crosslinker
is stable and robust. Moreover, stable and robust thiol-ene linkage between dopamine
93
acrylamide functional UCNPs and HRP did not affect the binding ability of aptamer to its
target CEM cells or the catalytic activity of HRP enzyme. Bioconjugates based on
reactions with nanomaterials have enormous potential in such fields as biology and
materials science. In particular, the superior selectivity and stability of thiol-ene click
reaction-based crosslinker will enable the engineering of multifunctional nanomaterial
bioconjugates, making this a powerful tool with broad applications in biosensing,
bioanalysis, bioimaging, drug delivery and theranostics.
94
Table 4-1. Detailed sequence information of HS-DNA and HS-Aptamer. Name Sequence
HS-DNA 5’-HS- TTT TTT TTT TAT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT AGA-3’
HS-Aptamer 5’-HS- TTT TTT TTT TAT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT
AGA-TAMRA-3’
95
Figure 4-1. Hydrophobic colloidal nanoparticles ligand exchange and thiol-ene click reaction- based bioconjugation.
Figure 4-2. H-NMR (500 MHz, d6-DMSO, δppm) of dopamine acrylate.
96
Figure 4-3. C-NMR (125MHz, d6-DMSO, δppm) of dopamine acrylate.
Figure 4-4. IR spectroscopy of UCNP capped with oleic acid (UCNP-OA, blakc), dopamine acrylate (Dop-Ac, blue), and acrylated UCNP after ligand exchange (UCNP-Dop-Ac, red).
3500 3000 2500 2000 1500 1000
0
20
40
60
80
100
Tra
ns
mit
tan
ce
(%
)
Wavelength (nm)
UCNP-OA
UCNP-Dop-Ac
Dop-Ac
97
Figure 4-5. Photographs of the UCNP in chloroform (a) before ligand exchange and water (b) after thiol-ene crosslinking with HS-DNA under 980 nm laser illumination, respectively. TEM images of UCNP in hexane (c) and water(d), iron oxide in hexane (e) and water(f), and manganese oxide in hexane (g) and water(f), before ligand exchange and after thiol-ene crosslinking with HS-DNA. Photo courtesy of author.
98
Figure 4-6. Photoluminescence of UCNP before ligand exchange and after thiol-ene
crosslinking with HS-PEG and HS-DNA.
Figure 4-7. Zeta-potential of UCNP after thiol-ene crosslinking with HS-PEG and HS-DNA.
400 500 600 700
Inte
nsit
y (
a.u
.)
Wavelength (nm)
UCNP
UCNP-PEG
UCNP-DNA
99
Figure 4-8. The fluorescence of UCNP-S-PEG (black) and UCNP-S-PEG-FITC (red) excited at 488 nm indicated a successful peglation on UCNP.
Figure 4-9. Stability test of UCNP thiol-ene click conjugation by agarose gel (left) and flow cytometry histograms of CEM (target) and Ramos (negative) cells incubated with aptamer and UCNP-S-Aptamer. Photo courtesy of author.
500 520 540 560 580 600
Inte
ns
ity
(a
.u.)
Wavenumber (nm)
UCNP-S-PEG
UCNP-S-PEG-FITC
100
Figure 4-10. Top: SDS-PAGE gel of UCNP-S-HRP, HRP and UCNP-HRP under UV-light and natural light; bottom: Enzymatic activity of UCNP-S-HRP via thiol-ene crosslinking. Photo courtesy of author.
101
CHAPTER 5 CONCLUSIONS
Surface Functionalization of Inorganic Colloidal Nanoparticles for Biochemical Applications
Inorganic colloidal nanoparticles have achieved great success over the last
decades. Various nanomaterials have been synthesized and wide applications have
been studied. Due to nanoscale size effect and unique physical and chemical
properties, nanomaterial conjugation with biomolecules provides even wider
applications than any of the component alone. For example, functional nucleic acid
conjugated nanomaterials offer enhanced properties, such as molecular recognition
ability of nanomaterials and nuclease digestion resistance. This doctoral research has
been focused on the surface functionalization of hydrophobic inorganic colloidal
nanoparticles for biochemical applications through a single-phase ligand exchange and
crosslinking strategy. Three major projects have been presented here: (1) Facile
surface functionalization of hydrophobic magnetic nanoparticles; (2) Ionic
functionalization of hydrophobic colloidal nanoparticles to form ionic nanoparticles with
enzymelike properties; (3) Thiol-ene click reaction based bioconjugation of hydrophobic
colloidal nanoparticles.
Facile and Efficient Surface Functionalization of Hydrophobic Magnetic Nanoparticles
Over the past decades, magnetic nanoparticles have been demonstrated wide
application in biochemical area such as bioanalytical detection, bioimaging, drug
delivery and cancer therapy. Magnetic nanoparticles synthesized in organic solvent at
high temperature gains advantages over that synthesized in aqueous phase. For
example, uniform and highly crystallized iron oxide nanoparticles can be obtained
102
without size sorting. The size of nanoparticles can be controlled by changing the
reaction temperature and reaction time. However, the application of magnetic
nanoparticles synthesized via thermalysis have been limited because of their poor
solubility in aqueous phase. Instead of using ligand encapsulation or two-phase ligand
exchange system, we developed a facile, efficient, and single-phase ligand exchange
strategy to transfer the hydrophobic magnetic nanoparticles to aqueous phase. Single-
phase environment significantly increased the ligand exchange process and efficiency.
By neutralize the nanoparticle surface functional group carboxylic acid with aqueous
sodium hydroxide, hydrophobic colloidal nanoparticles were not only transferred into
aqueous phase, but also maintained the carboxyl group function. Through EDC/NHS
coupling, the as-transferred aqueous magnetic nanoparticles were able to be modified
with small molecule dye, DNA aptamer, and natural enzyme. This facile surface
functionalization method enables hydrophobic magnetic nanoparticles further
biochemical applications such as bioimaging and targeted drug delivery and enzyme
immobilization.
Ionic Functionalization of Hydrophobic Colloidal Nanoparticles to Form Ionic Nanoparticles with Enzymelike Properties
Inorganic colloidal nanoparticles stabilized with a layer of hydrophobic surfactant
on their surface have limited their applications as biosensors in physiological conditions.
We have developed a facile surface functionalization method to transfer hydrophobic
magnetic nanoparticles to aqueous phase. However, various inorganic colloidal
nanoparticles which were synthesized in organic solvent at high temperature need facile
surface functionalization for further biosensor applications. In this work, we developed a
universal strategy to transfer different hydrophobic colloidal nanoparticles to aqueous
103
phase through ionization. Two pair replacement ligands which have stronger
nanoparticle binding affinity and can be ionized either by aqueous sodium hydroxide or
hydrochloric acid were used to transfer iron oxide, FePt, Pd, CdSe, and upconversion
nanoparticles to aqueous phase. For potential application as biosensors, those as
transferred aqueous colloidal nanoparticles were further studied to mimic hydrogen
peroxidase.
Enzyme-Linked ImmunoSorbent Assay (ELISA) has been widely used in
biochemical detection. Nanozyme (nanomaterials based artificial enzyme) has potential
to replace the horseradish peroxidase and overcome the limitation of hoseradish
peroxidase, such as poor stability in harsh environment (high temperature, extreme
acidic or basic pH). Nanozyme-linked immunosorbent assay offers opportunity to
develop reproducible high quality tool with maximal performance and achieve large
scale application and impact. Rational design of nanozymes allowing natural
horseradish peroxidase to be replace by nanozymes, which are cheaper and more
stable.
Thiol-ene Click Reaction-based Bioconjugation of Hydrophobic Colloidal Nanoparticles
Nanomaterials bioconjugation, combining nanomaterials and biomolecules,
enable nanomaterials wide applications in physiology research. In order crosslink
nanomaterials and biomolecules, such as dye, enzyme, and drug, various crosslinkers
have been developed. For example, EDC/NHS coupling can link carboxylic aicd and
amine groups to form a peptide. Maleimide can react with thiol to form a stable
thiolester. As a representative of click chemistry, azide-alkyne reaction has been widely
used in biomolecular labeling and cell surface modification. However, EDC/NHS
104
coupling usually has low crosslinking efficiency. The succinimide linkage of the
maleimide addition product is susceptible to hydrolysis, although the maleimide reaction
is rapid. Azide or alkyne functional groups are not available in native biomolecules. A
special introduction of these groups into proteins or DNA are necessary to take
advantage of azide-alkyne click reaction.
Click chemistry is a class of biocompatible reactions that join substrates to
biomolecules in a quick, selective, and high-yielding manner. In this project, we were
aim to take advantage of thiol-ene click chemistry to conjugate biomolecules including
small molecueles, DNA aptamer, and enzyme with nanomaterials to achieve large scale
biochemical applications of nanomaterials. By modifying the replacement ligand with
ene group, any thiol containing molecules can be conjugated with nanoparticles. A
cysteine selective conjugation with nanoparticles without changing the catalytic activity
was achieved through this thiol-ene click chemistry.
105
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BIOGRAPHICAL SKETCH
Yuan Liu was born in Bole, Xinjiang, China. He attended Harbin Institute of
Technology to study chemistry in 2007. Supervised by Dr. Gang Chen, his
undergraduate research focused on visible photocatalytic activities of nitrogen doped
In2TiO5 sensitized by carbon nitride. He won the 7th China Youth Science and
Technology Innovation Award. Then Yuan came to the United States in the fall of 2011
to work under the mentorship of Dr. Weihong Tan. He received his Doctor of Philosophy
degree in chemistry from the University of Florida in December of 2016.