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Nanomaterial Interfaces in Biology Methods and
Protocols 1st Edition Daniel F. Moyano Digital Instant
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Author(s): Daniel F. Moyano, Bradley Duncan, Vincent M. Rotello (auth.),
Paolo Bergese, Kimberly Hamad-Schifferli (eds.)
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Methods in
Molecular Biology 1025

Paolo Bergese
Kimberly Hamad-Schifferli
Editors

Nanomaterial
Interfaces in
Biology
Methods and Protocols
METHODS IN M O L E C U L A R B I O LO G Y ™

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School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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 Nanomaterial Interfaces
in Biology
Methods and Protocols

Edited by

Paolo Bergese
Dipartimento di Ingegneria Meccanicae Industriale, Università degli Studi di Brescia, Brescia, Italy

Kimberly Hamad-Schifferli
Department of Mechanical Engineering, Massachusetts Institute of Technology
Cambridge, MA, USA
Editors
Paolo Bergese Kimberly Hamad-Schifferli
Dipartimento di Ingegneria Meccanicae Department of Mechanical Engineering
Industriale Massachusetts Institute of Technology
Università degli Studi di Brescia Cambridge, MA, USA
Brescia, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-62703-461-6 ISBN 978-1-62703-462-3 (eBook)
DOI 10.1007/978-1-62703-462-3
Springer New York Heidelberg Dordrecht London

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Preface

The intersection of nanotechnology with biology has given rise to numerous ideas for new
ways to use nanotechnology for biological applications. Nanomaterials possess unique size-
and material-dependent properties, which make them attractive for improving regular bio-
medical fields, such as drug delivery, imaging, therapy, and diagnostics as well as next-level
developments, comprising activation/deactivation, mimicking, and implementation of bio-
molecular systems and functions. Consequently nanotechnology has held great potential
for novel and unique capabilities, and stirred the imagination of many scientists.
While nanotechnology and nanoscience has held great promise for revolutionizing
biology, it has been hindered by the fact that when nanomaterials are interfaced to biomol-
ecules or put into biological environments, many undesirable side effects result, such as
aggregation and nonspecific adsorption, which can compromise biological function or give
rise to negative biological responses. This can be largely attributed to a range of interface
and intermolecular interactions between the nanomaterials with the biomolecules as well as
the solvent that mediates their interactions. These interactions are difficult to control, pre-
dict, and prevent. Interface effects of inorganic surfaces have been a major issue historically,
manifesting as surface fouling of medical device implants and stents. Unfortunately, these
effects worsen or give rise to new and unexpected complications for nanoscale materials
because surface volume ratios are exponentially higher, and nanoscale surfaces have differ-
ent physical and chemical properties.
Therefore, despite the fact that we now have a high degree of control over the synthetic
properties of the nanomaterials, similar control over their interfaces to biology has yet to be
achieved. This is crucial as their biological interface ultimately determines their biological
identity and fate. In the last 10 years, the biological–nanomaterial interface has created
unprecedented challenges not only for finding useful ways to exploit nanomaterials in biol-
ogy but also in their unintentional consequences, such as environmental and toxicological
effects. For example surface fouling, nonspecific adsorption, or unexpected aggregation
and instability have prevented many of the exciting and early ideas of nanobiotechnology
from reaching fruition.
We believe a key pitfall that plagues this area is the difficulty in reproducing results,
where a huge amount of variability exists not only between different labs but also from day
to day in the same lab. This variability is almost never reported in peer-reviewed journal
papers, despite its criticality. Also, members of the nanotechnology community typically
come from chemistry, physics, and materials science, and historically are not accustomed to
providing stepwise protocols. Therefore, a handbook of detailed protocols and best prac-
tices for this field is critically needed.
While there have been some examples of individual protocols in protocol literature
such as Nature Methods, Current Protocols, Molecular Cloning, and on the National Cancer
Institute’s Nanotechnology Characterization Laboratory website, we believe that it is time
to provide a consolidated volume of step-by-step protocols in the tradition of Methods in
Molecular Biology. Methods in Molecular Biology has played a major role in providing
vi Preface

biological recipes and protocols, and in doing so has advanced biology in immeasurable
ways. By creating standards for everyone and making experimental techniques more acces-
sible, Methods in Molecular Biology has accelerated the discovery process. What used to be
difficult experimentally now can be done by nearly anyone. These new tools have benefited
the entire biology community, enabling huge leaps and bounds in scientific discovery and
applications.
Thus, we present this volume with the hopes of improving the utility of nanotechnol-
ogy as a tool to advance biological and medical sciences. While this volume is far from
comprehensive, we hope that it will serve the new and emerging community well, and
enable new capabilities and technologies that were not previously possible. The proposed
protocols predominantly deal with nanomaterials, covering many of the now classic sub-
jects, such as conjugation of nanoparticles to biomolecules and their applications in drug
delivery or imaging. Additionally, some chapters are dedicated to flat surfaces because most
of the methods for manipulating nanomaterials stem from those of flat surfaces, facilitated
by fabrication advances to shrink the dimensions of materials.
The volume is organized into three parts: (1) protocols describing synthesis, fabrica-
tion, and construction of bio-nanomaterial interfaces, (2) characterization protocols of bio-
nanomaterial interfaces, and (3) applications which utilize the bio-nanomaterial interfaces.
We would like to note that we are not including significant coverage of toxicology and the
unintended effects of nanomaterials. Due to the evolving scope of the toxicological studies
of nanomaterials, the current state of the art is still developing, and thus is difficult to cover
adequately in this volume of Methods in Molecular Biology. However, we hope that this col-
lection will aid this growing field by serving as a guide.
We gratefully acknowledge all of the authors contributing to this volume, as well as our
home institutions of the Department of Mechanical Engineering at MIT and the Department
of Mechanical and Industrial Engineering of Università degli Studi di Brescia. This work
was made possible by the UniBS-MIT-MechE faculty exchange program cosponsored by
the CARIPLO Foundation, Italy under grant 2008-2290.

Brescia, Italy Paolo Bergese

Cambridge, MA, USA Kimberly Hamad-Schifferli
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I MAKING BIO–NANO INTERFACES

1 Preparation of 2 nm Gold Nanoparticles for In Vitro
and In Vivo Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Daniel F. Moyano, Bradley Duncan, and Vincent M. Rotello
2 DNA Conjugation to Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Sunho Park
3 Conjugation of Nanoparticles to Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Marie-Eve Aubin-Tam
4 Water-Solubilization and Functionalization of Semiconductor
Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Christina M. Tyrakowski, Adela Isovic, and Preston T. Snee
5 Synthesizing and Modifying Peptides for Chemoselective Ligation
and Assembly into Quantum Dot-Peptide Bioconjugates. . . . . . . . . . . . . . . . . 47
W. Russ Algar, Juan B. Blanco-Canosa, Rachel L. Manthe, Kimihiro Susumu,
Michael H. Stewart, Philip E. Dawson, and Igor L. Medintz
6 Reliable Methods for Silica Coating of Au Nanoparticles . . . . . . . . . . . . . . . . . 75
Isabel Pastoriza-Santos and Luis M. Liz-Marzán
7 Surface Modifications by Polymers for Biomolecule Conjugation. . . . . . . . . . . 95
Laura Sola, Marina Cretich, Francesco Damin, and Marcella Chiari
8 Functionalization Protocols of Silicon Micro/Nano-mechanical Biosensors . . . 109
Francesca Frascella and Carlo Ricciardi

PART II CHARACTERIZING BIO–NANO INTERFACES

9 Stability and Aggregation Assays of Nanoparticles in Biological Media. . . . . . . 119
James Chen Yong Kah
10 Electrochemical Measurements of DNA Melting on Surfaces. . . . . . . . . . . . . . 127
Irina Belozerova, Dongbiao Ge, and Rastislav Levicky
11 Formation and Characterization of the Nanoparticle–Protein Corona . . . . . . . 137
Marco P. Monopoli, Andrzej S. Pitek, Iseult Lynch, and Kenneth A. Dawson
12 Electrophoretic Implementation of the Solution-Depletion Method
for Measuring Protein Adsorption, Adsorption Kinetics,
and Adsorption Competition Among Multiple Proteins in Solution. . . . . . . . . 157
Hyeran Noh, Naris Barnthip, Purnendu Parhi, and Erwin A. Vogler
viii Contents

13 Hyperspectral Microscopy for Characterization of Gold Nanoparticles

in Biological Media and Cells for Toxicity Assessment . . . . . . . . . . . . . . . . . . . 167
Christin Grabinski, John Schlager, and Saber Hussain
14 Immunocytochemistry, Electron Tomography, and Energy Dispersive X-ray
Spectroscopy (EDXS) on Cryosections of Human Cancer Cells Doped
with Stimuli Responsive Polymeric Nanogels Loaded with Iron
Oxide Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Roberto Marotta, A. Falqui, A. Curcio, A. Quarta, and Teresa Pellegrino

PART III IMPLEMENTING BIO–NANO INTERFACES

15 Zwitterion Siloxane to Passivate Silica Against Nonspecific
Protein Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Zaki G. Estephan and Joseph B. Schlenoff
16 Preparation and Characterization of DNA Block Copolymer Assemblies
Loaded with Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Xi-Jun Chen, Robert J. Hickey, and So-Jung Park
17 Polyaspartic Acid Coated Iron Oxide Nanoprobes for PET/MRI Imaging . . . 225
Taku Cowger and Jin Xie
18 Ligand Synthesis and Passivation for Silver and Large
Gold Nanoparticles for Single-Particle-Based Sensing and Spectroscopy . . . . . 237
Daniel Montiel, Emma V. Yates, Li Sun, Marissa M. Sampias, John Malona,
Erik J. Sorensen, and Haw Yang
19 Noncovalent Intracellular Drug Delivery of Hydrophobic Drugs on Au NPs. . . . . 251
Tennyson Doane and Clemens Burda
20 Modification of Carbon Nanotubes for Gene Delivery Vectors . . . . . . . . . . . . 261
Victor Ramos-Perez, Anna Cifuentes, Núria Coronas,
Ana de Pablo, and Salvador Borrós
21 Lipid-Based Nanoparticles as Nonviral Gene Delivery Vectors . . . . . . . . . . . . . 269
Daniele Pezzoli, Anna Kajaste-Rudnitski, Roberto Chiesa,
and Gabriele Candiani
22 Stabilizing Gold Nanoparticle Bioconjugates in Physiological Conditions
by PEGylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Joan Comenge and Víctor F. Puntes

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Contributors

W. RUSS ALGAR • Naval Research Laboratory, Washington, DC, USA

MARIE-EVE AUBIN-TAM • Delft University of Technology, Delft, The Netherlands
NARIS BARNTHIP • Division of Physics, Rajamangala University of Technology, Pathum
Thani, Thailand
IRINA BELOZEROVA • Polytechnic Institute of New York University, New York, NY, USA
JUAN B. BLANCO-CANOSA • Scripps Research Institute, La Jolla, CA, USA
SALVADOR BORRÓS • Institut Quimic de Sarria, Universitat Ramon Llull, Barcelona, Spain
CLEMENS BURDA • Case Western Reserve University, Cleveland, OH, USA
GABRIELE CANDIANI • INSTM (National Interuniversity Consortium of Materials Science
and Technology), Research Unit Milano Politecnico, Milan, Italy; Department of
Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano,
Milan, Italy
XI-JUN CHEN • Department of Chemistry, University of Pennsylvania,
Philadelphia, PA, USA
MARCELLA CHIARI • Consiglio Nazionale delle Ricerche, Istituto di Chimica del
Riconoscimento Molecolare, Milan, Italy
ROBERTO CHIESA • Department of Chemistry, Materials and Chemical Engineering
“Giulio Natta”, Politecnico di Milano, Milan, Italy
ANNA CIFUENTES • Institut Quimic de Sarria, Universitat Ramon Llull, Barcelona, Spain
JOAN COMENGE • Catalan Institute of Nanotechnology, Universitat Autònoma de
Barcelona, Barcelona, Spain
NÚRIA CORONAS • Institut Quimic de Sarria, Universitat Ramon Llull, Barcelona, Spain
TAKU COWGER • University of Georgia, Athens, GA, USA
MARINA CRETICH • Consiglio Nazionale delle Ricerche, Istituto di Chimica del
Riconoscimento Molecolare, Milan, Italy
A. CURCIO • Istituto Italiano di Tecnologia, Genoa, Italy
FRANCESCO DAMIN • Consiglio Nazionale delle Ricerche, Istituto di Chimica del
Riconoscimento Molecolare, Milan, Italy
KENNETH A. DAWSON • Centre for BioNano Interactions, School of Chemistry and Chemical
Biology, University College Dublin, Dublin, Ireland
PHILIP E. DAWSON • Scripps Research Institute, La Jolla, CA, USA
TENNYSON DOANE • Case Western Reserve University, Cleveland, OH, USA
BRADLEY DUNCAN • University of Massachusetts, Amherst, MA, USA
ZAKI G. ESTEPHAN • Florida State University, Tallahassee, FL, USA
A. FALQUI • Istituto Italiano di Tecnologia, Genoa, Italy
FRANCESCA FRASCELLA • Politecnico di Torino, Torino, Italy
DONGBIAO GE • Polytechnic Institute of New York University, New York, NY, USA
CHRISTIN GRABINSKI • Air Force Research Laboratory, Wright-Patterson Air Force Base,
Dayton, OH, USA
x Contributors

ROBERT J. HICKEY • Department of Chemistry, University of Pennsylvania, Philadelphia,

PA, USA
SABER HUSSAIN • Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton,
OH, USA
ADELA ISOVIC • University of Illinois at Chicago, Chicago, IL, USA
JAMES CHEN YONG KAH • National University Singapore, Singapore
ANNA KAJASTE-RUDNITSKI • Division of Regenerative Medicine, Stem Cells and Gene
Therapy, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific
Institute, Milan, Italy
RASTISLAV LEVICKY • Polytechnic Institute of New York University, New York, NY, USA
LUIS M. LIZ-MARZÁN • CIC biomaGUNE, Donostia - San, Sebastián, Spain
ISEULT LYNCH • Center for BioNano Interactions, School of Chemistry and Chemical
Biology, University College Dublin, Dublin, Ireland
JOHN MALONA • Princeton University, Princeton, NJ, USA
RACHEL L. MANTHE • Sotera Defense Solutions, University of Maryland,
College Park, MD, USA
ROBERTO MAROTTA • Istituto Italiano di Tecnologia, Genoa, Italy
IGOR L. MEDINTZ • U.S. Naval Research Laboratory, Center for Bio/Molecular Science and
Engineering, Washington, DC, USA
MARCO P. MONOPOLI • Center for BioNano Interactions, School of Chemistry and Chemical
Biology, University College Dublin, Dublin, Ireland
DANIEL MONTIEL • Princeton University, Princeton, NJ, USA
DANIEL F. MOYANO • University of Massachusetts, Amherst, MA, USA
HYERAN NOH • Department of Optometry and Vision Science, Seoul National University
and Technology, Seoul, South Korea
ANA dE PABLO • Institut Quimic de Sarria, Universitat Ramon Llull, Barcelona, Spain
PURNENDU PARHI • Department of Chemistry, Ravenshaw University, Cuttack, Orissa,
India
SO-JUNG PARK • Department of Chemistry, University of Pennsylvania,
Philadelphia, PA, USA
SUNHO PARK • Dankook University, Yongin-si, Gyeonggi-do, South Korea
ISABEL PASTORIZA-SANTOS • Departamento de Química Física, Campus Universitario,
Universidade de Vigo, Bilbao, Spain
TERESA PELLEGRINO • Istituto Italiano di Tecnologia, Genoa, Italy; National Nanotechnology
Laboratory of CNR-NANO, Lecce, Italy
DANIELE PEZZOLI • INSTM (National Interuniversity Consortium of Materials Science
and Technology), Research Unit Milano Politecnico, Milan, Italy
ANDRZEJ S. PITEK • Center for BioNano Interactions, School of Chemistry and Chemical
Biology, University College Dublin, Dublin, Ireland
VÍCTOR F. PUNTES • Catalan Institute of Nanotechnology, Universitat Autònoma de
Barcelona, Barcelona, Spain
A. QUARTA • Istituto Italiano di Tecnologia, Genoa, Italy; National Nanotechnology
Laboratory of CNR-NANO, Lecce, Italy
VICTOR RAMOS-PEREZ • Institut Quimic de Sarria, Universitat Ramon Llull,
Barcelona, Spain
CARLO RICCIARDI • Politecnico di Torino, Torino, Italy
VINCENT M. ROTELLO • University of Massachusetts, Amherst, MA, USA
MARISSA M. SAMPIAS • Princeton University, Princeton, NJ, USA
 Contributors xi

JOHN SCHLAGER • Air Force Research Laboratory, Wright-Patterson Air Force Base,
Dayton, OH, USA
JOSEPH B. SCHLENOFF • Department of Chemistry and Biochemistry, Florida State University,
Tallahassee, FL, USA
PRESTON T. SNEE • University of Illinois at Chicago, Chicago, IL, USA
LAURA SOLA • Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento
Molecolare, Milan, Italy
ERIK J. SORENSEN • Princeton University, Princeton, NJ, USA
MICHAEL H. STEWART • Division of Optical Sciences, U.S. Naval Research Laboratory,
Washington, DC, USA
LI SUN • Princeton University, Princeton, NJ, USA
KIMIHIRO SUSUMU • Naval Research Laboratory, Washington, DC, USA
CHRISTINA M. TYRAKOWSKI • University of Illinois at Chicago, Chicago, IL, USA
ERWIN A. VOGLER • Department of Materials Science and Engineering, The Pennsylvania State
University, University Park, PA, USA; Department of Bioengineering, The Pennsylvania
State University, University Park, PA, USA
JIN XIE • Department of Chemistry, University of Georgia, Athens, GA, USA
HAW YANG • Princeton University, Princeton, NJ, USA
EMMA V. YATES • Princeton University, Princeton, NJ, USA
 Part I

Making Bio–Nano Interfaces

 Chapter 1

Preparation of 2 nm Gold Nanoparticles for In Vitro

and In Vivo Applications
Daniel F. Moyano, Bradley Duncan, and Vincent M. Rotello

Abstract
Gold nanoparticles have been a versatile tool in recent years for the exploration of biological systems.
However, challenges with purification and adequate surface coverage limit the biocompatibility of gold
nanoparticles. Here, we describe a detailed procedure for the synthesis, purification, and functionalization
of biologically compatible gold nanoparticles for in vitro and in vivo studies.

Key words Gold nanoparticle, Biocompatibility, Purification

1 Introduction

Gold nanoparticle (AuNP) properties and applications continue to

be the focus of a variety of fields since their discovery in 1857 by
Michael Faraday [1]. Among these areas, avid research has been
pointed towards the creation of biocompatible nanoparticles [2].
The precise control introduced by the two-phase Brust–Schiffrin
method [3, 4] allowed more systematic studies of nanoparticle
properties to be performed. However, the use of tetra-n-
octylammonium bromide as the phase transfer catalyst limits bio-
logical applications due to the difficultly in separating this highly
cytotoxic chemical from the nanoparticles [5, 6]. Additionally, the
functionality of nanoparticle surfaces has been a prominent con-
cern due to the direct correlation of cytotoxicity with the function-
ality of the surface monolayer [7]. To further improve nanoparticle
utility, a fine balance between hydrophobic functionalization near
the metallic core to confer monolayer stability [8] and non-fouling
character provided by poly(ethylene glycol) moieties must be
achieved [9]. An excellent ligand base for biological applications is
23-mercapto-3,6,9,12-tetraoxatricosan-1-ol (Fig. 1) as its compo-
nents confer the desired stability and non-fouling properties [10].
This structure can be easily modified to provide selective binding

Paolo Bergese and Kimberly Hamad-Schifferli (eds.), Nanomaterial Interfaces in Biology: Methods and Protocols,
Methods in Molecular Biology, vol. 1025, DOI 10.1007/978-1-62703-462-3_1, © Springer Science+Business Media New York 2013

3
4 Daniel F. Moyano et al.

Fig. 1 Gold nanoparticle chemical structure. The ligand is composed of three parts, namely, the sulfur atom
(binding with gold), an alkyl chain (nanoparticle stability), and a tetraethylene glycol spacer (biocompatibility
and solubility)

making these nanoparticles a promising platform for systematic

biological studies [11]. Employing these concepts, this protocol
elaborates on the synthesis, purification, and functionalization of
gold nanoparticles with the biocompatibility required for in vivo
and in vitro studies.

2 Materials

1. Hydrogen tetrachloroaurate (III) hydrate.

2. Tetra-n-octylammonium bromide (TOAB).
3. 1-Pentanethiol.
4. Sodium borohydride.
5. 23-mercapto-3,6,9,12-tetraoxatricosan-1-ol, synthetized accord-
ing to the reported procedure [12].
6. 200 proof ethanol, acetone, hexanes, and acetonitrile.
7. 10,000 MWCO snakeskin pleated dialysis tubing membrane.
8. 3.0 cm Grade 1 filter paper circles.

3 Methods

All procedures are done at room temperature unless otherwise

specified.

3.1 Synthesis of Gold 1. Pour 1.0 g (2.5 mmol, 1 Eq) of hydrogen tetrachloroaurate
Nanoparticles (from (III) hydrate and 150 mL of Type I ultrapure water into a
the Brust–Schiffrin 1,000 mL round bottom flask. Stir slowly for 5 min using a
Method) [3] 2 in. egg-shaped magnetic stir bar until complete dissolution.
Solution should be clear yellow (see Note 1).
 Preparation of Biocompatible Gold Nanoparticles 5

2. Premix 2.1 g (3.8 mmol, 1.5 Eq) of tetra-n-octylammonium

bromide in 150 mL of toluene and add it to the gold solution
in the round bottom flask. Stir at maximum speed for 15 min
until the solution presents is a cloudy dark orange color
(see Note 2).
3. While continuing to rapidly stir, cover the round bottom flask
with a septum stopper and using a syringe add dropwise 0.7 mL
(5 mmol, 2 Eq) of 1-pentanethiol over the course of 15 min.
Stir until the solution presents a cloudy deep white color
(see Note 3).
4. Remove the stopper and quickly add a freshly prepared solu-
tion of 2.0 g (50 mmol, 20 Eq) of sodium borohydride in
10 mL of Type I ultrapure water. Stir the solution for 5 h
(see Note 4).
5. Using a separatory funnel, remove the water phase (colorless)
from the toluene solution (black). Evaporate the majority of
the solvent under reduced pressure at 40 °C (see Note 5).
6. Add 900 mL of 200 proof ethanol and mix until the solid is
completely dispersed. Store the flask at −20 °C.

3.2 Purification 1. Two days after the synthesis, the nanoparticles precipitate and
of Gold Nanoparticles a black solid is observed at the bottom of the flask. Carefully
remove the ethanol solution (brown color) without dispersing
the precipitate (see Note 6).
2. Add fresh 200 proof ethanol and mix the solution until the
nanoparticles are dispersed. Leave the solution at −20 °C until
precipitation is complete (see Note 7).
3. Repeat steps 1 and 2 until the ethanol solution is colorless
after precipitation (approximately five times) (see Note 8).
4. Remove the solvent and redisperse the precipitate in 10 mL of
200 proof ethanol. Sonicate the solution for 10 min.
5. Using a 3.0 cm grade 1 filter paper circle over a 25 mm filter
holder (No. 5 stopper) attached to a vacuum line, wash the
nanoparticles several times with 200 proof ethanol redispers-
ing the precipitate in each wash (see Note 8).
6. Recover the solid from the filter paper.
7. Dissolve the nanoparticles in a minimal amount of toluene
and analyze the pentanethiol-capped AuNPs using mass
spectrometry (LDI) paying particular attention for the pres-
ence of a peak at 466 m/z (TOAB). If the peak is present
repeat steps 5 and 6 until it is no longer observed (see Notes
9 and 10) [13].
8. Use transmission electron microscopy (TEM) to obtain the
average particle diameter and use nuclear magnetic resonance
(solvent—CDCl3) to confirm purity.
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 Politics - Exercise Book
Summer 2024 - Center

Prepared by: Instructor Miller

Date: July 28, 2025

Module 1: Ethical considerations and implications

Learning Objective 1: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 1: Diagram/Chart/Graph]
Learning Objective 2: Critical analysis and evaluation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Research findings and conclusions
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Learning Objective 4: Experimental procedures and results
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 4: Diagram/Chart/Graph]
Learning Objective 5: Problem-solving strategies and techniques
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Critical analysis and evaluation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 8: Current trends and future directions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Experimental procedures and results
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Module 2: Best practices and recommendations
Key Concept: Case studies and real-world applications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Historical development and evolution
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Learning outcomes and objectives
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 13: Diagram/Chart/Graph]
Note: Current trends and future directions
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Historical development and evolution
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Fundamental concepts and principles
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Comparative analysis and synthesis
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Historical development and evolution
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 19: Diagram/Chart/Graph]
Key Concept: Learning outcomes and objectives
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
References 3: Ethical considerations and implications
Remember: Statistical analysis and interpretation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Key terms and definitions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Experimental procedures and results
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Practical applications and examples
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Current trends and future directions
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 26: Diagram/Chart/Graph]
Note: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 27: Experimental procedures and results
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 28: Best practices and recommendations
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Key terms and definitions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 30: Diagram/Chart/Graph]
Results 4: Practical applications and examples
Practice Problem 30: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 31: Diagram/Chart/Graph]
Example 31: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 32: Diagram/Chart/Graph]
Note: Theoretical framework and methodology
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 33: Historical development and evolution
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 34: Ethical considerations and implications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Problem-solving strategies and techniques
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Assessment criteria and rubrics
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Fundamental concepts and principles
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 39: Diagram/Chart/Graph]
Important: Research findings and conclusions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Topic 5: Best practices and recommendations
Example 40: Fundamental concepts and principles
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 41: Ethical considerations and implications
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 42: Literature review and discussion
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Study tips and learning strategies
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 44: Diagram/Chart/Graph]
Key Concept: Statistical analysis and interpretation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 45: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Fundamental concepts and principles
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 47: Diagram/Chart/Graph]
Practice Problem 47: Statistical analysis and interpretation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Research findings and conclusions
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
References 6: Key terms and definitions
Definition: Fundamental concepts and principles
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
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