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Nanobiomaterials Development and Applications 1st
Edition Dong Kee Yi Digital Instant Download
Author(s): Dong Kee Yi, Georgia C. Papaefthymiou
ISBN(s): 9781439876428, 1439876428
Edition: 1
File Details: PDF, 87.48 MB
Year: 2013
Language: english
NANOBIOMATERIALS
DEVELOPMENT AND APPLICATIONS
Edited by

DONG KEE YI
GEORGIA C. PAPAEFTHYMIOU
NANOBIOMATERIALS
DEVELOPMENT AND APPLICATIONS
 Advances in Materials Science and Engineering

Series Editor
Sam Zhang

Aerospace Materials Handbook, edited by Sam Zhang and Dongliang Zhao

Biological and Biomedical Coatings Handbook: Applications, edited by Sam Zhang
Biological and Biomedical Coatings Handbook: Processing and Characterization,
edited by Sam Zhang
Hydroxyapatite Coatings for Biomedical Applications, edited by Sam Zhang
Nanobiomaterials: Development and Applications, Dong Kee Yi and
Georgia C. Papaefthymiou
Micro- and Macromechanical Properties of Materials, Yichun Zhou, Li Yang, and
Yongli Huang
NANOBIOMATERIALS
DEVELOPMENT AND APPLICATIONS
Edited by

DONG KEE YI
GEORGIA C. PAPAEFTHYMIOU

Boca Raton London New York

CRC Press is an imprint of the

Taylor & Francis Group, an informa business
CRC Press
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Contents
Series Preface............................................................................................................vii
Preface.......................................................................................................................ix
Editors..................................................................................................................... xiii
Contributors.............................................................................................................. xv

PART I Nanomaterials in Nanobiotechnologies:

Preparation, Characterization,
and Applications

Chapter 1 Bio-Inspired Magnetic Nanoparticles...................................................3

Georgia C. Papaefthymiou and Eamonn Devlin

Chapter 2 Nanoparticles for Bioimaging............................................................. 43

Hye Sun Park and Yong Taik Lim

Chapter 3 Biomedical Applications of Dendrimer Porphyrin

or Phthalocyanine................................................................................ 81
Woo-Dong Jang and Won-Gun Koh

Chapter 4 Polymeric Nanoparticles in Cancer Therapy.................................... 109

Heebeom Koo, Ji Young Yhee, Ick Chan Kwon,
Kwangmeyung Kim, and Ramesh Subbiah

Chapter 5 Carbon Nanotube Bioconjugates....................................................... 151

Monica Samal, Dong Kee Yi, and Shashadhar Samal

Chapter 6 Biocatalytic Nanosystems................................................................. 225

Jaehong Lim and Su Seong Lee

Chapter 7 Magnetically Induced Hyperthermia for Biomedical Applications...... 261

Michael Fardis, Ioannis Rabias, Georgios Diamantopoulos,
Eleni Karakosta, Danai Tsitrouli, Vassilios Tzitzios, and
Georgios Papavassiliou

v
© 2010 Taylor & Francis Group, LLC
vi Contents

PART II Soft Block Nanobuilding:

New Preparation Routes of Soft
Nanomaterials Using Biomolecules

Chapter 8 Engineered Biomolecules as Nanomaterials..................................... 287

Yun Jung Lee and Ki Tae Nam

PART III Nanomaterials and Bio-MEMS: Nano-

and Microscale Hybridization of
Materials and Applications

Chapter 9 Microfluidic-Based Polymer Scaffold Design for Tissue

Engineering....................................................................................... 341
Mohana Marimuthu and Sanghyo Kim

Chapter 10 Fabrication of Mobile Hybrid Microswimmers Using

Micro/Nanoparticles and Bacterial Flagella..................................... 361
U. Kei Cheang and Min Jun Kim

PART IV Nanotoxicity Studies and Applications

in Eco-Biosystems

Chapter 11 Environmental Applications of Nanomaterials................................. 385

Jaesang Lee, Byoung Chan Kim, Hun Je Cho, and Changha Lee

Chapter 12 Cytotoxicity of Biosynthesized Nanomaterials

and Functionalized Nanomaterials: Use in Therapy......................... 417
Murugan Veerapandian, Kyusik Yun, Ramesh Subbiah, and
Min-Ho Lee

© 2010 Taylor & Francis Group, LLC

Series Preface
ADVANCES IN MATERIALS SCIENCE AND ENGINEERING
Series Statement
Materials form the foundation of technologies that govern our everyday life, from
housing and household appliances to handheld phones, drug delivery systems,
­airplanes, and satellites. Development of new and increasingly tailored materials
is key to further advance important applications with the potential to dramatically
enhance and enrich our experiences.
The Advances in Materials Science and Engineering series by CRC Press/
Taylor & Francis Group is designed to help meet new and exciting challenges in
materials science and engineering disciplines.
The books and monographs in the series are based on cutting edge research and
development, and thus are up to date with new discoveries, new understanding,
and new insights in all aspects of materials development, including processing and
characterization and applications in metallurgy, bulk or surface engineering, inter-
faces, thin films, coatings, and composites, just to name a few.
The series aims at delivering an authoritative information source to readers in
academia, research institutes, and industry. The publisher and its series editor are
fully aware of the importance of materials science and engineering as the foundation
for many other disciplines of knowledge. As such, the team is committed to making
this series the most comprehensive and accurate literary source to serve the whole
materials world and the associated fields.
As series editor, I thank all authors and editors of the books in this series for their
noble contributions to the advancement of materials science and engineering and to
the advancement of humankind.

Sam Zhang

vii
© 2010 Taylor & Francis Group, LLC
Preface
Nanobiotechnology is a recently activated term describing the convergence of
­molecular biology and engineering. The combination of these disciplines over
the last decade has realized a new class of smart devices or systems for biological
and chemical analysis defined by improved specificity and sensitivity as well as
higher rates of molecular recognition compared with previous solutions. Advances
have been made in nanobiochip materials, biomimetic materials, nanocomposite
­materials, interface biomaterials, photocatalytic materials, nanomotors, nanobio-
sensors, and nano drug delivery systems, with enormous prospect in industrial,
defense, and medical applications of great societal impact. Such technological
advances are the direct outcome of the continuous exchange of ideas, which is
­taking place across the border between the biological and physical sciences in
many areas of nanoscience. This interdisciplinary exchange is based on the premise
that nanotechnology offers biology new tools, while biology offers nanotechnology
new types of functional materials. The ability to observe intracellular structures
with high selectivity and follow their dynamic behavior is the greatest challenge of
biology to nanoscience that drives the nanotechnological development of a variety
of bioimaging probes. Understanding the rotatory flagellar motor of bacteria and
adapting it to biotechnological needs is another challenge. Nanotechnology makes
use of biomimetic or bio-inspired processes for the production of nanosized materi-
als for applications not only in biology but also in a variety of seemingly unrelated
fields including nanoelectronics. On the other hand, the fruits of nanotechnology
are increasingly being applied to expanding areas of biomedical and therapeutic
processes. Nanotechnology is affording biomedicine new nanostructures and scaf-
folds for tissue engineering, nanoparticle probes for magnetic resonance imaging
(MRI), targeted drug delivery and hyperthermia cancer therapy, photonic band
structures, new optical imaging probes, and carbon nanotube–based technologi-
cal advances for drug and gene ­delivery, to name a few. Thus, nanobiotechnology
­constitutes “a bridge between nano and bio,” with nanoscale materials providing the
building blocks for the construction of the “bridge.”
The present volume of the Advances in Materials Science and Engineering series
provides an update on developments, and new findings and applications on many of
the topics mentioned above that comprise active areas of multidisciplinary bionano-
materials research. It presents 12 chapters contributed by an international team of
investigators, who are actively engaged in the forefront of research in their respec-
tive disciplines. These 12 chapters fall under 4 broad themes, and thus the book is
internally organized into 4 parts: Part I, “Nanomaterials in Nanobiotechnologies:
Preparation, Characterization, and Applications”; Part II, “Soft Block
Nanobuilding: New Preparation Routes of Soft Nanomaterials Using Biomolecules”;
Part III, “Nanomaterials and Bio-MEMS: Nano- and Microscale Hybridization of
Materials and Applications”; and Part IV, “Nanotoxicity Studies and Applications
in Eco-Biosystems.” Potential readers who will benefit from this volume include

ix
© 2010 Taylor & Francis Group, LLC
x Preface

advanced undergraduate and graduate students engaged in ­ nanobiomaterials

research, ­ industrial researchers, pharmaceutical innovators, m ­ edical and public
health personnel as well as environmental scientists and engineers. The reader will
come away with a better appreciation of the importance of this i­nterdisciplinary
field to biotechnology, biomedicine, and environmental remediation—all subjects
of importance to society at large. A brief description of the contents of each chapter
follows.
In Part I, Chapter 1, “Bio-Inspired Magnetic Nanoparticles,” reviews the
­biomineralization of iron in biological processes and the biomimetic synthesis of
magnetic nanoparticles within protein cages and viral capsids, block copolymers,
microemulsions, and silica shells, as well as the magnetic characterization of the
thus-derived magnetic nanosystems. Chapter 2, “Nanoparticles for Bioimaging,”
introduces semiconductor nanocrystals or quantum dots (QDs) and their photonic
properties and reviews their synthesis, surface modification, and applications in cell
labeling and targeting for medical applications in cancer diagnosis and therapy as
well as other novel applications. Chapter 3, “Biomedical Applications of Dendrimer
Porphyrin or Phthalocyanine,” discusses the design of dendritic materials and their
use in photodynamic therapy, the development of dendritic photosensitizer-loaded
micelles for photochemical delivery and as drug carriers, as well as diagnostic appli-
cations of the dendrimer porphyrin. Chapter 4, “Polymeric Nanoparticles in Cancer
Therapy,” focuses on the use of natural and synthetic polymer-based nanoparticles for
cancer therapy. Chapter 5, “Carbon Nanotube Bioconjugates,” presents an in-depth
discussion of the rapidly advancing area of carbon nanotube bioconjugation, reactiv-
ity, and functionalization for applications in cell imaging, p­ harmacokinetics, protein
and antigen transfection, nucleic acid delivery, tumor targeting, and cancer therapy.
Chapter 6, “Biocatalytic Nanosystems,” addresses the subject of enzyme immobili-
zation using nanomaterials, nanoparticle and nanoporous s­upports, novel nanosys-
tems for biocatalysis, nanoentrapment, single-enzyme nanoparticles, and biocatalytic
nanosystems for biosensing. Chapter 7, “Magnetically Induced Hyperthermia for
Biomedical Applications,” presents a lucid discussion of the physics of heat dissipa-
tion mechanisms of magnetic ferrofluids and magnetic hyperthermia instrumentation
for in vitro and in vivo studies in animal models.
In Part II, Chapter 8, “Engineered Biomolecules as Nanomaterials,” discusses
the manufacturing of functional nanomaterials using rod-shaped filamentous and
spherical or nearly spherical viruses.
In Part III, Chapter 9, “Microfluidic-Based Polymer Scaffold Design for Tissue
Engineering,” presents the design of polymeric scaffolds, microfluidic cell ­culture
systems, and bioreactors for tissue engineering. Chapter 10, “Fabrication of
­
Mobile Hybrid Microswimmers Using Micro/Nanoparticles and Bacterial Flagella,”
gives a fascinating discussion of biomimetic device fabrication and bio-inspired
­engineering and l­ ocomotion, using micro- and nano-sized particles and bacterial flagella.
In Part IV, Chapter 11, “Environmental Applications of Nanomaterials,” intro-
duces the area of nanotechnology for environmental remediation and decon-
tamination using nanomaterials and environmental biosensors, and discusses
the environmental impact of nanomaterials in terms of toxicity and impact
on ecosystems. Chapter 12, “Cytotoxicity of Biosynthesized Nanomaterials

© 2010 Taylor & Francis Group, LLC

Preface xi

and Functionalized Nanomaterials: Use in Therapy,” discusses the intra- and

­extracellular biosynthesis of nanoparticles by bacteria and their functionalization,
­cytotoxicity mechanisms, and potential use in therapy.
We thank all the authors and coauthors of these chapters for their valuable
­contributions to this volume and for their patience and dedication during all stages
of the preparation and production of this book. We especially thank Dr. Sam Zhang,
the editor of the Advances in Materials Science and Engineering series, for inviting
and encouraging us to edit the present volume on nanoscale materials applications in
bionanotechnologies. Last, but not least, special thanks are due to the staff at CRC
Press for their professional support, patience, and dedication in all phases of the
preparation of the book.

Dong Kee Yi
Department of Bionanotechnology
Gachon University
Sungnam City, Republic of Korea

Georgia C. Papaefthymiou
Department of Physics
Villanova University
Villanova, Pennsylvania

© 2010 Taylor & Francis Group, LLC

Contributors
U. Kei Cheang Eleni Karakosta
Department of Mechanical Institute of Advanced Materials,
Engineering & Mechanics Physicochemical Processes,
Drexel University Nanotechnology &
Philadelphia, Pennsylvania Microsystems
National Centre for Scientific Research
Hun Je Cho “Demokritos”
Water Environmental Center Attiki, Greece
Korea Institute of Science and
Technology Byoung Chan Kim
Seoul, Republic of Korea Water Environmental Center
Korea Institute of Science and
Eamonn Devlin Technology
Institute of Advanced Materials, Seoul, Republic of Korea
Physicochemical Processes,
Nanotechnology & Microsystems Kwangmeyung Kim
National Centre for Scientific Research Biomedical Research Institute
“Demokritos” Korea Institute of Science and
Attiki, Greece Technology
Seoul, Republic of Korea
Georgios Diamantopoulos
Institute of Advanced Materials, Min Jun Kim
Physicochemical Processes, Department of Mechanical
Nanotechnology & Microsystems Engineering & Mechanics
National Centre for Scientific Research and
“Demokritos” School of Biomedical Engineering,
Attiki, Greece Science & Health Systems
Drexel University
Michael Fardis Philadelphia, Pennsylvania
Institute of Advanced Materials,
Physicochemical Processes, Sanghyo Kim
Nanotechnology & Microsystems Department of Bionanotechnology
National Centre for Scientific Research Gachon University
“Demokritos” Gyeonggi-do, Republic of Korea
Attiki, Greece
Won-Gun Koh
Woo-Dong Jang Department of Chemical and
Department of Chemistry Biomolecular Engineering
Yonsei University Yonsei University Seoul
Seoul, Republic of Korea Seoul, Republic of Korea

xiii
© 2010 Taylor & Francis Group, LLC
xiv Contributors

Heebeom Koo Jaehong Lim

Biomedical Research Institute Institute of Bioengineering and
Korea Institute of Science and Nanotechnology
Technology The Nanos
Seoul, Republic of Korea Singapore

Ick Chan Kwon Yong Taik Lim

Biomedical Research Institute Department of Analytical Science
Korea Institute of Science and and Technology
Technology Chungnam National University
Seoul, Republic of Korea Daejeon, Republic of Korea

Changha Lee Mohana Marimuthu

School of Urban and Environmental Department of Bionanotechnology
Engineering Gachon University
Ulsan National Institute of Science and Gyeonggi-do, Republic of Korea
Technology
Ulsan, Republic of Korea Ki Tae Nam
Department of Materials Science
Jaesang Lee and Engineering
Water Environmental Center Seoul National University
Korea Institute of Science and Seoul, Republic of Korea
Technology
Seoul, Republic of Korea Georgia C. Papaefthymiou
Department of Physics
Min-Ho Lee Villanova University
Medical IT Technology Villanova, Pennsylvania
Korea Electronics Technology
Institute Georgios Papavassiliou
Seongnam, Republic of Korea Institute of Advanced Materials,
Physicochemical Processes,
Su Seong Lee Nanotechnology & Microsystems
Institute of Bioengineering and National Centre for Scientific Research
Nanotechnology “Demokritos”
The Nanos Attiki, Greece
Singapore
Hye Sun Park
Yun Jung Lee Department of Analytical Science and
Department of Energy Engineering Technology
Hanyang University Chungnam National University
Seoul, Republic of Korea Daejeon, Republic of Korea

© 2010 Taylor & Francis Group, LLC

Contributors xv

Ioannis Rabias Vassilios Tzitzios

Institute of Advanced Materials, Institute of Advanced
Physicochemical Processes, Materials, Physicochemical
Nanotechnology & Microsystems Processes, Nanotechnology &
National Centre for Scientific Research Microsystems
“Demokritos” National Centre for Scientific
Attiki, Greece Research “Demokritos”
Attiki, Greece
Monica Samal
Department of Bionanotechnology Murugan Veerapandian
Gachon University Department of Bionanotechnology
Seoul, Republic of Korea Gachon University
Gyeonggi-do, Republic of Korea
Shashadhar Samal
Department of Chemistry Ji Young Yhee
S.B.R. Govt. (Autonomous) Women’s Biomedical Research Institute
College Korea Institute of Science
Berhampur, India and Technology
Seoul, Republic of Korea
Ramesh Subbiah
University of Science Dong Kee Yi
and Technology Department of Bionanotechnology
Daejeon, Republic of Korea Gachon University
and Seoul, Republic of Korea
Center for Biomaterials
and Kyusik Yun
Nanomaterials Center Department of Bionanotechnology
Korea Institute of Science Gachon University
and Technology Gyeonggi-do, Republic of Korea
Seoul, Republic of Korea

Danai Tsitrouli
Institute of Advanced Materials,
Physicochemical Processes,
Nanotechnology & Microsystems
National Centre for Scientific Research
“Demokritos”
Attiki, Greece

© 2010 Taylor & Francis Group, LLC

Editors
Dong Kee Yi received his BS in 1996 from the Seoul National University, MS
in 1998 from POSTECH, and PhD in materials science and engineering in 2003
from Gwangju Institute of Science and Technology (GIST), Korea. He was a post-
doctoral fellow at Brown University and at the Institute of Bioengineering and
Nanotechnology in Singapore from 2003 to 2005. He worked as a senior scientist in
Samsung Advanced Institute of Technology from 2005 to 2007. Since 2007, he has
been working as a faculty member at the Department of Bionanotechnology, Gachon
University, Korea.
Professor Yi serves on the editorial board of ISRN Nanotechnology and writes
reviews for journals from leading scientific societies including American Chemical
Society, Royal Society of Chemistry, and American Institute of Physics. He has
been involved in the fields of nanomaterial preparation and application, ­bioimaging,
and colloid science for the past 12 years. He has authored and coauthored more
than 60 peer-reviewed international journal articles and worked as an inventor for
32 ­international patents.
Professor Yi works on research/technology evaluation and advisory panels for the
governments of China, Korea, and Romania. He has also worked as a consultant to
industrial organizations in Korea. He was featured in the first edition of Who’s Who
in Asia and Who’s Who of Emerging Leaders (2007). Since 2004, he has been fea-
tured in Who’s Who in the World and 2000 Outstanding Intellectuals of the Twenty-
First Century.

Georgia C. Papaefthymiou, a graduate of Columbia University in New York,

is a professor of physics at Villanova University, Villanova, Pennsylvania, and is
a visiting research professor at the National Centre for Scientific Research (NCSR)
Demokritos, Athens, Greece. She is the recipient of a Marie Curie Chair of
Excellence award from the European Union with the Institute of Materials Science
of Demokritos and a CAPES award from the Ministry of Education of Brazil with
the Brazilian Center for Research in Physics in Rio de Janeiro. Her research interests
are in condensed matter physics encompassing areas in (1) cluster science and the
transition from molecular to bulk ­behavior with increasing cluster size; (2) nanoscale
magnetism and f­ undamental studies and applications to nanotechnology and nano-
medicine; (3) multiferroic materials; and (4) iron-containing proteins. She is the
author or coauthor of over 150 publications that include peer-reviewed articles, book
chapters, reviews, conference proceedings, and opinion pieces. She is on the board
of editors of the Journal of Materials; peer reviewer for multiple physics, chemistry,
and materials j­ournals; and grant reviewer for the National Science Foundation and
the Department of Energy of the United States and for Aristeia of Greece.

xvii
© 2010 Taylor & Francis Group, LLC
Part I
Nanomaterials in
Nanobiotechnologies:
Preparation, Characterization,
and Applications

© 2010 Taylor & Francis Group, LLC

 1 Bio-Inspired Magnetic
Nanoparticles
Georgia C. Papaefthymiou
Villanova University

Eamonn Devlin
National Center of Scientific Research “Demokritos”

CONTENTS
1.1 Introduction....................................................................................................... 3
1.2 Biomineralization of Iron in Biological Processes............................................ 4
1.2.1 Ferrihydrite Grown within Apoferritin.................................................4
1.2.1.1 The Challenge of Nanoparticle Nucleation and Growth........5
1.2.1.2 Particle Nucleation and Growth in the Ferritins..................... 6
1.2.2 Magnetite Grown within Membrane Vesicles.......................................8
1.3 Biomimetic Magnetic Nanoparticles............................................................... 10
1.3.1 Ferrite Nanoparticles Grown within Block Copolymer
Nanoreactors.......................................................................................11
1.3.2 Preparation of Magnetic Nanoparticles Using Microemulsions......... 13
1.3.3 Magnetic Nanoparticles Grown within Protein Cages........................ 15
1.3.4 Magnetic Nanoparticles Grown within Viral Capsids........................20
1.3.5 Maghemite Core/Silica Shell Nanoarchitectures................................ 22
1.4 Magnetic Characterization of Iron-Based Magnetic Nanoparticles................24
1.4.1 Isolated Magnetic Nanoparticles.........................................................26
1.4.2 Interacting Magnetic Nanoparticles.................................................... 31
1.5 Conclusion....................................................................................................... 36
Acknowledgments..................................................................................................... 37
References................................................................................................................. 37

1.1 INTRODUCTION
Materials science explores the relationship between the structure and properties
of materials. Borrowing techniques and practices from solid-state chemistry and
­physics for the synthesis and characterization of materials, it has developed as a
separate scientific area. Originally, it had no affinity with biology, but the advent and
integration of nanoscience and nanotechnology into materials science has changed
this dramatically [1,2]. Nanotechnology deals with the manipulation of matter at the
atomic and molecular levels for the bottom-up synthesis of materials. The synthesis

3
© 2010 Taylor & Francis Group, LLC
4 Nanobiomaterials: Development and Applications

of supramolecular structures in nature is the archetypical example of such processes.

Today materials science draws inspiration from biology and strives to reproduce
or mimic biological processes in the synthesis of materials. In the specific case of
magnetic nanoparticles, the subject of this chapter, it is the ubiquitous process of
biomineralization that has been particularly inspiring [3,4].
Through the process of biomineralization nature integrates hard inorganic ­materials
with the soft organic world of biology. Living organisms can provide chemical envi-
ronments that control the nucleation and growth of unique mineral phases, forming
exceptional nanocomposite materials, such as bone or seashell. Bone is the archetypi-
cal biological nanocomposite, consisting of hard apatite, a form of calcium phosphate,
and soft organic tissue, the protein collagen—the nanoscale plate-like crystals of apa-
tite being dispersed within the collagen fiber matrix [5,6]. The outstanding mechanical
properties of bone do not derive simply from those of the component materials, but
depend on how they are arranged and how this arrangement is altered under stress. For
example, bone is twice as strong in compression as in tension or torsion. This is due to
the fact that collagen does not act as glue, but more like a net in which the apatite par-
ticles are trapped and allowed to move against each other. In the biomineralization of
bone, end-use functionality dictates the formation of a nanostructured material, as the
mechanical properties of bone cannot be reproduced by bulk apatite, that is, its nano-
composite structure is essential to its function. There exists a growing interest in the
processes by which such complex and intricate hierarchical biomineral architectures
can be reproduced in the laboratory for the production of functional materials, without
the harsh high-temperature and high-pressure requirements of solid-state chemistry.
In the case of magnetite biomineralization of magnetotactic bacteria, the ­formation
of a chain of single-domain magnetic particles (MPs) within membrane vesicles is
essential for the construction of a biomagnetic compass [7,8]. Nano-ferrihydrite
biomineralization in the ferritins derives from the metabolic need for iron [9–12].
Time and again biological examples show that nature produces unique structures
with end-use functionality in mind. This is also the goal of materials science today.
In this chapter we explore bio-inspired routes to iron-based magnetic nanoparticle
formation. To appreciate this rapidly growing field, we must first review some gen-
eral basic principles encountered in the biogenesis of magnetic nanoparticles.

1.2 BIOMINERALIZATION OF IRON IN BIOLOGICAL PROCESSES

Biomineralization of iron in biological processes is encountered in two major areas:
the biomineralization of ferrihydrite in the ferritins for iron metabolic purposes
and the biomineralization of magnetite in magnetosomes for magnetoreception and
navigation in microbial and higher living organisms.

1.2.1 Ferrihydrite Grown within Apoferritin

The availability of iron for metabolic purposes requires a mechanism for the efficient
and reversible storage of iron. Nature has solved this problem by forming a dense
solid particle within the ferritin molecule, shown in Figure 1.1, where a large number
of Fe atoms are stored [9–12].

© 2010 Taylor & Francis Group, LLC

Bio-Inspired Magnetic Nanoparticles 5

Iron stored
as mineral
inside ferritin

FIGURE 1.1 Ribbon representation of the ferritin molecule. A polymeric shell consisting of
24 polypeptide subunits encapsulates a particle of biomineralized ferrihydrite. Shell dimen-
sions: inner diameter 7 nm; outer diameter 12 nm.

Ferritins represent a family of proteins that are widespread in biological ­systems.

Although there are differences among them, their overall similarities are more
important than their differences. They are all large multicomponent proteins that
self-assemble to form molecular cages, within which a hydrated ferric oxide is
­mineralized. The mammalian ferritin cage, also known as apoferritin, is a hetero-
polymer consisting of 24 amino acid chains of two types: H (heavy) and L (light).
It has an inner diameter of ~7 nm and an outer diameter of ~12 nm. In the inte-
rior cavity of the protein cage it forms a micellar core of hydrated iron(III) oxide
(­ferrihydrite), with various amounts of phosphate inclusions. It was first described by
V. Laufberger in 1937 [13] as a protein isolated from horse spleen, which contains
about 20% iron. The nanocomposite state of ferrihydrite in ferritin affords a high
surface-area-to-volume ratio, that is, an increased proportion of surface iron sites
enhancing the iron storage and release processes. The formation of ferritin is a clas-
sic example of nature’s response to the challenging problem of the nucleation and
growth of nanoparticles for the efficient packing of iron atoms.
1.2.1.1 The Challenge of Nanoparticle Nucleation and Growth
In a homogeneous medium, particle nucleation is generally hindered due to the
­energetically demanding process of interface formation at the boundary of the nucleated
phase, the nascent particle surface. The nucleation energy barrier can be overcome kinet-
ically in supersaturated solutions where encounters or collisions between atoms or mol-
ecules are frequent. One must consider the change in Gibbs free energy, ΔG, that results
from nucleation [14], as in all types of phase transitions. Bonding is energetically favored,
as particle formation liberates ΔGv of energy per unit volume. However, the creation of an
interfacial boundary consumes ΔGs free energy per unit interfacial area. For the forma-
tion of a spherical cluster of radius r, the overall change in Gibbs free energy is given by
4p 3
DG = - r DGv + 4 pr 2DGs . (1.1)
3

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6 Nanobiomaterials: Development and Applications

Interfacial energy

Gibbs free energy (ΔG)

rthermo
rkin
Particle radius

Volume free energy

FIGURE 1.2 Particle nucleation and growth: change in volume, surface, and total Gibbs
free energy, ΔG, as a function of particle radius. The kinetic and thermodynamic critical radii
are indicated.

The first term gives the energy gained in creating the volume of the particle, thus this
is the term that drives nucleation, while the second term gives the energy lost due to
surface tension at the interface, and thus is the term that hinders nucleation. Figure 1.2
gives a sketch of the dependence of various terms on r.
For very small particles, ΔG is positive, due to the high surface-area-to-volume
ratio, making nucleation thermodynamically unfavorable. The graph of ΔG as a
function of r reaches a maximum when d ( DG ) dr = 0. The maximum occurs at
2DGs
rkin = , (1.2)
DGv
which defines the kinetic critical radius for nucleation. For r > rkin , further growth of
the nucleated cluster is kinetically favored as ΔG decreases with increasing particle
size. The radius rthermo above which ΔG becomes negative defines the thermodynamic
critical radius. The particle, once nucleated, is thermodynamically driven to continu-
ous growth and the formation of bulk material. Surface passivation with terminal
ligation is needed to arrest the growth at the nanoscale.
In heterogeneous nucleation, the positive energy associated with surface tension at
phase boundaries is greatly reduced since the presence of nucleation sites eliminates
the particle/solution interface in the process of particle nucleation. Thus, e­ nergetic
considerations for heterogeneous nucleation are less demanding, and nucleation can
be more readily initiated.

1.2.1.2 Particle Nucleation and Growth in the Ferritins

Ferritin provides (1) the nucleation sites for iron polymerization, (2) the means to
promote supersaturation conditions within the confined space of the protein cage,
and (3) the terminal ligation needed to stabilize the nanoparticles. The molecular
properties of the amino acid cage are designed to induce ferrihydrite formation in

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Bio-Inspired Magnetic Nanoparticles 7

the interior of the protein rather than in the bulk solution. This is achieved by the
­presence of negatively charged glutamic acid residues, the nucleation sites, which
line the interior wall of apoferritin with COO− ions that attract Fe2+ ions and lead to
double-layer formation with an increased density of iron ions at the interior protein
surface [15]. The molecular cage also functions as a catalyst for the oxidation of
Fe2+ → Fe3+ at a binuclear iron-binding site known as the ferroxidase center [16],
using O2 or H2O2 as the oxidant [17], and induces the hydrolytic polymerization of
iron to form the ferrihydrite core, according to reactions as follows:

2Fe 2+ + O2 + 4H 2O Æ 2Fe(O)OH( core ) + H 2O2 + 4H + , (1.3)

2Fe 2+ + H 2O2 + 2H 2O Æ 2Fe(O)OH( core ) + 4H + , (1.4)

4Fe 2+ + O2 + 6H 2O Æ 4Fe(O)OH( core ) + 8H + . (1.5)

X-ray crystal structure determination of ferritin by Pauline Harrison and coworkers in

1991 [18] indicated that between amino acid chains or subunits there are small pores,
or channels, of about 0.3 nm diameter, through which ions and small molecules can
travel. These channels play a crucial role in ferritin’s ability to uptake and release iron
in a controlled fashion. They are of two types: fourfold channels are formed at the
intersection of four subunits, while threefold channels are formed at the intersection of
three subunits. The two types of channels have different properties, and thus perform
different functions. The walls of the threefold channels are lined with charged, polar
(hydrophilic) amino acids such as aspartate and glutamate, while the walls of a four-
fold channel are lined with nonpolar (hydrophobic) amino acids, such as alanine and
leucine. The polarity of the threefold channels facilitates interaction with the Fe2+ ions.
In contrast, the nonpolar nature of the fourfold channels cannot facilitate ion passage
between the interior and the exterior of the protein. It is believed that the function of
the fourfold channels is the transport of electrons into and out of the cavity, allowing
oxidation or reduction of iron to take place on the mineral core surface.
Ferrihydrite, or hydrous ferric oxide, can be precipitated directly from o­ xygenated
iron-rich aqueous solutions as a fine-grained, defective nanomaterial with varying
degrees of crystallinity. Its powder x-ray diffraction (XRD) pattern can change from
two broad scattered bands, in its most disordered state, to a maximum of six strong
lines, in its most crystalline state [19], as shown in Figure 1.3. The six-line form
corresponds to the nominal chemical formula FeOOH•0.4H2O; however, the exact
structure is fundamentally indeterminate as the water content is variable.
In native ferritin, the iron is stored as ferrihydrite with the inclusion of various
amounts of phosphate with the general chemical formula [FeO(OH)]8[FeO(H2PO4)].
One of the most readily (commercially) available source of ferritin for experimenta-
tion is that which is extracted from equine spleen, or horse spleen ferritin (HoSF).
Figure 1.4 shows a transmission electron microscopy (TEM) micrograph of an
in vivo produced HoSF sample. The dark spots correspond to the electron-rich iron
ferritin core with a diameter of ~7 nm, consistent with the internal size of the protein
shell, which maintains the hydrous iron oxide particles isolated from each other,

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8 Nanobiomaterials: Development and Applications

2.51 Å

2.25 Å 1.481 Å

1.98 Å 1.73 Å
3.3–3.2 Å
4.5 Å 2.59 Å
(a)

1.49 Å

(b)

15 23 31 39 47 55 63 2θ°

FIGURE 1.3 Six-line (a) and two-line (b) ferrihydrite XRD patterns, CuKα radiation.
(Reproduced with kind permission of the Mineralogical Society of Great Britain and Ireland from
the paper by Drits, V. A., et al., Structural model for ferrihydrite, Clay Miner., 28, 185–207, 1993.)

40 nm

FIGURE 1.4 TEM micrograph of native in vivo produced HoSF ferritin. (Sample purchased
from Sigma-Aldrich.)

preventing coagulation. The in vivo produced biomineral core may be removed

from the protein to obtain apoferritin, allowing subsequent in vitro reconstitution
of the core under controlled laboratory conditions in the absence of phosphates.
Through this process, “reconstituted ferritins” with differing degrees of iron loading
can be prepared. The XRD spectra of the derived cores indicate the formation of
­ferrihydrite, with the degree of crystallinity increasing with particle size [20].

1.2.2 Magnetite Grown within Membrane Vesicles

Magnetite is the most common of the known iron oxide biominerals. Biologically
precipitated magnetite was first identified by H. Lowenstam in 1962 [21] in the
denticle capping of chitons (primitive marine mollusks) in which magnetite
­

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Bio-Inspired Magnetic Nanoparticles 9

precipitation and tooth formation [22] proceed through the biochemically ­controlled
reduction of ferrihydrite. The mineral hardens the major lateral teeth of chitons,
enabling them to scrape surface and embedded algae from hard marine rocks
for food. Unlike ferrihydrite, which is a common product of both biological and
inorganic processes, magnetite is naturally formed inorganically only at elevated
temperatures and pressures in igneous and metamorphic rocks. Yet, organisms
ranging from bacteria to vertebrates are capable of forming magnetite under ambi-
ent c­ onditions. By natural selection, the chitons have found a route to biochemically
mediate the transformation of ferrihydrite to magnetite at atmospheric temperatures
and ­pressures. Biological magnetite deposits have also been identified with another
entirely different biological function, “magnetoreception,” which is the ability of
living organisms to sense the polarity and inclination of the Earth’s magnetic field
[23]. Some bacteria, honey bees, homing pigeons, and migratory fish are known to
possess this sense.
Magnetotactic bacteria [7,8] synthesize membrane-enclosed intracellular crys-
talline MPs, called magnetosomes, comprised primarily of iron oxides or, in rare
cases, iron sulfides. Magnetosomes are nanometer-sized, magnetic mineral crystal
deposits enveloped by a stable membrane that contains some lipids and proteins,
often referred to as the membrane vesicle. They are aligned to form chains within
the bacterium, creating a biomagnetic compass that enables the bacterium to orient
in the Earth’s magnetic field, a phenomenon known as “magnetotaxis.” These are
microaerobic bacteria, endowed with flagella that allow them to swim and migrate
along oxygen gradients in aquatic environments. They were first reported in 1975
by the microbiologist Richard P. Blakemore [24]. Studies have revealed that mag-
netic bacteria tightly control the synthesis of their own magnetite, mediated by the
magnetosome membrane that has a distinct biochemical composition and contains
specific magnetosome membrane proteins (MMPs) [25,26]. Since 1975, a variety
of strains have been found to exist in marine and freshwater habitats. Figure 1.5
shows TEM micrographs of magnetosomes and bacteria representing three dif-
ferent strains of typical magnetotactic bacteria. Note that magnetosomes can be
isolated from bacteria with intact magnetosome membranes surrounding the MPs,
as indicated in Figure 1.5c, where the magnetosome membranes, indicated by the
arrow, are clearly visible.
Detailed studies of the crystal structure of the magnetite particles within the mag-
netosomes indicate that they contain highly crystalline magnetite nanoparticles of
cubo-octahedral shape, yielding superior magnetic properties. Overall, magnetosome
crystals have high chemical purity, narrow size distribution, and species-specific mor-
phologies, unattainable in inorganically precipitated magnetite. These features point
to magnetosome formation under strict biological control [25], a ­process known as
“biologically controlled mineralization.”
The archetypical core/shell architectural model of the ferritin molecule and bacte-
rial magnetosomes is presently widely being used in materials science in bio-inspired
routes for the formation and application of magnetic nanoparticles. Recent efforts
focus on the use of protein cages and viral capsids as space-confined reaction ves-
icles for the formation of magnetic nanoparticles, but various other polymeric and
inorganic shells are also being explored in core/shell nanoarchitectures. In the case of

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10 Nanobiomaterials: Development and Applications

(a) (b)

0.25 μm
0.5 μm

MM
(c)

100 nm

FIGURE 1.5 TEM micrographs of typical magnetotactic bacteria and ­magnetosomes.

(a) Magnetotactic spirillum (MV-4) with a flagellum at each end of the cell and a chain of
­electron-dense, magnetite-containing magnetosomes along the long axis of the cell. (From
Bazylinski, D. A. and R. B. Frankel, Magnetosome formation in prokaryotes, Nat. Rev.
Microbiol., 2004. 2: pp. 217–230. Reprinted with permission from Nature Publishing Group
Nature Reviews Microbiology, copyright 2004.) (b) TEM micrograph of thin-sectioned
­magnetic cells of Magnetotactic spirillum (MS-1). The chains of crystals within the cell are
clearly visible. (From Frankel, R. B., et al., Magnetite in freshwater magnetotactic ­bacteria,
Science, 1979. 203: pp. 1355–1356. Reprinted with permission of AAAS.) (c) Isolated
­magnetosomes from Magnetospirillum gryphiswaldense. Arrow indicates the ­magnetosome
membrane (MM). (From Schüler, D., Molecular analysis of a subcellular compartment:
The magnetosome membrane in Magnetospirillum gryphiswaldense, Arch. Microbiol., 2004.
181: pp. 1–7. With kind permission from Springer Science+Business Media.)

magnetic nanoparticles, the shell carries out the additional function of steric isolation
of the magnetic nanoparticles to minimize magnetic interactions between particles
and thus prevent particle agglomeration, an important issue in ferrofluid preparations
for biomedical applications [27].

1.3 BIOMIMETIC MAGNETIC NANOPARTICLES

Broadly, we define “biomimetic,” as any synthesis procedure in which hard inor-
ganic nanoparticles are incorporated within soft polymeric matrices, surfactants, or
polypeptide cages, in order to address the critical issues of size confinement, surface
terminal ligation, and dispersion. We then expand our definition of “biomimetic”
to include other biocompatible core/shell magnetic nanoparticles such as ­those
­encapsulated in silica.

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Bio-Inspired Magnetic Nanoparticles 11

1.3.1 
Ferrite Nanoparticles Grown within
Block Copolymer Nanoreactors
The rich magnetic behavior of spinel ferrites stems from the presence of two
­magnetic sublattices that originate from the magnetic interaction between cations,
with magnetic moments situated in tetrahedral and octahedral coordination sites,
strongly exchange-coupled to produce ferrimagnetic ground states. Theoretically,
they can be obtained from magnetite Fe3O4 by the substitution of Fe2+ by another
transition metal. Solid solutions of the form TxFe3–xO4 (where T = Cu2+, Co2+, Mg2+,
Zn2+, Ni2+, etc.) can be prepared [28,29]. Such substitutions into the parent structure
of Fe3O4 modify the incipient magnetic exchange interactions between and within
sublattices and, therefore, the overall magnetic behavior of the system. Microphase
separation of diblock copolymers results in heterogeneous structures with lamel-
lar, cylindrical, or spherical microdomains, as shown in Figure 1.6 [30,31]. These
microdomains can serve as nanoreactors, or space-constrained reaction vesicles,
analogous to protein cages and membrane vesicles, for the nucleation and growth of
magnetic nanoparticles.
Figure 1.7 shows a schematic diagram of CoFe2O4 nanoparticle formation by
self-assembly within a diblock copolymer matrix consisting of polynorbornene
­
(NOR) and polynorbornene-dicarboxylic acid (NORCOOH), with a repeat unit ratio
of 400/50, synthesized using ring-opening metathesis polymerization (ROMP) [32].
FeCl3 and CoCl2 were used as precursors (polymer:FeCl3:CoCl2 = 1:25.0:12.5 mol).
The metal salts were introduced while the polymer was in solution, before the micro-
phase separation of the two blocks could occur. Rapid diffusion and attachment
of the metal salts to the polymer, due to their high affinity for the COOH groups,
resulted in the dispersion of the metal salts within the copolymer matrix [28].
A solid film was subsequently formed via static casting over a period of 3 days.
The films were then washed with NaOH and H2O. The FeCl3 and CoCl2 salts reacted
with NaOH and water within the NORCOOH nanoreactors. As a result, CoFe2O4
nanocrystals were formed within the self-assembled NORCOOH nanospheres of the
diblock copolymer matrix. Figure 1.8 shows the TEM micrograph of the resulting

0%–21% 21%–34% 34%–38% 38%–50%

Increasing volume fraction of minority component

FIGURE 1.6 Microphase separation of diblock copolymers into heterogeneous structures

with lamellar, cylindrical, or spherical microdomains.

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12 Nanobiomaterials: Development and Applications

Cl−
Fe3+
Microphase
COOH FeCl3 COO− + separation
H 2+
Co
CoCl2 Film formation
COOH COO− +
H
Cl−

Block copolymer solution in THF

Block copolymer matrix

Fe3+
−
Cl−
COO COO− Co
2+ NaOH
H+ H+ Cl−

H2O
COOH Fe3+ COONa
CoFe2O4 CoFe(OH)4 COONa −
COOH COONa COONa OH Co2+
COOH COONa COONa
OH−

FIGURE 1.7 Schematic of formation of CoFe2O4 nanoparticles within the diblock c­ opolymer.
(Reproduced from Papaefthymiou, G. C., et al., Magnetic and structural characterization of
CoFe2O4 nanoparticles encapsulated within block copolymer films, Rev. Adv. Mater. Sci., 10,
306–313, 2005, with kind permission of Advanced Study Center Co. Ltd.)

30 nm

FIGURE 1.8 Morphology of block copolymer films: highly dispersed CoFe2O4 nanopar-
ticles, oval in shape and of average diameter d of 9.6 ± 2.8 nm, are observed. (Reprinted
with permission from Ahmed, S. R., et al., Magnetic properties of CoFe2O4 nanoparticles
synthesized through a block co-polymer nanoreactor route, Appl. Phys. Lett., 2002. 80(9):
pp. 1616–1618. Copyright 2002, American Institute of Physics.)

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Bio-Inspired Magnetic Nanoparticles 13

cobalt ferrite nanoparticles [28]. The particles are well separated by the ­intervening
polymer, but they are not of uniform size or shape, with an effective average ­diameter
of 9.6 nm and a standard deviation of ±2.8 nm.

1.3.2 Preparation of Magnetic Nanoparticles Using Microemulsions

Emulsions are mixtures of two immiscible or unblendable liquids, such as polar
and nonpolar solvents, usually referred to as “water and oil” mixtures, depicted
in Figure 1.9. Such mixtures form colloidal systems in which both the dispersed
and continuous phases are liquid. For the emulsion to be formed, rigorous shak-
ing of the mixture is necessary (Figure 1.9b). Due to high interfacial tension, the
colloidal phase is unstable, and, over time, phase separation occurs, as depicted
in Figure 1.9c. If surfactants are added to the mixture, the surfactant molecules
­self-assemble at the interface between the two immiscible phases, where they intro-
duce a degree of continuity, reducing the interfacial tension and, thus, stabilizing
the colloid (Figure 1.9d).
Microemulsions are clear, stable, isotropic liquid mixtures of oil, water, and
surfactant. In contrast to ordinary emulsions, due to the presence of surfactants,
microemulsions form upon simple mixing of the components and do not require
the high shear conditions generally used in the formation of ordinary emulsions.
The two basic types of microemulsions are (1) direct, oil dispersed in water (o/w),
and (2) reversed, water dispersed in oil (w/o), determined by the relative amounts of
the two phases present in the mixture. Figure 1.10 depicts the interface structure in

Oil

Water

(a) (b)

(c) (d)

FIGURE 1.9 (a) Mixture of two immiscible liquids, (b) formation of emulsion through
rigorous shaking, (c) subsequent phase separation after cessation of shaking, and (d) stable
microemulsion in the presence of surfactants indicated by the solid circle drawn at the inter-
face of the two phases.

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14 Nanobiomaterials: Development and Applications

Oil
Water
Hydrophobic tail
Hydrophilic head

Water Oil

Hydrophobic tail
Hydrophilic head

(a) (b)

FIGURE 1.10 Depiction of interface characteristics in (a) reversed and (b) direct
microemulsions.

a water–oil–surfactant mixture in a microemulsion. It is oil in water or water in oil

droplets stabilized by just a monolayer of surfactant. Thus, even in small quantities,
surfactants markedly affect the surface characteristics of the dispersed phase.
Relative to bulk solutions, microemulsions can be used to add extra control
over chemical reactions. Chemical reactions to produce magnetic nanoparticles
can be carried out within the microscopic pools of water confined in reversed
­microemulsions. Both metallic and metal oxide nanoparticles have been prepared
through this method. The uniform nanoreactors impose kinetic and hydrodynamic
constraints on particle formation, limit particle size, and lead to the production of
monodispersed nanoparticles, in ways that mimic the protein cage of ferritin. Due
to Brownian motion the droplets undergo frequent collisions, during which they
have the ability to exchange the content of their water pools. Afterward, the droplets
separate back into independent droplets of the same size. This makes it possible for
chemical reactions to take place between the compounds solubilized in the different
droplets. In addition, chemical reactants may be exchanged between the exterior and
interior of the droplets, similarly to ferritin and lipid bilayer function in biological
cells. Metal magnetic nanoparticles can be formed by mixing two reversed micro-
emulsions with the same water content, one containing the metal salt to be reduced
and the other the reducing agent, such that the reduction reaction takes place exclu-
sively within the droplets as they exchange solute molecules.
As an example, we mention the synthesis of cobalt magnetic nanoparticles in
reversed microemulsions done by Pileni and coworkers [33]. They mixed two Na(AOT)
[sodium bis(2-ethylhexyl) sulfosuccinate] microemulsion solutions of similar water
content. The first contained Co(AOT)2 (derived from CoCl2) within its water pool,
while the second contained the reducing agent NaBH4 (sodium b­ orohydride) within
its water pool. The mixing resulted in the formation of colloidal particles of m­ etallic
cobalt. Figure 1.11 shows the TEM patterns obtained and the h­ istogram of the diam-
eter size distribution. Spherical nanoparticles, well separated from each other by the
enclosed surfactant layer, are seen. The particles spontaneously form hexago­nally
packed 2D networks, or superlattices, due to their narrow size distribution. The sur-
factant coating mimics the protein shell of ferritin in ­constraining the ­particles to

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Bio-Inspired Magnetic Nanoparticles 15

(a)

21 nm
30
(b)
25
Frequency (%)

20

15

10

0
0.5 2.1 3.6 5.2 6.8 8.3 9.9 11.5
Diameter (nm)

FIGURE 1.11 Monodispersed Co nanoparticles produced in a reversed m ­ icroemulsion s­ olution:

(a) TEM micrograph and (b) particle size distribution, d = (5.8 ± 0.5) nm. (Reprinted with per-
mission from Petit, C., et al., Cobalt nanosized particles organized in a 2D ­superlattice: Synthesis,
characterization, and magnetic properties, J. Phys. Chem. B, 1999, 103: pp. 1805–1810. Copyright
1999 American Chemical Society.)

a well-defined size and providing terminal ligation to passivate the ­surface. Assuming
a log-normal distribution, analysis of the histogram in Figure 1.11b indicates ­particles
of mean diameter d = (5.8 ± 0.5) nm.
While microemulsions are obtained from three-component mixtures of water,
organic solvents, and surfactants, two-component mixtures of just water and sur-
factants result in the formation of surfactant aggregates, or micelles, with the polar
heads pointing outward toward the water and the hydrophobic tails inward. Mixtures
of organic solvents and surfactants also produce surfactant aggregates or inverse
micelles. In many synthetic processes involving magnetic nanoparticles, surfactants
are simply added to the reaction mixture in order to provide surface passivation, bio-
compatibility, and steric separation of the encapsulated nanoparticles. The resulting
core/shell structures replicate those of ferritin, in the sense that an inorganic solid
particle is encapsulated within an organic coat. Thus, such structures are generally
considered to be biomimetic.

1.3.3 Magnetic Nanoparticles Grown within Protein Cages

Under reducing conditions and in the presence of iron chelators, ferritin can be
depleted of its ferrihydrite biomineral core and reconstituted under conditions favor-
ing the synthesis of magnetic iron oxide phases, magnetite or maghemite. In 1992,

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16 Nanobiomaterials: Development and Applications

I II

Native ferritin Apoferritin Magnetoferritin

FIGURE 1.12 Schematic depicting the synthesis route for the formation of ­magnetoferritin.
(See text.) (From Meldrum, F. C., et al., Magnetoferritin: In vitro synthesis of a novel m
­ agnetic
protein, Science, 1992. 257: pp. 522–523. Reprinted with permission of AAAS.)

Stephen Mann and coworkers [34] demonstrated for the first time the synthesis of
such ferrimagnetic phases within ferritin via this process, schematically depicted in
Figure 1.12.
In their experiment, the iron was removed from native HoSF by dialysis, under
a nitrogen atmosphere, against thioglycolic acid (HSCH2COOH) in sodium ace-
tate (CH3COONa) buffer at pH 4.5 (step I in Figure 1.12). The resulting apoferritin
­solution was buffered at pH 8.5 and maintained at a temperature of 55–60°C under
argon in a water bath. Fe2+ solution, prepared by the dissolution of ferrous ammo-
nium sulfate, (NH4)2Fe(SO4)2•6H2O in deaerated water, was added slowly in small
increments along with small amounts of air to produce slow oxidation (step II in
Figure 1.12). The data confirmed that the protein cage remained intact and that the
MPs were formed within the protein shell [34].
This seminal experiment established the use of apoferritin as a confined reaction
vesicle, or nanoreactor, for the production of iron oxide nanoparticles. The process
takes advantage of the unusual stability of the apoferritin shell at elevated tempera-
tures (60°C) and pH (8.5) needed for the synthesis of magnetite, as ­low-temperature
methods failed to produce magnetite in the presence of the protein shell. It is believed
that magnetite production within the ferritin cage proceeds in an analogous way to that
of ferrihydrite, that is, through the controlled oxidation of Fe2+ ions at the ­ferroxidase
center and subsequent migration and nucleation at the COO−-lined ­interior surface
of the protein cage. Just as in the case of ferritin biomineralization in vivo, this
­process is presumably favored over the competing reaction in bulk s­ olution because
of the catalytic oxidation of Fe2+ by ferritin and the increased ­iron-ion ­concentration
within the protein interior cavity. This differentiation between “inside” and “outside”
is essential to the effective in vivo functioning of the protein and is also central to
the ferritin nanotemplating approach for the synthesis of nanophase materials within
the apoferritin shell.
The synthesis of magnetoferritin is now well established, having been repro-
duced by other investigators at various degrees of iron loading, and its magnetic
properties have been widely studied [35]. Fitting of superparamagnetic data for
ferritin and magnetoferritin to the Langevin function indicates roughly a core with
a magnetic moment of μc ~ 300 μB for ferritin [36], as opposed to μc ~ 13,100 μB
for magnetoferritin, corresponding to a magnetoferritin particle core containing

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Bio-Inspired Magnetic Nanoparticles 17

of the order of 12,000 Fe atoms [35]. The large difference in magnetic moment
is due primarily to the fact that the ferrihydrite core is antiferromagnetic, with
the particle moment arising only from noncompensated spins at its surface. In
contrast, the core of magnetoferritin comprises magnetite and/or maghemite, both
structures being ferrimagnetic with noncompensated spin sublattices throughout
the particle volume.
To date, many other magnetic phases have been synthesized within a­ poferritin,
further supporting the notion that the reactions are not specific to iron and that
the electrostatic properties of the protein play a significant role in the process of
mineralization. In addition to metal oxide and hydroxide phases [35], metallic
­
­magnetic nanoparticles of Ni and Co have been produced within horse spleen apofer-
ritin [37], as indicated schematically in Figure 1.13. Once the Ni2+ and Co2+ ions have
entered the protein cavity, they can react with NaBH4, which is small enough to pass
through the threefold channels and enter the apoferritin interior. Thus, the divalent
metal ions, attached to the interior wall of the apoferritin cage, undergo reduction to
zero valence to produce metallic magnetic nanoparticles. Metal–alloy nanoparticles
have also been produced within apoferritin, such as FePt or CoPt [38] as well as
binary phase Fe/Co oxides [39].
Mammalian ferritin belongs to a “ferritin superfamily” of similar but distinct
protein cages occurring in bacterial life forms that are capable of sequestering
iron. These afford additional supramolecular templates for bio-inspired materials
synthesis. Among these, Dps [deoxyribonucleic acid (DNA)-binding proteins from
starved cells] proteins have been extensively investigated and used in the biomi-
metic synthesis of magnetic nanoparticles. Dps proteins possess a shell structure of
12 identical amino acid subunits, as opposed to the 24 heteropolymer subunits of
the canonical ferritins. They are smaller than mammalian ferritins possessing an

Apoferritin II
(0) M

(1) (2)

MII—Apoferritin M0—Apoferritin

FIGURE 1.13 Schematic representation of apoferritin-encapsulated Ni and Co metal

nanoparticles: MII = Ni2+ or Co2+, M0 = Co or Ni. (0) Addition of MII, (1) Dialysis and
­chromatography. (2) Addition of NaBH4. (From Galvez, N., et al., Apoferritin-encapsulated Ni
and Co superparamagnetic nanoparticles, J. Mat. Chem., 2006, 16: pp. 2757–2761. Copyright
2006 The Royal Society of Chemistry, reproduced with permission.)

© 2010 Taylor & Francis Group, LLC

18 Nanobiomaterials: Development and Applications

Exterior surface Interior cavity

8 nm

12 nm

(a)

5 nm

9 nm

(b)

FIGURE 1.14 Ribbon representation of the exterior surface and the interior cavity of
HuHF (a) and Dps protein from Listeria innocua (b). (Reprinted from Biochim. Biophys.
Acta, 1800, Uchida, M., et al., The ferritin superfamily: Supramolecular templates for
­materials ­synthesis, 834–845, Copyright 2010, with permission from Elsevier.)

exterior diameter of ~9 nm and a central cavity with diameter of ~5 nm. Figure 1.14
gives a ribbon representation of in vitro produced h­ omopolymer, recombinant
human apoferritin consisting of 24 H-chains (HuHF) and that of Dps protein [40].
Dps proteins were first identified in the bacterium Escherichia coli in 1992, and
since their discovery homologous structures have been found in other bacteria and
archaea.
The 3D crystal structure of Dps proteins exhibits two types of threefold sym-
metry channels, one of which is lined with hydrophilic amino acids that can
facilitate the entrance of cations to the interior cavity of the protein. As in the
case of mammalian ferritin, once the iron ions are inside the cavity, they are elec-
trostatically attracted to the negatively charged surface of the interior wall where
the mineralization reactions can be initiated. The electrostatic surface of Listeria
innocua Dps is similar to that of canonical ferritins, with clusters of glutamic
acid residues lining the surface with negatively charged COO− ions that facilitate
iron cluster nucleation. The oxyhydroxide mineral core formed within the Dps
cavity can accommodate up to 500 Fe atoms, as opposed to 4500 Fe atoms in
mammalian ferritin. Investigators have used the Dps cage to form mineralized
γ-Fe2O3 nanoparticles. Treatment of the protein at pH 8.5 and 65°C with 400 Fe2+

© 2010 Taylor & Francis Group, LLC

Bio-Inspired Magnetic Nanoparticles 19

40 nm 40 nm

(a) (b)

FIGURE 1.15 TEM micrograph of the Listeria innocua Dps cage mineralized with
γ-Fe2O3 stained with uranyl acetate (a) and unstained (b). (From Allen, M., et al. Protein
cage constrained synthesis of ferrimagnetic iron oxide nanoparticles. Adv. Mater., 2002,
14, 1562–1565. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with
permission.)

per protein and stoichiometric amounts of H 2O2 resulted in the formation of a

homogeneous solution with deep brown coloration. TEM micrographs of nega-
tively stained proteins with uranyl acetate, shown in Figure 1.15a, indicate that
the mineralized iron oxide phase is contained within the L. innocua Dps cage.
The iron oxide mineral particle diameter of 4.1 ± 1.1 nm for the unstained pro-
teins, shown in Figure 1.15b, correlates well with the inner diameter of the Dps
protein cage [41].
Furthermore, the reaction of Co2+, instead of Fe2+, with H2O2 at pH 8.5 at an
elevated temperature of 65°C results in the formation of Co3O4 within the Dps pro-
tein cage, while the same reaction carried out at 23°C forms Co(O)OH within the
cage, indicating that the temperature at which the reaction is performed can influ-
ence the nature of the mineralized phase within the protein [41]. As the formation of
many inorganic phases requires reactions that take place at elevated temperatures,
often higher than most proteins can tolerate, investigators have sought to isolate
protein templates from thermophilic (heat-loving) and hyperthermophilic bacte-
rial and archaeal organisms found in hot springs, in acidic soils, and near volcano
vents. Such organisms were first discovered in the 1960s by Thomas Brock and have
successfully expanded the temperature range of biomimetic magnetic nanoparticle
­synthesis. For example, a ferritin-like protein has been isolated from the anaerobic
marine bacterium Pyrococcus furiosus [42], which lives in thermal springs where
temperatures can reach 120°C. The structure of this protein is homologous to other
ferritins and the synthesis of maghemite within this ferritin cage has been demon-
strated [42]. Its wide temperature stability opens up the possibility of biomimetic
synthesis at temperatures unattainable with other biomolecular templates. As the
number of protein cages isolated from hyperthermophilic organisms increases, so
too will the variety and size of magnetic nanoparticles that can be synthesized using
biomimetic approaches.

© 2010 Taylor & Francis Group, LLC

20 Nanobiomaterials: Development and Applications

1.3.4 Magnetic Nanoparticles Grown within Viral Capsids

In addition to the ferritins, nature provides us with a large selection of protein
cages in the form of viral capsids, which can also function as nanoreactors for
the biomimetic self-assembly of nanomaterials. Increasingly, genetic engineering
techniques are being applied to the design of mutant protein cages, with elec-
trostatic properties conducive to the self-assembly of magnetic nanomaterials.
Figure 1.16 gives examples of space-filling images of viral capsids and protein
and enzyme cages [43]. These include capsids of both plant and bacterial viruses.
Non-viral protein cages include the canonical ferritins, Dps proteins, heat shock
proteins (Hsps), and the enzyme lumazine synthase. In their biological functions,
these architectures serve diverse roles from nucleic acid storage and transport in
viruses, chaperon function to prevent protein misfolding and denaturation in Hsps,
iron sequestration in the ferritins, and enzymatic action in lumazine ­synthase.
However, they all share the same architectural principle in being assembled from
a limited number of polypeptide subunits to form robust nanocages; they also
share the ability to have their functionality altered through both chemical and
genetic means.

(a) (b) (c)

(h)
(e) (f ) (g)
15 nm
(d)

FIGURE 1.16 Space-filling images of protein cage architectures with various (exterior)
diameters, d: (a) Cowpea mosaic virus (d = 31 nm); (b) Brome mosaic virus (d = 28 nm);
(c) Cowpea chlorotic mottle virus (d = 28 nm); (d) MS2 bacteriophage (d = 27 nm); (e) luma-
zine synthase (d = 15 nm); (f) ferritin (d = 12 nm); (g) small heat shock protein (d = 12 nm);
(h) Dps protein (d = 9 nm). Mosaic viruses are plant viruses that make the leaves of infected
plants acquire a specked appearance, while the MS2 bacteriophage is a virus that infects the
bacterium Escherichia coli. (From Uchida, M., et al. Biological containers: Protein cages as
multifunctional platforms. Adv. Mater., 2007, 19, 1025–1042. Copyright Wiley-VCH Verlag
GmbH & Co. KGaA. Reproduced with permission.)

© 2010 Taylor & Francis Group, LLC

Bio-Inspired Magnetic Nanoparticles 21

The discovery of viruses dates back to 1892, with Dmitri Ivanovsky’s description
of a nonbacterial pathogen infecting tobacco plants, leading to the discovery of the
tobacco mosaic virus in 1898 by Martinus Beijerinck. Since then, over 5000 viruses
have been described in detail; they are the most abundant type of biological entity.
The viral capsids, once depleted of their genomic content, can be engineered to pos-
sess negatively charged interior walls, and thus electrostatically attract Fe2+ ions and
induce the hydrolytic polymerization of iron within their interior cavities in a way
analogous to that of the ferritin protein cage.
Viral capsids are presently among the most extensively studied biological
templates in the biomimetic synthesis of inorganic nanoparticles. Due to their
well-defined architectures and readily accessible genetic information, they afford
unlimited possibilities for structural modification via genetic engineering. Their
interior space is normally reserved for their genome, where in their native state
the viral capsids act as host containers for nucleic acid storage and transport.
However, purified viral coat protein subunits can be easily assembled in vitro into
empty virions even in the absence of RNA or DNA, and can therefore be utilized
as constrained reaction vesicles. In 1998, investigators Douglas and Young [44]
pioneered the approach of using viral capsids for the synthesis of size-constrained
inorganic and organic polymer species. In their experiments, they used Cowpea
chlorotic mottle virus (CCMV) as a model system for reversibly gated entrapment
of inorganic minerals. CCMV is an icosahedral virus composed of 180 identical
subunits forming a protein cage with an inner diameter of 18 nm and an outer
diameter of 28 nm (Figure 1.16a). This affords a cavity diameter approximately
twice as large as that of ferritin, defining a new upper limit for the crystal size
of entrapped minerals. The exact size of the capsid depends on the pH of the
medium, as it undergoes a pH-dependent reversible swelling behavior. The capsid
increases its dimensions by about 10% compared to the non-swollen form when
the pH of the medium is raised to ≥6.5. In its swollen (open) state, the CCMV
­capsid provides 60 open pores of about 2-nm diameter each that allow small
external guest molecules to diffuse into the internal space [44]. Such dynamic
structural transitions induced by chemical switches are common in many virions
providing unique molecular gating mechanisms to control the containment and
release of entrapped materials—a property important in the design of targeted
drug d­ elivery agents in bionanotechnology.
X-ray structural analysis indicates that the basic arginine and lysine amino acids
line the interior wall of the CCMV capsid, providing a large positive charge. The
positive charge is necessary for the native virus to package and condense the anionic
viral genome. This is opposite to the case of ferritin where the interior wall is lined
with acidic glutamic acid, which provides a negative charge to the interior protein
cage wall. Thus, the native capsid exhibits an electrostatic attraction for negatively
charged species in the bulk solution, unlike ferritin. Indeed, the first material crys-
tallized within the viral capsid was accomplished by the electrostatic attraction
and subsequent oligomerization of aqueous molecular tungstate ( WO42-) ions to
form macromolecular complexes of paratungstate (H 2W12O42 10-
). Young and Douglas
­demonstrated, however, that it was possible to alter the chemical properties of the
protein cage through rational design without disturbing the overall architecture of

© 2010 Taylor & Francis Group, LLC

22 Nanobiomaterials: Development and Applications

the capsid [45]. They accomplished this by genetically engineering the protein to
replace the basic residues of the native capsid with glutamic acid, forming a mutant
capsid, with the electrostatic characteristics of the interior wall changed from cat-
ionic to anionic. The altered electrostatic character of the interior of the protein
favors strong interaction with ferrous ions, which promotes oxidative hydrolysis and
formation of size-constrained iron oxide particles within the viral capsid in a fashion
similar to that of apoferritin.
The electrostatically altered viral protein cage catalyzed the rapid oxidation of
Fe2+, leading to the formation of iron oxide magnetic nanoparticles. Specifically, the
purified mutant proteins were treated with aliquots of Fe2+ at pH 6.5 and allowed to
oxidize in air. Single and double mineralization processes, with loading factors of
2000 and 6000 Fe atoms per protein cage, were carried out, resulting in the forma-
tion of spherical particles with core diameters of (8.2 ± 1.6) nm and (24 ± 3.5) nm
[45]. High-resolution TEM images indicated that the resulting nanoparticles were
single-crystalline lepidocrocite.
Biotechnological processes borrowed from protein engineering ­techniques,
such as screening combinatorial peptide libraries using phage display, are
­currently being explored for the identification of peptide sequences with high
­specificity toward particular inorganic materials and in some cases with high
specificity toward unique crystal faces. Phage display, first described by
George P. Smith in 1985, is a laboratory technique for the study of protein–protein,
­protein–­peptide, and protein–DNA interactions that use bacteriophage (viruses
that infect ­bacteria) to connect proteins with the genetic information that encodes
them. One of the most common bacteriophage used in phage display is the fila-
mentous M13 ­bacteriophage. Identification of peptide sequences that promote the
nucleation of specific crystallographic phases is extremely useful in the direct
synthesis of magnetic nanoparticles with desired properties and crystal ­structure,
e.g., the hard magnetic FePt and CoPt nanoparticles in their high anisotropy
­face-centered tetragonal (fct) phase, also known as the L10 phase, without the
need for postsynthesis ­h igh-temperature annealing. Unlike previous examples,
in this approach, biological interactions control the nucleation of nanoparticles
with no isomorphous complement in nature. For example, the identification of
a specific peptide sequence from screening the M13 bacteriophage engineered
into the protein cage of MjHsp from the thermophilic archaeon Methanococcus
jannaschii directs the peptide-specific recognition synthesis of L10 CoPt nanopar-
ticles, which exhibit ­room-temperature ferromagnetism [46]. Eliminating the
high-temperature annealing step would greatly simplify the production of hard
magnetic nanoparticles suitable for device applications.

1.3.5 Maghemite Core/Silica Shell Nanoarchitectures

Surfactant and protein coats render magnetic nanoparticles biocompatible. There
are also biocompatible inorganic materials, further extending the scope and range
of bio-inspired magnetic nanoparticle synthesis. Silica is just such a material, being
nontoxic and water soluble. Yi et al. [47] produced γ-Fe2O3 magnetic nanoparticles
encapsulated within silica shells of finely tuned thicknesses. Thermal decomposition

© 2010 Taylor & Francis Group, LLC

Bio-Inspired Magnetic Nanoparticles 23

160
(311)
140 (440)
120
100
Intensity (a.u.)

80 (511)
(400)
60 (220)
(422)
40
20
0

20 30 40 50 60 70
(a) 2θ (°)

(b)

FIGURE 1.17 (a) XRD pattern and (b) TEM images of oleic acid-covered γ-Fe2O3 ­nanocrystals.
Scale bar: 10 nm. (Reprinted with permission from Yi, D. K., et al., Nanoparticle a­ rchitectures
templated by SiO2/Fe2O3 nanocomposites, Chem. Mater., 2006, 18: pp. 614–619. Copyright
2006 American Chemical Society.)

of iron pentacarbonyl [Fe(CO)5] in the presence of oleic acid produced monodispersed

metal iron particles. Controlled oxidation using trimethylamine oxide [(CH3)3NO] as a
mild oxidant produced highly crystalline γ-Fe2O3 particles. Figure 1.17 gives XRD and
TEM characterization of the resulting highly uniform, oleic acid-covered, γ-Fe2O3 mag-
netic nanoparticles. XRD patterns confirm the presence of Fe2O3. The TEM character-
ization indicated monodispersed nanoparticles of average ­diameter d of (12.5 ± 0.2)
nm, forming highly ordered 2D superlattices on the TEM grid, as seen in Figure 1.17b.
Uniform, silica-coated maghemite (Fe2O3/SiO2) nanoparticles were then prepared
using the oleic acid-covered monodispersed γ-Fe2O3 nanocrystals. Silica coating on the
γ-Fe2O3 nanoparticles was performed through the formation of water-in-cyclohexane

© 2010 Taylor & Francis Group, LLC

24 Nanobiomaterials: Development and Applications

Igepal
SiO2/Fe2O3
Fe2O3
nanoparticle

Rattle-type
TEOS HCl SiO2/Fe2O3
nanoball

Reverse microemulsion TEOS/C18TMS HCl

Hollow SiO2
nanoball

Mesoporous SiO2 coating

FIGURE 1.18 Schematic of the synthesis of MP/SiO2/MS nanoarchitectures. C18TMS,

octadecyltrimethoxysilane; MP, magnetic particle; MS, mesoporous silica; SiO2, solid s­ ilica;
TEOS, tetraethyl orthosilicate. (Reprinted with permission from Yi, D. K., et al., Nanoparticle
architectures templated by SiO2/Fe2O3 nanocomposites, Chem. Mater., 2006, 18: pp. 614–619.
Copyright 2006 American Chemical Society.)

reverse microemulsion, as schematically depicted in Figure 1.18. The Fe2O3/SiO2

core–shell nanoparticles were then used as a platform for deriving higher ­nanoparticle
architectures.
The silica coating was tunable between ~1.8 and ~30 nm, as shown in Figure 1.19a–d.
Subsequently, a second layer of mesoporous silica (MS) of 10 and 21 nm thickness
was applied to the 25-nm silica-coated nanoparticles to form MP/SiO2/MS nanoar-
chitectures (Figure 1.20a and b).
Well-controlled particle size and morphology are important for the application
of SiO2-coated magnetic nanoparticles in biomedical applications, such as ­magnetic
resonance imaging, magnetic relaxometry, magnetic cell separation, and drug
­delivery [47]. By introducing an MS coating and by creating rattle-type silica balls
with magnetic nanoparticles, applications in catalysis and chemical sensing and
separations may be explored [47]. For biomedical applications, we note that the MS
coating could be loaded with pharmaceuticals or DNA for targeted drug delivery or
gene transfection therapies, respectively.

1.4 MAGNETIC CHARACTERIZATION OF
IRON-BASED MAGNETIC NANOPARTICLES
Detailed magnetic characterization of bio-inspired magnetic nanoparticles is of utmost
importance in the development of biocompatible magnetic agents for various applica-
tions in biomedicine and biotechnology. The magnetic properties of nanometer-sized
particles and their assemblies are dominated by finite-size effects [48] and superpara-
magnetism and interparticle interactions [49] that can affect their quality and suit-
ability for various applications. Thus, in-depth magnetic characterization of prepared
nanostructures and their assemblies is an essential component to their development.

© 2010 Taylor & Francis Group, LLC

Bio-Inspired Magnetic Nanoparticles 25

5 nm

(a) (b)

(c) (d)

FIGURE 1.19 TEM micrographs of ~12.5 nm γ-Fe2O3 oleic acid-encapsulated particles

­covered with solid silica shell. Shell thicknesses from 1.8 to 30 nm were achieved: (a) ~1.8 nm,
(b) ~4 nm, (c) ~9 nm, and (d) ~25 nm. Arrows indicate asymmetric silica coating. Scale
bar: 20 nm. (Reprinted with permission from Yi, D. K., et al., Nanoparticle architectures
­templated by SiO2/Fe2O3 nanocomposites, Chem. Mater., 2006, 18: pp. 614–619. Copyright
2006 American Chemical Society.)

For iron-based superparamagnetic particles, the combination of two techniques, 57Fe

Mössbauer spectroscopy and superconducting quantum interference device (SQUID)
magnetometry, has been proven especially powerful [50].
57Fe Mössbauer spectroscopy [51–53] probes specific atomic sites within the

nanocrystalline lattice of the particles through the hyperfine interactions of the 57Fe
nucleus with its electronic surroundings and yields atomic detail on the particles’
magnetic and electronic properties. SQUID magnetometry probes the total mag-
netic moment, or superspin, of the nanoparticle via its interaction with an applied
magnetic field. Furthermore, for investigations of dynamic nanomagnetism, the

© 2010 Taylor & Francis Group, LLC

Exploring the Variety of Random
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Title: The Antelope Boy; or, Smoholler the Medicine Man

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*** START OF THE PROJECT GUTENBERG EBOOK THE ANTELOPE

BOY; OR, SMOHOLLER THE MEDICINE MAN ***
 THE ANTELOPE BOY;
OR,

SMOHOLLER, THE MEDICINE-MAN

A TALE OF INDIAN ADVENTURE AND MYSTERY.

BY GEORGE L. AIKEN.

NEW YORK.
BEADLE AND ADAMS, PUBLISHERS,
98 WILLIAM STREET.

Entered according to Act of Congress, in the year 1873, by

FRANK STARR & CO.,
In the office of the Librarian of Congress, at Washington.
 CONTENTS
I The Surveyors’ Camp 9
II The Arrow Message 14
III Smoholler’s Fiend 19
IV Smoholler’s Angel 24
V The Scouting Party 28
VI Finding the Trail 32
VII A Desperate Encounter 35
VIII The Prophet-Chief 39
IX Conjuration 42
X Oneotah 46
XI A Silvan Repast 50
XII The Tree-Ladder 54
XIII Multuomah 59
XIV The Chief’s Bride 63
XV The Old Hunter’s Idea 67
XVI Holding a Council 70
XVII The Boy Embassadors 75
XVIII The White Lily 80
XIX On the Way 84
XX Oneotah’s Memories 88
XXI The Mystic Cavern 91
XXII The Search is Ended 95

9
 THE ANTELOPE BOY;
OR,

SMOHOLLER, THE MEDICINE-MAN.

 CHAPTER I.
THE SURVEYORS’ CAMP.

The surveying party were camped upon the banks of the Columbia
River, a short distance from the mouth of its confluent, the Yakima.

This party consisted of the two surveyors—Owen Blaikie, a bluff,

middle-aged Scotchman, long since “naturalized” to this country, and
Cyrus Robbins, a shrewd young Yankee, twelve United States
soldiers under command of Lieutenant Charles Gardiner, detailed
expressly from the nearest fort to protect the surveying party from
predatory bands of Indians, an old hunter, generally known under
the name of “Gummery Glyndon,”—his prefix of Montgomery having
suffered this abbreviation at the hands of his associates—whose duty
it was to act as guide, and keep the surveyors supplied with fresh
meat; and two boys, the chain-bearers of the expedition.

These boys merit more than a passing notice here, as they are
destined to play conspicuous parts in the events which were to
follow the advance of the surveying party into the country of the
Yakimas.

There was this peculiarity about them, that they were first cousins,
and were both called Percy—Percy Vere and Percy Cute.

But despite their relationship and the similarity of their surnames,

there was very little resemblance between the two.

Percy Vere was a slender youth, graceful and active, with a frank,
honest face, and regular features, his hair being a dark 10
chestnut, thick and curly, and his eyes a clear hazel, giving
evidence of courage and decision of character in their glances. He
looked quite picturesque in his coarse suit, with the trowsers tucked
into high-topped boots, and his crispy curls straggling from beneath
his broad-leafed felt hat.

Percy Cute was full a head shorter, and his figure was decidedly
dumpish. He had a fat, good-natured face, light flaxen hair, and a
laughing blue eye. Indeed, a grin appeared to be the prevailing
expression of his features. He was sluggish-looking, and appeared
like one who would not put forth exertion unless compelled to do so.
He was dressed after the fashion of his cousin and comrade, with
heavy boots, coarse trowsers, a striped shirt, with a broad collar, and
a kind of roundabout, which was short for a coat, and too long for a
jacket; and like him, he wore a revolver in a belt buckled around his
waist, the pistol resting convenient to hand, upon his right hip, while
on the left side the handle of a bowie knife made itself conspicuous.

All in this party carried arms, for the service was one of danger, and
at any moment the emergency for their use might arise.

The boys were quite favorites in the party, the first by his frank,
manly bearing, and accommodating spirit, and the other by his
unvarying good nature, and the drollery in which he was so fond of
indulging. His humor appeared to be inexhaustible, and his quaint
manner of giving vent to it was irresistible.

In fact, Percy Cute had, at a very early age, been forcibly impressed
by the antics of a clown in a circus, and his great delight had been
to play clown from that eventful moment.

The culinary department of the expedition was attended to by a

colored individual who combined the two functions of cook and
barber for the party. He was a jolly little darky, but terribly afraid of
the Indians. The fear of his life was that he might have his “wool
lifted”—as the old hunter phrased it—before he got out of the
wilderness. But he had one consolation even in this apprehension:
he had, like a great many other barbers, invented a HAIR
RESTORATIVE, which he considered infallible.
“Never you mind, boys,” he would tell the soldiers, “if de 11
Injines does gobble us, an’ lift our ha’r, as Gummery says, I
can make it grow ag’in—hi yah-yah! I jist kin!”

Whereupon he would exhibit a small bottle in a mysterious manner,

adding, “Dar’s de stuff dat can do it—you bet!” And then he would
consign it to his pocket again.

This assurance afforded much amusement to the “high privates” of

the party, who made a standing joke of the Professor’s Hair
Restorative—for Isaac Yardell had prefixed the word “Professor” to
his name when he was a tonsorial artist in Chicago, before the spirit
of adventure had seized upon him and led him after gold among the
mountains of Montana.

Gummery Glyndon had brought in an antelope. Some of the soldiers

had captured a few fish from the river, a fire had been built in the
center of the camp, and preparations were going on briskly for the
evening meal.

In this Isaac had four assistants, he having contrived to transfer the

drudgery of his office, with true Ethiopian cunning, to others. A
colored servitor will always shirk all the work he can. Thus two of
the soldiers, a German named Jacob Spatz—Dutch Jake, was his
camp name—and one Irishman, Cornelius Donohoe—Corney for
short—were always available for services at meal-time, and the two
boys—the Percys—collected the wood for the firing. By this
arrangement Isaac had little to do but the cooking, which he
performed to the entire satisfaction of the party.

Even the rough old hunter—Glyndon—a gaunt, grizzly man of fifty

years of age, bestowed his meed of praise upon him.

“It don’t matter what I bring in,” he told Lieutenant Gardiner, “game,
fish or fowl—antelope, mountain sheep, or b’ar meat, that Ike can
just make it toothsome. These darkies take to cooking, ’pears to me,
just as naturally as ducks do to water.”
Ike had only one grievance in the camp, Percy Cute was continually
playing jokes upon him. Such little pranks as putting powder in his
pipe, nipping at the calves of his legs and imitating a dog’s growl,
and grasping his wool at night, and shouting a war-whoop in his ear,
had a damaging effect upon Ike’s temper, and he vowed deadly
vengeance. But his vengeance never extended beyond a chase 12
after Percy Cute with a ladle, with the laudable intention of
administering a severe spanking; but in these onslaughts the
redoubtable Isaac always came to grief; for, just as he would
overtake the flying youth, Cute, with a nimbleness that his sluggish
look and dumpy figure never led any one to expect, would suddenly
fall upon his hands and knees, and pitch his pursuer over him. But
as Isaac invariably alighted upon his head, he received no injury
from these involuntary dives. A shout of laughter would herald his
defeat, and he would pick himself up, and return to his camp-kettle,
in a crest-fallen manner, swearing to himself until every thing got
blue around him, and vowing that he would “fix him de next time,
suah!”

These little episodes enlivened the camp, and nobody enjoyed them
better than Gummery Glyndon. The old hunter had, generally, a
morose look upon his seamed and weather-beaten countenance,
and his hatred of every thing in shape of an Indian was well known.

Nor was the cause of that hatred a secret. He had been the victim of
one of those forest tragedies so frequently enacted upon the frontier.
It was the old story which has been told so often, and will be
repeated until the extermination of the red-man—which has been
going on slowly but surely for years—is completed.

While absent upon a hunting and trapping expedition, his cabin had
been surprised, his wife and only child, a little girl some three years
of age, cruelly murdered, and their mutilated remains consumed in
the fire that destroyed his home.
A blackened ruin was all that was left of the spot that was so dear to
him, and he found himself alone in the world, with only one thought
in the future—vengeance upon the murderers.

In the drear solitude of that heart-sickening scene, and beside the

ashes of all that he had treasured in the world, he breathed that
vow of vengeance, which the lips of so many bereaved settlers in
the Far West have sent up to heaven—death to the destroyers.

That was fifteen years before the time in which I introduce him here.
In all those years he had pursued the Indians with a deadly 13
malignity. He had taken part in every Indian war that had
broken out, and the number of his victims had been many.

As the years passed away this feeling of vengeance grew fainter, and
though he never spared an Indian who came against him with
hostile intent, yet he did not go out of his way to seek for them, as
he had done. The Yakimas were supposed to be the destroyers of
his home and family, and against that nation he cherished an
undying enmity. Yet circumstances had led him away from their
country, to the hunting-grounds of the Apaches, with whom he had
many encounters.

He had gladly accepted the service that would take him back to the
land of the Yakimas. In all these years he had gained experience as
a guide, in wood-craft, and as an Indian-fighter. No hunter of the
plains bore a better reputation for skill, prudence, and knowledge of
the Indians than Gummery Glyndon.

His face bore a somewhat morose expression, as I have said, but he

was far from being a morose man. Indeed, there was quite a fund of
dry humor in his disposition, which was an agreeable surprise to
those who judged the man by his saturnine countenance.

Percy Cute was a particular favorite of his, and none in the party
enjoyed the boy’s drolleries more than he did. Indeed, both the boys
were prime favorites with him, and often accompanied him upon his
hunts. He looked upon them in the light of proteges, as he had got
them their places in the expedition.

He had met them at Fort Benton, where they had come from Omaha
up the Missouri river, on one of the steamboats that ply on that
stream, and was rather surprised to hear what had brought them
there.

Though partly led by a spirit of adventure, they had a mission, and

one of some importance.

14
 CHAPTER II.
THE ARROW MESSAGE.

Percy Vere explained this mission to the old hunter. His father had
been missing for years. He was an eccentric character, and professed
spiritualism, astrology, ventriloquism, and kindred sciences, dabbling
a little in magic and chemistry. In fact, he was a universal genius—a
jack-of-all-trades, and not doing well with any.

Percy’s mother was a woman of ability and good sense, a first rate
milliner, and her industry kept the wolf, which the father’s
eccentricities brought to the door, away. In other words, she was
obliged to support herself and son, and often furnish money to the
genius, who could not make it for himself with all his diverse talents.

He did not appear to be able to concentrate his forces so as to

produce any good from them. He was full of wild theories and
startling speculations, but he failed signally whenever he attempted
to put them to an application.

His wife expressed her opinion of him freely one day, and told him
she could no longer expend her savings in his wild schemes. He
replied that it was the fate of genius to be misunderstood, that he
was destined to be a great man, and she would live to see it; and
having uttered this ambiguous prophecy, left her.

He did not return the next day, or the next—a year passed away
without bringing Guy Vere home. His wife became alarmed at his
prolonged absence. She reproached herself with being too harsh
with him and having driven him away from her. He was a handsome
man, and she had cherished a warm affection for him, which his
eccentricities had not destroyed. She feared that she had driven him
to commit suicide. But no tidings came of his death.
She was obliged to keep her little millinery shop going for the
support of herself and son, and her sister’s child, who being left an
orphan, fell to her care. This was Percy Cute—who was just 15
one year younger than his cousin, his mother having been so
pleased with the name of her sister’s child, that she had bestowed it
upon her own.

The little shop prospered, and the boys grew in years. Mrs. Vere
could not drive the image of her husband from her mind. If she
could have satisfied herself that he was dead, she would have been
more content, but she could not do that.

The impression among Guy’s neighbors when he was at home, was

that he was not in his right mind—“Luny,” they called him.

But many years passed away before she got any tidings of the
missing man, and then it came in a very vague shape.

Percy Vere got an Omaha Herald one day, which had been sent as
an exchange to a St. Louis paper, and in it was the advertisement of
an astrologer who called himself “Professor Guy.”

He took it home to his mother, and said to her, “That’s father!”

These words put her all in a flutter. She took the paper and scanned
the advertisement eagerly.

“What makes you think so?” she asked.

“Father’s name was Guy, and he was a ‘professor’ of astrology!”

She smiled. “He was a professor of almost everything.”

“Suppose I go and see if it is my father,” he suggested.

She pondered over this.

“Would you know him, do you think?”

“Oh, yes, if the picture you have in your locket is any thing like him.”

“It was when it was taken.”

She took out the locket, which she wore constantly around her neck,
sprung it open, and regarded the two portraits it contained
earnestly, for it held her miniature likeness as well as his.

“I have not changed much,” she said, “and perhaps he has not,
either. I should really like to know if he is alive. Suppose I was to
write to this Professor Guy?”

Percy, who was a bright youth, shook his head dissentingly.

“If he is staying away of his own accord, it is no use to write 16

to him to come back,” he replied.

She breathed a sigh. “I suppose not,” she said.

“But if I was to go after him and have a talk with him, I might
prevail upon him to come back.”

Mrs. Vere was impressed by these words, but she answered: “How
could I trust you so far away from home?”

He smiled, and drew himself proudly up.

“Don’t you think I am big enough to take care of myself?”

She surveyed his tall, graceful figure, with a mother’s pride, saying:

“Perhaps; but you are so young.”

“I’m seventeen, and I feel quite a man.”

“But I don’t like to trust you so far from home alone.”

“Oh! I needn’t go alone; Percy can go with me.”

Mrs. Vere laughed.

“A great protection he would be—another boy like yourself!” she

cried. “There, there—let us not talk any more about it.”

But they did talk about it upon several occasions afterward, and Mrs.
Vere’s desire to hear from her missing husband overcame all other
considerations, and she consented to Percy’s request to go in search
of him. She thought that the sight of his boy would induce him to
return home.

Her business had proved prosperous, as I have said, and she was
able to fit out the boys in good style. She hung the locket that
contained her own and husband’s likeness around her son’s neck,
and bade him a tearful “good speed.”

The boys took passage upon a steamboat bound for Omaha, and
steamed up the Big Muddy, as the Missouri is called by the dwellers
on its banks, and reached that ambitious city in due season.

Upon making inquiries, Percy Vere learned that Professor Guy had
found Omaha dull for the exercise of his profession, and had joined
a party of adventurers—a mixture of hunters and gold-seekers—and
gone with them to Fort Benton.

The very eccentricity of this proceeding was a convincing proof to

Percy that this Professor Guy was indeed his father So he 17
wrote to his mother, and then he and Percy Cute sailed up the
river in one of the light-draught steamboats.

They reached Fort Benton without misadventure, but here, instead

of being at the end of their journey, they found it was just the
starting-point. The party to which the Professor had attached himself
had taken the trail that led into the wilderness, and it was necessary
to follow it, or abandon the search.
Percy Vere chose the former alternative, for he could never think of
the latter, and Percy Cute was always of his way of thinking—in fact,
thinking was irksome to his sluggish nature.

“I just tumble to any thing you say,” he told his cousin. “Follow your
leader—that’s my maxim. You lead and I’ll follow. Say! we might
have some high old fun among the Injuns, and bears, and things.
Let’s invest in a revolver and bowie-knife, and travel on our muscle!”

So Percy Vere, filled with a true spirit of boyish adventure, wrote his
intentions to his mother, and he and Cute made their preparations
for a journey into the wilderness.

At this juncture of affairs they made the acquaintance of the old

hunter, Gummery Glyndon. They told him their story, (or rather
young Vere did, for he was the spokesman on all occasions) and he
promised to aid them, and fulfilled his promise by attaching them to
the surveying party, though in the capacity of chain bearers; but the
boys did not mind that.

Such an opportunity to penetrate into the Indian country was not to

be neglected, and the first Percy, who was treasurer, wished to
husband their means, for there was no telling how long their search
might last, or whither it would lead them.

They made rapid journeys at first, as a portion of the “Northern

Pacific Railroad” had already been surveyed, and they were to take it
up at, or near, that point, where it was to connect in a south-easterly
direction with the “Union Pacific.”

As they passed the different Government forts their escort was

changed, until they were joined by Lieutenant Gardiner and his
squad, from Fort Walla Walla. He was to remain with them until they
were through the Yakima country.

Hitherto their journey had led through the land of the Nez 18
Perces, who were a friendly tribe, and they had been
undisturbed; but when they made this new camp Gummery Glyndon
told them they might now expect trouble from the Indians.

“There’s three tribes through here,” he said, “and there ain’t much
choice between ’em. There’s the Cayuses, the Yakimas, and the
Umatillas—a pesky set of murdering thieves the lot of ’em. They all
belong to the great Snake Nation, I believe—red sarpints, every
mother’s son of ’em.”

When he returned from his hunt he told them that he had seen
“Indian sign.”

“There’s Injuns watching us, and we shall hear from them,” he said.
“We’ll have to keep a sharp watch to-night, or they’ll stampede our
animals.”

The lieutenant and the surveyors did not neglect this warning. They
had great confidence in the old hunter’s judgment.

When the supper was disposed of the camp was placed in as good a
condition of defense as the locality would permit. The ground had
been well selected; it was a little grove on the river’s bank, a kind of
oasis among the cliffs, which rose beetling upon either side,
precipitously, and, apparently, inaccessible. These cliffs were some
distance—a long rifle-shot—from the little grove, and a kind of rocky
valley lay between them, devoid of vegetation in many places, where
the hard rocks cropped up. Through this valley must the foe come,
or else risk their necks, or a plunge into the river, by attempting to
skirt the cliffs.

The horses belonging to the party were secured in the grove. In the
center of the grove, in a kind of natural fireplace formed by the
rocks, the fire had been built, and its red embers were still glowing.
Two sentinels were posted at either extremity of the camp. Around
the fire the hunter, the surveyors, and the lieutenant were stretched
in easy attitudes, enjoying their pipes of tobacco—the great luxury
of the wilderness.
A short distance from them the two boys reclined upon a mossy
bowlder, listening to their conversation.

The sun had sunk, and the glorious twilight of that western 19
land was upon them. The scene was of calm tranquillity. But
that tranquillity was broken in a singular manner.

There came a hurtling sound in the air, and an arrow descended,

apparently from the heavens, and stuck quivering in the turf at
Lieutenant Gardiner’s head.

All started and grasped their weapons, instinctively, for the trusty
rifles were close at hand.

“An attack?” cried Gardiner.

“No—a message. See, there’s a scroll upon the arrow,” answered

Gummery. “Read it.”

He threw some brush upon the coals which speedily burst into a
flame. Lieutenant Gardiner undid the scroll of bark from the arrow,
and spread it open. It contained characters which he had no
difficulty in deciphering, for they were written in English.

“White men, begone! If you advance further into the land of the
Yakimas, certain destruction awaits you.

“Smoholler, the Prophet.”

 CHAPTER III.
SMOHOLLER’S FIEND.

“What does this mean?” added Lieutenant Gardiner, having read this
singular scroll aloud.

“A game of bluff!” answered the irrepressible Percy Cute. “Let’s see

him, and go two better!”

“It’ll be more than a bluff game,” rejoined Gummery Glyndon,

shaking his head gravely. “This means business. It’s a notice to quit,
and if we don’t take it, these Injuns will do their best to put us out.”

“Rub us out entirely, I guess you mean,” cried Surveyor Robbins,

laughingly. “But we won’t take the back track on such a notice as
that. Who is this Smoholler?”

“Yes, that’s what I want to know,” chimed in Blaikie and Lieutenant

Gardiner.

“I have heard tell of him, though I never met him,” replied 20

Glyndon. “He’s a great gun among the Injuns hereabouts. He’s
a kind of red Brigham Young—calls himself a Prophet, and has
started a new religion among the red-skins.”

“What is this religion like?”

“That’s more than I can say; though, from what I’ve heard, there
appears to be a deal of trickery about it. He’s a great Medicine-man,
and can raise the Old Boy, generally. He has his familiar fiends, and
makes ’em appear to his followers whenever he likes. He works
miracles, and all that sort of thing. And when he predicts the death
of any one, they just go, sure pop, at the time mentioned.”

“A singular man, this,” remarked Lieutenant Gardiner, thoughtfully.

“He’s more smart than sing’lar; he just keeps these benighted
heathen right under his thumb. They don’t dare to say their souls
are their own when he’s around.”

“Where did he come from?”

“He is said to be a Snake Indian of the Walla Walla tribe. He started

a village on the river, above here, at a place they call Priest’s Rapids,
and his followers increased like magic. He is said, by the Nez Perces,
to have a couple of thousand of believers, renegades from all the
other tribes in this region, and he can put three hundred fighting
men in the field, and then the Cayuses, Yakimas and Umatillas all
stand in dread of him, and wouldn’t dare to do any thing else but
join him in a war against the whites if he called on ’em. I believe
he’s got a reg’lar stronghold at Priest’s Rapids.”

“Is it named so on his account?” asked Robbins.

Glyndon shook his head dubiously.

“I s’pose so, but I couldn’t say for sure. I don’t know the place; was
never up there.”

“What kind of a place is it—did you ever hear?”

“Oh, yes. It is north of the Oregon line, and is a great place for
salmon-fishing. The Injuns have a great time catching ’em in the
season.”

“This Smoholler, then, is a kind of independent chief among the

other tribes?”

“Yes; and his tribe is a conglomeration of all the other tribes, and
the pick of ’em, too. They are called Smohollers by the other 21
Injuns, but there’s Cayuses, Yakimas, Umatillas, Modocs,
Snakes, and Piutes amongst them.”
“A mongrel set!”

“But tough customers to deal with.”

Lieutenant Gardiner turned to Percy Vere.

“You and your chum send the sentinels in to me, and take their
places—young eyes are sharp.”

The two boys, who had been listening attentively to this

conversation, obeyed at once, and the two sentinels soon appeared
before the lieutenant. But they had not seen any one approach the
camp, and were surprised to hear that an arrow had been shot into
it.

Gummery Glyndon surveyed the nearest cliff critically. Its base was
about a stone’s throw from where he sat. The rising moon threw a
silvery radiance upon its peak, disclosing an irregularity near its top,
that looked like a cavity in its face, though it might have been only a
shadow.

“It’s my opinion the arrow came from there,” he exclaimed, giving

utterance to this thought suddenly.

All eyes were turned in the direction indicated.

“But how could any one get up there? A cat couldn’t climb that. It’s
as steep and as smooth as a wall.”

“Just you wait,” returned the old guide, coolly. “If this Smoholler is
the kind of man he’s said to be, we ain’t done with him yet. Just
keep your weather eye peeled in the direction of that cliff, and have
your rifles handy. That arrow was only the commencement. I saw
plenty of Injun sign to-day, and there may be a hundred of
Smoholler’s braves beyond there. I opine that he is not going to let
us travel much further into this country, if he can help it.”
“But, man, what harm does our surveying do him?” asked Blaikie.

“He don’t want any railroad through this country—all Injuns are
down on railroads—sp’ils their hunting-grounds, and settles up the
country. And the white settlers settle the Injuns. We’ve had a
genteel notice to leave, and if we don’t take it, we’ll have ’em
swarming round us like enraged hornets.”

“You would not advise a retrograde movement?” asked Lieutenant

Gardiner.

“Who said any thing about taking the back-track?” somewhat 22

tartly rejoined Glyndon. “Did I? I never saw Injuns enough to
back me down yet.”

The lieutenant laughed, as he added:

“The suggestion of a backward movement came from me,” he said,

“and by so doing I am not afraid to have my courage called into
question. Discretion is said to be the better part of valor. We appear
to have reached a critical position here. Our party is small—nineteen
in all, counting the two boys. If the Indians oppose us in force—and
from what Glyndon says it seems that this Indian Prophet Smoholler
can put three hundred warriors in the field—shall we be justified in
advancing against such odds?”

The surveyors looked at Glyndon, but he was silent, gazing

reflectively at the cliff, upon whose summit the moonbeams now
played in a fantastic manner.

“I confess I don’t like the idea of retreating,” said Blaikie. “I don’t

want to be turned back by such a scarecrow as that.”

“No more do I,” added Robbins.

“I don’t say go back, and I don’t say go on,” replied Glyndon, in his
deliberate manner; “but I say, just hold on for a while here, where
we are, until we can see how the cat jumps.”

“How long will it be before the feline animal indulges in her

gymnastic exercise, do you think?” asked Robbins.

“Before you can smoke another pipe,” answered Glyndon. “I have an

idea that something is going to happen right away—kind o’ feel it in
my bones. Get the men ready, leftenant—there’s no telling what is—
Hello! it’s coming! Fireworks—by king!”

The amazement of the old hunter was shared by the whole camp,
and the two boys came running in from their posts.

“See—see—look there!”

A strange fire issued from the face of the cliff, disclosing a little shelf
or platform, backed by a cavity. From this cavity the fire came forth
with crimson luster, and rose colored smoke rolled upward toward
the heaven, obscuring the moon-rays.

The entire force of the whites clustered in front of the grove, 23

clutching their rifles, and gazing with wondering eyes upon
this singular sight, and exclamations burst spontaneously from their
lips.

“Ach Gott! what ish dat?” cried the Dutch private.

“It’s a volcayano!” explained the Irishman.

“It’s the debble’s fireplace!” mumbled Isaac, and his teeth chattered
together with superstitious awe.

“It’s some of Smoholler’s deviltry!” said Glyndon.

The fire grew in intensity, and then a dark body seemed to grow up
in the midst of it. A black, unearthly figure of a man, with eyes of
fire, a tongue of flame, and livid horns projecting from his head, of a
deep-red color.

“The devil!” was the cry that burst from the lips of the astonished
whites.

He held what appeared to be a thunderbolt in his hand, and

suddenly launched it like a javelin at the astonished gazers. It
whizzed past Isaac’s head, singeing his wool in its passage, and
exploding at his heels, and the tonsorial professor sprawled upon his
back with one heart-rending yell that evinced his firm belief that he
had received his quietus.

“Fiend or man, I’ll have a try at him!” cried Glyndon, and he took a
rapid sight along the barrel of his rifle, and fired at the apparition on
the cliff.

Two other rifles echoed his, for Blaikie and Robbins had impulsively
followed his example. The three rifles sent forth their contents, and
the smoke clouded their vision for a moment. But following the
reports came an unearthly, soul-curdling laugh, and then something
pattered down among them like heavy drops of rain.

Robbins stooped and picked up a round object that struck at his

feet.

“Good heavens! here’s my bullet sent back to me!” he cried.

These words sent a thrill through every heart. Isaac, still lying curled
up in a heap where he had fallen, uttered a plaintive howl.

Percy Cute went to him.

“Are you dead, Ike? If you are, say so, and tell us where you would
like to be buried,” he said.

Isaac sat up on end, resenting this question.

“Glory!” he cried. “S’pose de debble had shot you, how would 24
you like it?”

“Well, if I warn’t hurt any more than you are, I shouldn’t mind it
much. Singed your wool a little, but your Hair Restorer will fix that
all right, you know.”

A roar of laughter followed this remark, and in the midst of it Isaac

scrambled sheepishly to his feet.
 CHAPTER IV.
SMOHOLLER’S ANGEL.

When the smoke of the rifles cleared away the fiend had vanished
from the cliff, and the crimson light had died away. The silvery
beams of the moon played hide and seek among the projections and
depressions of the cliff’s peak.

The gazers rubbed their eyes. What they had seen appeared to
them already like a fantastic dream. But a new vision awaited them,
a new wonder was to be presented to their eyes.

Another light began to glow from the cliff, but this time it was of a
bluish tint, and the smoke that arose from it was white and fleecy.
And this light grew dense, as the other had done, and assumed a
form and shape—a shape of ethereal loveliness.

As the other vision thrilled the beholders with a kind of supernatural

awe, so did this one excite their wondering admiration. It bore the
shape they supposed an angel would wear.

The face was that of a girl, angelic in its beauty. Her long black hair
floated in wavy masses upon her neck and shoulders, and was
confined upon the forehead by a golden coronet in the center of
which gleamed a diamond star, which emitted scintillating rays of
light. Her arms and legs were bare, revealing their faultless
perfection, and the alabaster purity of her skin. Her only garment
was a long white tunic, of some snowy, fleecy fabric, confined at the
waist by a golden cestus, which was studded with large rubies 25
glittering with blood-red rays.

This angelic vision held in her right hand a kind of glittering dart. For
a minute she transfixed their wondering gaze, then hurled the dart
into their midst.
The fire around her grew more vivid, the volume of white smoke
increased in density, obscured her figure from view, and then began
to roll away. When the light of the fire faded and the smoke lifted
from the face of the rock, the platform was vacant, the lovely vision
had disappeared.

The surveying party gaze inquiringly into each other’s faces.

Lieutenant Gardiner expressed the general opinion by asking the
hunter, Glyndon:

“What do you think of that?”

Glyndon shook his head dubiously.

“Did you ever see a girl as pretty as that one was?” he asked.

“Well, no, I can’t say that I ever did,” the lieutenant admitted, with a
smile; “and if she is a human I should like to become better
acquainted with her.”

“All women have something angelic about them,” said Glyndon,

reflectively, and his voice had a strange touch of pathos to it as he
spoke—“particularly when they are good and true women. I knew
one once—an angel couldn’t have had a better disposition, and she
—” His voice broke here. “Well, well, the murdering red-skins sent
her to heaven before her time!” he resumed, huskily. “And our little
one went with her. Perhaps it was best so—but I’ve often thought I
could have stood it better if she had been spared. Do you know,
leftenant—it was an odd idea, but when I looked at that bright spirit-
angel or whatever it was—up on the cliff yonder—I thought to
myself, my little girl, maybe, looks just like that up in heaven.”

The hunter turned away his head and wiped his eyes with the back
of his bony hand. His hearers respected his grief for they knew the
story of Glyndon’s bereavement.