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Nano-inspired Biosensors for
Protein Assay with Clinical
Applications
Nano-inspired
Biosensors for
Protein Assay with
Clinical Applications

Edited by

Genxi Li
State Key Laboratory of Pharmaceutical Biotechnology,
Department of Biochemistry, Nanjing University,
Nanjing, P. R. China
Laboratory of Biosensing Technology, School of Life Sciences,
Shanghai University, Shanghai, P. R. China
Elsevier
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List of Contributors

Ya Cao Center for Molecular Recognition and Biosensing, School of Life Sciences,
Shanghai University, Shanghai, P. R. China
Guifang Chen Center for Molecular Recognition and Biosensing, School of Life
Sciences, Shanghai University, Shanghai, P. R. China
Chang Feng State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Tao Gao Center for Molecular Recognition and Biosensing, School of Life Sciences,
Shanghai University, Shanghai, P. R. China
Chao Li State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Hai Shi State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Liu Shi State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Lei Wang State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Shuai Wu State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Jingjing Xu Center for Molecular Recognition and Biosensing, School of Life
Sciences, Shanghai University, Shanghai, P. R. China
Juan Zhang Center for Molecular Recognition and Biosensing, School of Life
Sciences, Shanghai University, Shanghai, P. R. China
Jing Zhao Center for Molecular Recognition and Biosensing, School of Life
Sciences, Shanghai University, Shanghai, P.R. China
Ji Zheng State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, P. R. China
Nandi Zhou School of Biotechnology and the Key Laboratory of Industrial
Biotechnology, Ministry of Education, Jiangnan University, Wuxi, P. R. China
Xiaoli Zhu Center for Molecular Recognition and Biosensing, School of Life
Sciences, Shanghai University, Shanghai, P. R. China

xi
Preface

Biosensors are developing so rapidly, even faster than expected. One main
reason is the employment of various kinds of nanomaterials and the assem-
blies of many types of nanostructures for the fabrication of biosensors.
Therefore, the interaction between the recognition element and the analyte
can be more favorable, the sensing layers can be more easily designed, and
the measureable signal can be amplified, etc. While more and more biosen-
sors are proposed, they must also perform with satisfactory sensitivity, selec-
tivity, specificity, and accuracy. In the meantime, the fabricated biosensors
have demonstrated their clinical applications. For instance, with the develop-
ment of proteomic technologies, a lot of disease marker proteins have been
discovered for many kinds of diseases, so detection methods are required to
be developed towards the assay of the newly discovered disease markers due
to the high importance and potential application in the diagnoses of these
diseases. Meanwhile, the proposed methods are expected to be simple, rapid,
sensitive, and cost-effective, thus they can be feasibly employed for the diag-
noses and for the evaluation of the treatment of these diseases. Among the
methods, biosensor-based techniques may live up to the expectations, and
more and more biosensors reported in these years have shown their practical
or potential applications in clinical diagnosis and the evaluation of the dis-
ease treatment. Remarkable progress has been made over the years on the
design and fabrication of sensing systems for disease marker detection with
clinical applications. So, it is highly required to write a book that is focused
on “nano-inspired biosensors for protein assay with clinical applications.”
On the one hand, colleagues, especially the young scientists and the graduate
students, in the research area of analytical chemistry and biosensors can
quickly know the recent advance of protein analysis and the new methodol-
ogy in designing new kinds of biosensors. On the other hand, for those col-
leagues from the area of nanotechnology or biosensors, it may provide an
opportunity for them to know the possible application of their research
results in medical science. Besides, for those readers from the area of diag-
nosis, this book may provide an opportunity for them to know the frontier of
the diagnostic methodology. I hope we have prepared a handbook for the

xiii
xiv Preface

young scientists and the graduate students in the research fields of analytical
chemistry, biosensors, biotechnology, and nanotechnology. This book may
also provide valuable information for the students majoring in material
science, molecular science, biomedical engineering, basic and clinical
medicine, etc.

Genxi Li
Acknowledgments

Firstly, I would like to thank George Knott, an Acquisitions Editor from

Elsevier, for his invitation to write this book. When George asked me
whether I would be interested in writing a book for Elsevier, I gave a posi-
tive answer without hesitation. One reason is that I noticed it was highly
required to write a book on biosensors. Although there have been several
books published on biosensors, it is necessary to present another one that is
focused on the employment of various kinds of nanomaterials and the assem-
blies of many types of nanostructures for the fabrication of biosensors. On
the one hand, nano-inspired biosensors are developing so rapidly. On the
other, more and more biosensors are proposed towards the assay of disease
marker proteins and many sensors have demonstrated their clinical applica-
tions. So, an in-time summary is urgently needed. Another reason is that I
have written lots of contributions to books about biosensors, such as
“Encyclopedia of Sensors,” “Nanomaterials for Biosensors,” “Biosensors and
Molecular Technologies for Cancer Diagnostics,” “Electrochemical Analysis
of Proteins and Cells,” “Engineering in Translational Medicine,” etc.; how-
ever, these books were not published by Elsevier, although I have published
so many research papers with Elsevier. Therefore, the kind invitation by
George Knott provides me a good opportunity to publish a book with
Elsevier, which is really appreciated. Secondly, I would like to thank
Kathryn Morrissey, who is also an Acquisitions Editor from Elsevier, for her
kind help and encouragement. Although I have great interest to write a book
for Elsevier, I have also realized that it requires extensive work to write a
book. Moreover, besides the daily work on research and teaching, I am so
busy with so many other obligations. So, I had planned to give up the plan
to write this book. Without the kind help from Katy, this book would not
have appeared. Thirdly, I would like to thank Tasha Frank, a Senior
Editorial Project Manager from Elsevier, for her kind help and support. She
is really an excellent EPM. Without her help, everything related to this book
would not have gone forward satisfactorily and successfully. Finally, I would
like to thank my coworkers. In the last several months, preparation of the
manuscript for this book has been the first priority in my labs both in
Nanjing and Shanghai. Without their involvement, this great job would not
have been completed in due course. Although the names of some coworkers
have been listed as coauthors in the chapters, their help should be still

xv
xvi Acknowledgments

appreciated. Certainly, special thanks should go to my coworkers whose

names are not listed as coauthors. Please allow me to say my thanks to them,
they are Wenxin Chai, Hong Chen, Huinan Chen, Tianshu Chen, Tingjun
Chen, Chengjie Duan, Yiwei Han, Yunfei Liu, Jianyang Lu, Dongsheng
Mao, Chaoli Mu, Yanxia Wang, Lan Xue, and Yi Yang.

Genxi Li
Introduction
Jing Zhao and Guifang Chen
Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University,
Shanghai, P. R. China

I.1 THE PRINCIPLE OF BIOSENSOR

With the tremendous development in biochemistry and molecular biology, a
bioassay that provides methods for accurate and efficient measurement of
specific analytes in a biological sample has attracted increasing attention.
Based on the biosensing principle, a new type of device has been developed
that can translate the amount of target molecules into a quantifiable signal to
detect analytes for different purposes, such as drug analysis, disease diagno-
sis, and biomolecule quantification. The device is called a biosensor, which
reports the quantifiable, exclusive, and specific signal for biomolecular inter-
actions (Perumal and Hashim, 2014).
In 1962, Clark and Lyons reported the first biosensor by immobilizing a
glucose oxidase on the surface of an amperometric oxygen electrode with
the assistance of a semipermeable dialysis membrane, which was used for
the direct quantification of glucose in a biological sample. According to the
definition from IUPAC, a biosensor is a self-contained integrated device
based on specific biochemical reactions, which is regulated by isolated bio-
logical macromolecules (e.g., enzymes, immunosystems, tissues, organelles,
or whole cells) and can detect chemical compounds using different signal
outputs (e.g., electrical, thermal, or optical signals). A biosensor is one sub-
type of chemical sensors, and is composed of two basic elements as a recep-
tor for chemical recognition and a physicochemical transducer for signal
reporting (Ali et al., 2017; Mohanty and Kougianos, 2006). A receptor is a
recognition system to identify specific molecules based on various intermo-
lecular interactions, thereby ensuring a high degree of selectivity for analyte
identification; a transducer, which is also known as a detector or a sensor,
transfers interaction information from recognition events to a
detectable signal, thereby ensuring a high degree of sensitivity for analyte
measurements.

xvii
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xviii Introduction

I.1.1 Bioreceptor
The receptor of biosensor, known as a bioreceptor, usually recognizes the
target analyte based on biomolecular interactions by making use of various
biological molecules (e.g., protein, DNA, cell, tissue) as recognition ele-
ments, which is the difference between biosensor and other types of chemical
sensors. The biological recognition element is an essential component in the
fabrication of a biosensor, which is able to sense both biological and nonbio-
logical targets in a given sample. According to the principle of biomolecular
interaction, bioreceptors are generally divided into two categories: biocata-
lytic recognition elements and bioaffinity recognition elements. Biocatalytic
elements rely on specific recognition of a catalyst and substrate that is
described as a “lock-and-key” system, while bioaffinity elements make use
of the bioaffinity interaction that is naturally observed in the biological
system.

I.1.1.1 Enzyme
An enzyme is one typical biocatalytic recognition element, which has been
extensively studied and used in biosensor fabrication (Wilson and Hu, 2000).
An enzyme is a macromolecular biological catalyst, which catalyzes the con-
version of a specific substrate to a new product. An enzyme is quite specific
to its substrate for the complementary binding site. The chemoselective,
regioselective, and stereospecific interaction with substrates ensures high
selectivity of enzyme as a bioreceptor. Specifically, the chemical reaction
between an analyte and an enzyme is based on the equation:
E
S 1 S0 ! P 1 P0
where S and S’ are the substrates of an enzyme, which are also the analytes;
while P and P’ are the products after enzymatic catalysis. Sensitive and
selective monitoring of analyte S is usually realized through the measure-
ment of quantitative changes of another substrate S’ or the generation of
reaction product P or P’, which is also achieved by monitoring the redox
state of an active center of the enzyme or direct electron transfer between
enzyme and the transducer. A cascade reaction using multiple enzymes pro-
motes extensive use of enzyme-based receptors, in which a product of the
first enzymatic reaction is a substrate of another enzyme for different detec-
tion purposes (Fu et al., 2012; Wang et al., 2009).

I.1.1.2 AntibodyAntigen
An immunosensor is a good example of a biosensor using a bioaffinity rec-
ognition element as a receptor, and it is based on the antigenantibody inter-
action (Conroy et al., 2009). An antibody is a Y-shaped immunoglobulin,
which is generated from B cells of the adaptive immune system of an
 Introduction xix

organism. Each antibody contains a particular epitope to allow recognition

and binding of the antigen with high-affinity and selectivity, which is known
as a target analyte. In this sense, identification of the analyte is realized
based on its interaction with the antibody using an immobilized biocomplex-
ing agent as a bioreceptor, while antigenantibody complexion could be
monitored by combining with different transducers after reaching a binding
equilibrium (Kokkinos et al., 2016; Mauriz et al., 2016; Yin et al., 2010).
However, the antibody suffers from several intrinsic limitations in applica-
tion. For example, preparation of the antibody is very complicated and rela-
tively expensive; an antibody is not stable and is easily disrupted by the
external environment and surface modification; an antibody with high immu-
nogenicity does not easily penetrate tissues for in vivo studying.

I.1.1.3 Cell and Tissue

A cell is the fundamental unit of living organisms with dimensions between
1 and 100 μm, which is also recognized as an independent function and
structure unit in vivo. A cell is surrounded by a plasma membrane, which
contains several organelles and various biological molecules inside, such as
electrolytes, proteins, and nucleic acids. Tissue is composed of several cells
with similar properties, and thus performs a specific function in vivo. In biol-
ogy, a tissue is a state of being between a cell and an organ. No matter
whether inside the cell or in the tissue, a lot of living activities are taking
place constantly. Both enzymes and receptor proteins maintain their native
structure and function to the greatest extent. Therefore, cells and tissues as
aggregations of different enzymes, antibodies, and receptors are directly used
as a natural source of recognition element for bioreceptors (Gui et al., 2017).

I.1.1.4 DNA and Aptamer

DNA is the most popular bioreceptor for the fabrication of a biosensor in the
recent decades (Dhiman et al., 2017; Saidur et al., 2017; Ye et al., 2018).
DNA hybridization is a natural bioaffinity system in organisms. Two or
more complementary nucleic acid strands are intertwined with each other
through noncovalent and sequence-specific interactions in a thermodynamic
manner. DNA hybridization is highly selective based on hydrogen bonds
between A and T or G and C. Probe DNA hybridizes with target DNA, the
analyte, according to precise WatsonCrick base pairing. Meanwhile, the
introduction of a DNA-assisted signal amplification strategy greatly
improves the signal production, acquisition, and amplification, thereby pro-
moting development of biosensors using DNA as a bioaffinity element.
In 1990, the Gold lab and Szostak lab reported the discovery of RNA
ligand for specific binding to target molecules, separately (Ellington and
Szostak, 1990; Turek and Gold, 1990). The nucleic acid ligand was then
named as an aptamer by Szostak, which originated from Latin word “apto”
xx Introduction

meaning “to fit.” Two years later, DNA ligand was screened as another type
of aptamer, which overcame the shortcomings of unstable RNA (Bock et al.,
1992). Since then, aptamer, especially DNA aptamer, replaces antibody as an
appealing bioreceptor. Aptamer exhibits the comparable binding affinity and
selectivity to antibodies, and also displays several advantages beyond antibo-
dies, including high thermostability, low immunogenicity, easy to undertake
large-scale synthesis, and modification. Unlike in vivo preparation of an anti-
body, an aptamer is screened from a large random DNA or RNA library
through in vitro selection process SELEX (systematic evolution of ligands
by exponential enrichment). Recently, improved SELEX of cell-SELEX and
tissue-SELEX have been developed in order to maintain the native folding
status of protein as that in vivo (Mi et al., 2010; Sefah et al., 2010).
A wide variety of molecules have been demonstrated as targets of apta-
mer from small molecules and proteins to whole cells. Aptamer-based bio-
sensors, known as aptasensors, are realized in different ways according to
the structural properties of the binding aptamer (Dhiman et al., 2017; Lan
et al., 2017b; Zhou et al., 2014). First, the aptamer is able to bind with a tar-
get and thus changes the external conditions (e.g., the mass, steric hindrance,
and refractive index) near the transducer surface upon a single binding event.
In this model, an aptamer interacts with the target molecule and forms a
composite structure similar to an antibodyantigen complex. Second, bind-
ing with the target molecule induces conformational changes of an aptamer,
and conformational change reduces the distance between the electrochemical
tag and electrode surface or fluorophore and quencher, thereby inducing
responses of electrochemical or fluorescent signal. Third, the target molecule
and complementary DNA compete to bind with an aptamer, and arouse sig-
nal changes from conformational switching induced by strand displacement.
Compared to conformational changes upon target binding, conformational
switching from strand displacement induces much greater changes in dis-
tances, thereby increasing signal response and reducing background interfer-
ence. Fourth, redesign of aptamers is employed to develop biosensors due to
their high ability for synthesis and modification. On one hand, a whole apta-
mer could be divided into two separate parts, while the split aptamers could
collaborate to work as a whole aptamer when binding with a target. On the
other hand, different aptamers could be combined together through synthesis
of a long nucleic acid strand, in which aptamers work to identify different
target, simultaneously.

I.1.1.5 Small Molecule Ligand

Binding of a small molecule to a protein is involved in several important
physiological processes in living organisms (Scott et al., 2016). The most
well-known example is reversible binding of oxygen and hemoglobin, which
plays a critical role in oxygen transportation through the circulation system.
 Introduction xxi

Binding of oxygen and hemoglobin stabilizes the mutual conformational adap-

tion for transportation purposes. Besides the example, small moleculeprotein
interactions are widely found at different stages of life based on conformational
accommodation, including enzymeinhibitor interaction, enzymesubstrate
interaction, and ligandreceptor interaction. Small moleculeprotein interac-
tion is a stable and high-affinity binding like antibodyantigen, while small
molecules benefit from low synthesis cost and increased chemical stability,
thereby providing new insight for bioreceptors.
One common use of small molecules is to detect enzymatic activity, in
which a small molecule is a native or artificial substrate or inhibitor of a tar-
get enzyme (Cao et al., 2010; Su et al., 2016). This detection is based on the
native function of enzymes in biochemical processes. The discovery of a
small molecule-ligand that binds to the target receptor with high affinity and
satisfactory specificity not only provides the opportunity for revealing the
function of protein in vivo, but also offers more choice for molecular recog-
nition and drug discovery. In this sense, the native small molecule ligand is
becoming a new type of recognition element in biosensing (Kubota and
Hamachi, 2015; Low et al., 2008; Pode et al., 2017). For example, native
ligand of folate receptor (FR), folate, is one typical small molecule-based
recognition element with high binding affinity (KdB1029 M). Because FR
emerges as a potential malignant tumor-selective target, folate is widely used
to detect FR-related tumors (Low et al., 2008). Besides limited native ligand,
synthetic binders are also extensively investigated with development of
supramolecular chemistry and molecular recognition. Generally, small mole-
cule binders are able to specially recognize a particular substructure, surface
“hot-spot,” or active center of the target protein (Pode et al., 2017). In most
studies, the interaction of a small molecule and target protein may induce
conformational changes of either the small molecule probe or target protein,
thereby arousing signal changes for quantitative measurement.
Besides small molecule chemical compounds, small molecule peptide
ligand is another appealing recognition element in the fabrication of biosen-
sors (Liu et al., 2015; Puiu and Bala, 2018; Reverdatto et al., 2015). Both
the peptide ligand and antibody are composed of amino acids, but the anti-
body needs a complex spatial structure to maintain native activity. Compared
to whole protein (e.g., antibody), a peptide ligand with biological simplicity
has low synthesis cost, high chemical and thermal stability, easy for large-
scale production, and modification. Short peptides originating from the
in vivo system were firstly used as recognition elements (Pavan and Berti,
2012). For example, RGD peptide that binds to integrin on the cell surface is
the most extensively used ligand for cancer detection. Afterward, artificial
peptides are selected from a random peptide library using phage-, ribosome-,
or mRNA-display strategies, which is quite similar to aptamer screening.
Therefore, the peptide ligand from in vitro selection is also called a peptide
aptamer. Due to the high stability of the peptide ligand, neither modification
xxii Introduction

nor immobilization has any influence on specificity and binding activity of

the peptide ligand, which is superior to antibody. Moreover, a peptide ligand
can bind to a specific site of the target and thus regulate the orientation of
the immobilized target, which is critical to promote communication of
immobilized protein and transducer for signal response.

I.1.1.6 Immobilization of Bio-Receptor

A biosensor translates the interfacial information of biological interaction
into a chemical or physical signal, which is used for quantitative measure-
ment of an analyte in a biological sample. Immobilization of the recognition
element on the transducer is a critical step to obtain interfacial information
upon a biological recognition event. Since the first GOD-based biosensor,
bioreceptors have been preferably immobilized on a transducer surface via
desired modification. In order to maintain the native biological activities of a
bioreceptor, several immobilization techniques have been developed with
high biocompatibility, stability, and conductivity, especially for electrochem-
ical biosensors (Bhakta et al., 2015; Gao et al., 2015; Gauchet et al., 2006;
Levicky et al., 1998; Li et al., 2014; Pramanik et al., 2012; Trilling et al.,
2013; Yang et al., 2012; Zhang et al., 2017). The immobilization methods
are divided into two categories: noncovalent immobilization and covalent
immobilization. Membrane encapsulation is an example of noncovalent
immobilization approach since the development of the first biosensor (Gao
et al., 2015). In the model of the first biosensor, GOD was modified on the
surface of an electric transducer (the electrode) by trapping it in membrane
materials. The membrane material thus became a hot spot in the develop-
ment of different generations of biosensors, which should be highly stable,
environmentally friendly, and suitable for signal conductivity. Biological
macromolecules and biocompatible polymers were firstly used for membrane
coating, including fish sperm DNA, polyethylene glycol (PEG), poly diallyl-
dimethylammonium chloride (PDDA), Nafion, and chitosan (Trilling et al.,
2013). Later, with the development of nanotechniques, nanomaterials
become a new-generation membrane material for bioreceptor immobilization,
benefiting from large surface-to-volume ratio, high surface energy, catalytic
activity, and adsorption ability (Bhakta et al., 2015; Zhang et al., 2017).
Different modification techniques also develop with the update of membrane
material, such as layer-by-layer, solgel, ionic liquids, and electrostatic
adsorption. Compared to noncovalent immobilization, covalent immobiliza-
tion is becoming more popular in recent years. Self-assembled monolayer
(SAM) technique is the most widely used technique for surface immobiliza-
tion of receptors, which presents increased stability, reproducible activity,
and is a much easier method to control the orientation of biomolecules on
the transducer interface (Levicky et al., 1998; Li et al., 2014; Pramanik
et al., 2012). The interaction of thiol group and gold surface is one
 Introduction xxiii

well-known covalent attachment procedure. Coating the transducer with a

gold thin film provides an available interface for the capture of biorecognition
elements containing a free thiol group, especially for thiol-functionalized probe
DNA and protein with cysteine. Another common covalent binding method
is based on condensation reaction, which exhibits better applicability.
Carboxyl group functionalized transducer surface can be combined with amino
group-containing recognition biomolecules through the reaction catalyzed
by 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine (EDC) and
N-hydroxysuccinimide (NHS). The immobilization of a bioreceptor is usually
based on the structure and function of recognition elements. For example,
antibody has a high requirement for native structure and function, which is
usually immobilized on a carboxylic group functionalized surface through a
condensation reaction. Compared to an antibody, there are more choices for
immobilization of peptide ligand on the transducer. Additional cysteine or
functional free thiol group help immobilization of peptide ligand through Au-S
interaction, while carboxylic group and amino group involved in peptide
ligand facilitate immobilization through covalent interaction. Moreover,
chemoselectivity and supramolecular interaction are also utilized to immobilize
peptide ligand based on the nature of amino acids, such as arginine and
phosphate group, aromatic amino acid and cucurbituril (Gauchet et al., 2006;
Yang et al., 2012).

I.1.2 Transducer
A bioreceptor for recognition and interaction with a target and a transducer
for signal production make up an integrated biosensor. A transducer trans-
lates recognition information from a bioreceptor mediated biological interac-
tion event into a readable signal for target detection. Based on different
signal sources, electrical, mechanical, magnetic, and optical biosensors are
most developed for biological measurements. Sufficient signal for biosensing
is generally produced in a manner of either label-free or chemical labeling.
For label-free detection, the analyte of interest usually has an intrinsic tag
for useful signal output, otherwise chemical labeling is required for efficient
distinguishing target from other interferes.

I.1.2.1 Electric Transducer

Since the first biosensor in 1962, the electrical biosensor is the most exten-
sively studied biosensor by making use of a chemically modified electrode
as a transducer. Electrochemical techniques provide various measurement
modes and thus extend the applications for different biosensing purposes,
including amperometry, potentiometry, electrical impedance spectroscopy,
field-effect transistor, and conductometry. Specifically, amperometry traces
current changes from redox reaction of an electro-active substance on the
xxiv Introduction

electrode surface using a conventional three-electrode system (Moreira et al.,

2017). A three-electrode system consists of a working electrode, a reference
electrode, and an auxiliary electrode. The common working electrodes are
noble metal-based electrode (e.g., platinum electrode, gold electrode, and sil-
ver electrode) and carbon-based electrode (e.g., glassy carbon electrode,
pyrolytic graphite electrode, and carbon paste electrode), which are easy and
stable for modification and reproduction. A reference electrode provides a
stable and well-known potential, such as saturated calomel electrode
(E 5 10.241 V) and silver chloride electrode (E 5 10.197 V). An auxiliary
electrode (e.g., platinum wire), also known as a counter electrode, constitutes
a complete circuit along with a working electrode as well as balances the
reaction on the surface of working electrode. The resulting current is propor-
tional to the amount of electro-active species on the electrode surface, which
is the best choice for target quantification. Potentiometry is based on poten-
tial differences between analyte and other references where there are no
obvious currents for target tracing (Tarasov et al., 2016). Ion-selective elec-
trode (ISE) is a typical transducer for such measurement by making use of
an ion-selective membrane for target identification (van de Velde et al.,
2016). The transducer converts ionic activity into a specific potential signal,
which is proportional to the logarithm of ionic activity based on the
NernstDonnan equation. Electrical impedance spectroscopy (EIS) is
another commonly used technique in electrochemical biosensor fabrication
(Bahadir and Sezginturk, 2016). EIS monitors the current flowing over sam-
ples under a given voltage, and electrical impedance is presented by voltage-
to-current ratio in the range from 10 kHz to 10 MHz. In general, the binding
of analyte and bioreceptor prevents electron transfer between electro-active
species and electrode surface for either steric hindrance or charge changes,
resulting in changes of electrical impedance. The field-effect transistor
(FET) regulates the electrical behavior of the device using an electric field,
which is also applied to trace ions with the assistance of an ion-selective
membrane (Sarkar et al., 2014). Conductometry is able to monitor enzymatic
reactions and biological recognition events through measurement of electro-
lytic conductivity, especially when using microelectrodes (Kucherenko et al.,
2015).

I.1.2.2 Optical Transducer

An optical biosensor is another appealing biosensor today due to low signal-
to-noise, excellent sensitivity, and fast response. The optical biosensor is
based on the development of different spectroscopic techniques, such as UV-
vis spectroscopy, fluorescent spectrometry, Raman spectroscopy, surface
plasmon resonance, and nuclear magnetic resonance. The natural properties
of analytes enable label-free, real-time, and simultaneous detection using
optical biosensors (Feng et al., 2014; Yan et al., 2018). Fluorescence
 Introduction xxv

spectrometry is the most powerful technique for fabrication of an optical bio-

sensor (Ma et al., 2016). Fluorescence spectrometry describes an optical
property of a substance that emits a certain wavelength of light after absorb-
ing energy from an external light source. Usually, the wavelength of emitted
light is longer than that of excitation light, ascribed to energy loss in the
luminescence process. The emitted light is called fluorescence. However,
most biological analytes do not have the fluorescent properties, so labeling
of fluorescent tags is necessary for biosensing. In this case, the information
of a biological recognition event is converted to a fluorescent signal for
quantification of the desired concentration of target molecules.
Chemiluminescence is another optical method to quantify the emitted light
for target measurements, but the energy resource for substrate absorption is a
chemical reaction while not external light as that for fluorescence (Roda
et al., 2016). Chemiluminescence is able to monitor specific biochemical
reactions at a sensor surface, presenting extremely high sensitivity with the
use of only a simple instrumentation. Similar to a fluorescent biosensor,
chemical labeling is sometimes required to characterize the reaction between
an analyte and a recognition system in chemiluminescence.
Electrochemiluminescence is an electrogenerated chemiluminescence, which
has attracted increasing attention in recent years (Chen et al., 2017b).
Different from chemiluminescence, electrochemiluminescence absorbs the
energy from an electrochemical reaction during the application of potential
in the solution. It is a highly sensitive and specific technique for the combi-
nation of the advantages of chemiluminescence and electrochemical tech-
nique, which has low background signal and is easy to control. Surface
plasmon resonance (SPR) originates from a particular optic phenomenon of a
nonradiative electromagnetic surface wave, which is a useful, label-free, and
real-time tool to identify the interaction of analyte and bioreceptor by tracing
the variations in the refractive index (Masson, 2017). However, label-free
detection mode limits the sensitivity of SPR biosensors. For example, SPR
can only distinguish the binding with a biological molecule that is larger
than 2 kDa, while smaller molecules cannot arouse sufficient signal readout
after recognition event. Besides, infrared spectroscopy, nuclear magnetic res-
onance, and surface-enhanced Raman spectroscopy have also been active
optical methods in recent years (Castro et al., 2014; Henry et al., 2016;
Neubrech et al., 2017).

I.1.2.3 Magnetic Transducer

A magnetic biosensor makes use of the magnetic properties involved in
interaction of the analyte and bioreceptor system (Lee et al., 2015; Zheng
et al., 2016). However, only a few biological molecules possess magnetic
properties except some iron-containing or metal-cluster proteins, thus chemi-
cal labeling is always needed for such a biosensor. Superparamagnetic
xxvi Introduction

nanoparticles are an important signal resource to detect targets of interest

after labeling on a certain recognition element. Much slower spinspin
relaxation (T2) time of nanoparticles promotes the characterization for excel-
lent contrast using magnetic resonance imaging (MRI). In the meantime,
magnetoresistive material is another signal resource for the fabrication of a
magnetic biosensor. The ultrathin ferromagnetic film facilitates construction
of a multilayered structure by overlaying a functionalization surface. The
interaction of magnetically labeled analyte and sensor surface is presented
through current changes under a given magnetic field. Besides the need for
chemical labeling, false-positive results from nonspecific adsorption on
nanoparticles always limit the application of magnetic biosensors. Even so, a
magnetic biosensor is still considered as a useful tool for biosensing as it is
easily incorporated with microfluidic systems.

I.1.2.4 Mechanical Transducer

In a mechanical biosensor, mechanical force and motion are utilized to trace
analyte concentration. Microcantilever and quartz crystal microbalance
(QCM) are two well-known examples of mechanical biosensors (Afzal et al.,
2017; Gopinath et al., 2015). Microcantilever measures the adsorption of tar-
get biomolecules by tracing either static deflection or change of oscillation
frequency, which is quite similar to atomic force microscopy. However,
QCM monitors frequency changes of a quartz crystal resonator upon adsorp-
tion of an analyte at a crystal surface. Although these mechanical biosensors
are always limited by sensitivity, they enable specific detection in a label-
free manner, and possess the advantages of easy fabrication and
functionalization.

I.1.3 Signal Amplification

One big challenge for a biosensor is to detect biomarkers at an extremely
low level in a given biological sample. Although good cooperation of biore-
ceptor and biotransducer enables direct measurement of target biological
molecules through signal readout, lack of sensitivity always leads to failure
in detection of targets of low abundance. Therefore, two strategies are com-
monly used to enhance detection sensitivity: target-based amplification and
signal-based amplification (Goggins and Frost, 2016). Target-based amplifi-
cation facilitates continuous generation of target molecules through a specific
cycle process. For example, PCR is a typical target-based amplification,
which amplifies only a few copies of target genes across several orders of
magnitude through a thermal-cycle process. However, the generation process
limits the extensive application of target-based amplification, which is only
suitable for amplified studies of target nucleic acid to date. In contrast, signal
amplification is much more universal than the target-based amplification
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 Introduction xxvii

strategy in the fabrication of a biosensor; this directly magnifies readable sig-

nals for achieving goals of ultrasensitive detection in biological samples.

I.1.3.1 Catalyst-Assisted Signal Amplification

Catalyst-assisted signal amplification is the most common signal amplifica-
tion strategy in the fabrication of a biosensor (Gianneschi et al., 2005;
Scrimin and Prins, 2011; Willner et al., 2008). Besides being a bioreceptor,
an enzyme is also a popular element in the design of a signal amplification
strategy. An enzyme is a natural catalyst involved in almost all metabolic
processes in living organisms, which accelerates the conversion of thousands
of substrates into a large number of products. If the product is a source of an
output signal, enzymatic catalysis generates a multitude of signal molecules
for enhancement of detection sensitivity. Enzyme-linked immunosorbent
assays (ELISA) is a good example of enzyme-based signal amplification
(Wei et al., 2016). Horseradish peroxidase (HRP)-labeled antibody is an
important source of detectable signal for ELISA, which catalyzes oxidation
of substrates (e.g., OPD, TMB) to generate an amplified amount of readable
optical signals. As a result, ELISA achieves a quite low detection limit at
pM level, which is a great progress when compared to that based on a single
signal labeling. For achieving high efficiency in signal amplification, high
stability, catalytic efficiency, and low cost of enzyme are usually required.
However, most enzymes are not stable for modification and only work in a
mild environment for the characteristics of native protein. Only a few
enzymes are suitable for signal labeling at present, such as HRP and alkaline
phosphatase (ALP). Besides, catalytic activity of an enzyme may decrease
dramatically beyond its optimal environment (e.g., temperature and pH).
Therefore, the limited choices of catalytic protein and strict requirement of
reaction environment restrict the application of enzyme-based signal
amplification.
Inspired by the fact that RNA (ribozymes) could work as a catalyst in a
biological system, it was believed that nucleic acid might have the potential
to be a catalyst. In 1994, the first single-stranded DNA with catalytic activity
was reported, which was named as DNAzyme (Breaker and Joyce, 1994).
Artificial DNAzyme is obtained from large-scale chemical synthesis with
low cost, and exhibits high thermal stability that is not susceptible to the
external environment and is able to catalyze substrates even at high tempera-
ture. The most appealing DNAzyme is reported by Sen et al., which is a
hemin-binding DNA strand and could work as a mimic of a peroxidase
(Travascio et al., 1998). Guanine-rich single-stranded DNA binds with hemin
and thus forms a quadruple structure. Accordingly, hemin-binding DNA
complex then exhibits increased peroxidase activity toward catalytical reac-
tions. Compared to native peroxidase, DNAzymes are much more flexible
and stable for structural design and surface modification, which could be
xxviii Introduction

divided into two or even four parts for different purposes. In this sense, arti-
ficial DNAzyme becomes a powerful substitute for native enzymes in design
of catalyst-based signal amplification strategy (Kosman and Juskowiak,
2011; Liang et al., 2017b; Willner et al., 2008).
Besides, nuclease-assisted target recycling is also an appealing strategy
for signal amplification in recent years (Gerasimova and Kolpashchikov,
2014; Miao et al., 2015; Yan et al., 2014). Nuclease catalyzes hydrolysis of
a phosphate diester bond within nucleic acids. With the cyclic enzymatic
reaction, thousands of amplified signals are produced in a few hours or even
several minutes. In nuclease-assisted target recycling strategy, a target-
binding event initiates nuclease-assisted cyclic digestion of reporter nucleic
acid probes, releasing an increased amount of readout signal molecules for
enhanced detection sensitivity. To realize the purpose, nucleases that selec-
tively digest a specific strand of duplex DNA or single-stranded DNA are
used, including Exonuclease III (Exo III), DNase I, T7 exonuclease, and
nicking enzymes. Exo III is a type of exonuclease that catalyzes the removal
of mononucleotides from the 3’-terminal of duplex DNA. Accordingly, a sig-
nal labeled at the 3’-terminal is released by Exo III-catalyzed digestion,
while another intact strand with a redundant terminal is protected from Exo
III-catalyzed digestion and then facilitates cyclic enzymatic digestion of the
reporter strand (Zuo et al., 2010). Similar to Exo III, T7 exonuclease cata-
lyzes the removal of mononucleotides from the 5’-terminal of duplex DNA,
which also promotes signal amplification with recycling of intact catalyst
DNA (Chen et al., 2013). DNase I is a nonspecific exonuclease that prefera-
bly digests single-stranded DNA, which usually helps to reduce background
signal for biological detection (Lu et al., 2010). Unlike the abovementioned
nucleases, nicking enzyme belongs to endonuclease family. Different from
the traditional restriction endonuclease that cleaves duplex DNA at a recog-
nition site; nicking enzyme only cuts one strand of duplex DNA, and leaves
another intact DNA for cyclic enzymatic reaction (Cao et al., 2012).
Nuclease-assisted signal amplification can be conducted at a constant tem-
perature with quite high detection efficiency, as the detection limit of aM
could be achieved within one hour. With the use of nuclease-assisted signal
amplification, a target molecule is not limited to nucleic acid, as the binding
of different biomolecules (protein or small molecules) to aptamer could also
be dissociated for enzymatic digestion reactions.

I.1.3.2 DNA Amplification—Assisted Signal Amplification

DNA-based target amplification enhances sensitivity of bioanalysis by
greatly increasing the actual amount of target DNA. Although the abovemen-
tioned PCR is the most mature technique of DNA amplification, the thermal
cycle that requires an expensive instrument to control temperature for differ-
ent steps always restricts its application. In this case, isothermal DNA
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given to the efforts of the Commission to enlighten the people as to
our mission, but having patiently awaited the termination of
business we returned to our search for the bead-work, only to find
that the finer specimens could not by any process of cajolery be
secured. Money meant nothing to the hillmen and we had no
substitutes in the way of gewgaws to offer them. The only one of us
who succeeded in getting a really good suit was Miss Anne Ide, and
her success was the result of a curious incident. She met a chieftain
gorgeously arrayed, and at a venture tried upon him the Samoan
greeting and a Samoan song which she had learned in her childhood
when her father was Chief Justice of the Samoan Islands. To her
great surprise the Bogobo answered and seemed greatly pleased. He
had already had conveyed to him the fact that the only thing the
ladies wanted was bead clothing, so he indicated to Miss Ide that he
would present to her his coat and pants, and without further ado,
and much to her astonishment, he began to divest himself of these
garments which she accepted with delight. The incident awakened
natural curiosity on our part as to the relation between the
Polynesian language of Samoa and the vernacular of the hill tribes
around the Davao gulf.
From Davao we proceeded on our journey around Mindanao,
sailing out into the open Pacific and up to the province of Surigao in
the northeast corner of the island.
 PICTURESQUE BEAD-BEDECKED
BOGOBOS OF THE DAVAO COUNTRY

The town of Surigao lies six miles up a swamp-bound, sluggish

river and we experienced, as we so often did in the whole course of
the trip, a sense of being in uncharted and therefore dangerous
waters. We embarked in a launch when the tide was high and had no
trouble in getting up to the village, but we were earnestly entreated
by the officer in charge of the launch to hurry with our business in
order that we might start back before the tide went out. He assured
us that it would be very difficult, if not impossible, to cross the bar at
the mouth of the river at low tide. His entreaties were in vain. The
Commissioners were engaged in interviews with Surigao citizens
which they could not or would not cut short, so the ladies and
children, having seen everything and met everybody, went back to
the landing and sat in the launch patiently waiting while the daylight
slowly disappeared. The launch captain was visibly agitated, and told
us time and again about what a hard time we were sure to have
getting back to the Sumner. And he was quite right.
The launch was not large enough to accommodate the entire party
so it towed a cutter which also was fairly well loaded. When the men
finally arrived, full of explanations and good-natured apologies, it
was pitch dark, but, being optimists, we shoved off into the river,
feeling sure that the fears of our commanding officer were
groundless.
After steaming merrily along for a few miles, becoming more and
more confident all the time, we suddenly got a shaking bump and
found ourselves fast in the mud. It didn’t take so long, however, to
get afloat again, and we were just congratulating ourselves that the
captain’s bug-bear of a sandbar was behind us when we felt a violent
impact followed by a terrifying sensation as if the keel were grinding
over rocks.
The captain swore softly and said something about striking “the
ruins of that old Spanish bridge,” then hurried forward to see what
damage had been done. The people in the cutter, riding the short
waves in our wake, were thoroughly alarmed and were clamouring to
know what had happened to us. We couldn’t tell them, but it
sounded very much as if we had torn the whole bottom out of the
launch. The engine had stopped; it was inky dark; the children all
began to cry; and, to add further discomfort to the situation, it began
to rain in torrents. The launch swayed sickeningly this way and that,
then the engine started again, whereupon came a most furious clatter
aft. There is no denying that it made us blanch with fear, but it
proved to be only a blade of the propeller which had been bent and
was striking the boat with each revolution.
Three times more we slid into the mud; the last time we stuck and
no effort that could be made would get us out, so we were forced to
abandon the launch and wedge ourselves altogether into the little
cutter. You may picture for yourself the scene of men, women and
children, in the rain and with no light save the faint flicker of
lanterns, dropping off a big launch into a small rowboat over an inky
stream supposed to be filled with crocodiles.
When we reached the mouth of the river the captain began to show
signs of nervousness, though he had been entirely self-controlled
throughout the worst of our troubles. We couldn’t see where we were
going, but we could distinctly feel that the open bay lay not far ahead
of us. What we wanted was to have the Sumner’s searchlight turned
on our path, but the only thing we had with us with which to convey
this desire to the ship’s officers were red rockets,—the last resort of
the sailor in distress. There was nothing else to do; the launch
captain began firing them off, and a weirder scene than was revealed
by their momentary glare can hardly be imagined. They produced the
desired effect, however, and in less than ten minutes a great shaft of
light, straight from the bridge of the Sumner, was sweeping the
banks of the river and bay shore and affording us just the kind of
assistance we required.
But that was not the end. Less than half way to the Sumner we met
a lifeboat, equipped with all the paraphernalia for rescuing us from a
watery grave, and manned by an excited crew in oilskins, who, under
the sharp commands of an almost frantic officer, were pulling in mad
haste for the river’s mouth. When they saw us they lapsed into a state
of utter disgust. They turned and rowed sadly back to the ship, and
afterward I overheard them exchanging very definite opinions as to
the possible future of a sailor who would burn red rockets when all
he wanted was a searchlight.
After calling at Cagayan Misamis, Dapitan, Iloilo, San José Antique
and Capiz, we made straight for Cebu. Cebu is, in rivalry with Iloilo
and next to Manila, the most important town in the Philippine
Islands. It is a receiving station for exports from all parts of the
southern islands and is altogether what is known as a “live” town. It
is the capital of the province of the same name which consists of a
single long island some two thousand square miles in area and with a
population (at that time) of nearly seven hundred thousand.
At Cebu we were rejoined by Chief Justice Arellano, who had left
us sometime before to go back to Manila. We were greatly interested
in his account of the effect of Aguinaldo’s capture and subsequent
treatment. The erstwhile insurgent leader was still in prison, but his
prison was made an honourable abode where he was permitted to be
with his family and to receive his friends. The mass of the people
would not, for a long time, believe he really had been captured. They
thought the report was an American fabrication to delude them and
to destroy their faith in Aguinaldo’s anting-anting,—or magic charm
against defeat. The shattering of that faith gave vast impetus to the
general peace movement and, though a few hundred rifles and
several insurrecto officers were still unaccounted for, and though
occasional outbreaks and the activities of marauding bands of
outlaws continued for a considerable length of time, the actual
organised insurrection had suffered a complete collapse.
The Commission kept Cebu on tenterhooks for a time as to
whether the condition of order in the province was such that they
could go on with the establishment of government there, and it was
interesting to watch the effect of this uncertainty. To be included in
the general organisation became at once the warmly expressed wish
of a majority of the people, and there was great excitement
throughout the town. Eventually Justice Llorente, of the Supreme
Court of Manila, a member of our party, and himself a Cebuano, was
appointed Governor of the province under the simple American
form, and because of his integrity and real patriotism, because of the
high regard in which he was held by the people, and because of the
enthusiasm and complete faith with which he entered upon his
duties, it was hoped that he would be able soon to lead his province
into the sensible paths marked out for it.
With Cebu and the problems of Cebu behind us, we felt that our
long trip was nearly finished. Bohol, Leyte, Samar, Albay, the
Camarines and Sorsogon, each in its turn brought us nearer to our
comfortable homes in Manila and to relaxation, for which we were
beginning to long.
Each district expected us to give them at least a day for business
and an evening for festivity, but this was not always possible. At
Sorsogon we found a veritable riot of decoration, with fine arches
and many flags and every indication that the town had spared no
effort to make our visit there a memorable event. In the evening,
beside the banquete and baile, there was to have been a torchlight
procession, with a triumphal car and a Filipino maiden as the
Goddess of Liberty. It was a great pity that we couldn’t stay, but we
had to sail that afternoon for Boak, so the programme had to be
advanced several hours.
The extraordinary car, or float, which had undoubtedly cost weeks
of skilled workmanship, came forth into the blistering sunlight
bearing the pretty brown girl in tinsel and white muslin, her long,
black hair almost wholly enveloping her as she held aloft the
flickering symbol of Enlightenment. It was a Filipino adaptation of
the “sacred torch” which we had ourselves been carrying throughout
the islands, and I felt that its production was a fitting climax to our
laborious progress.
Two days later when we landed in Manila, after organising
Marinduque and Batangas, we were able to look back upon a singular
experience, an expedition perhaps unique in history, with which was
ushered in a new era, not to say a new national existence, for the
people of the Philippine Islands.
 CHAPTER IX
THE WILD MEN’S COUNTRY

I should like to say here, by way of explanation, which may or may

not be necessary, that I am not trying in this narrative to pose as a
woman endowed with an especial comprehension of such problems
of state as men alone have been trained to deal with. I confess only to
a lively interest in my husband’s work which I experienced from the
beginning of our association and which nothing in our long life
together, neither monotony, nor illness, nor misfortune, has served
to lessen; and it would be practically impossible for me to write a
record of memories in which he did not figure very largely.
In the settlement of American control in the Philippine Islands Mr.
Taft, first as President of the first legislative Commission and, later,
as Civil Governor, had to contend with a varied and complex
resistance which it would be difficult for one not experienced in
politics to comprehend. If it had been Filipino resistance only it
would have been fairly easy to overcome, but Filipino resistance was
indirectly sanctioned and directly assisted by a strong opposition in
the United States to what seemed to us who were on the ground to be
the only sensible and really patriotic measures possible under the
circumstances.
For reasons which I have tried to convey, as clearly as I am able,
my husband was not in favour of a continuation of military rule in
the Islands beyond the time when military activity was imperative,
nor was he in favour of abandoning a problem which grew daily more
difficult and more complicated. So he and his colleagues persisted in
the tremendous task of settling a whole people under a sane and
sensible form of government.
 PHILIPPINE NON-CHRISTIANS. A
BONTOC IGORROTE (TOP, LEFT,) A
MORO AND TWO KALINGA CHIEFS
(WITH SHIELDS)

The trip through the southern islands was particularly valuable to

them in that it gave them first-hand, working knowledge of existing
conditions in every province. They immediately set about revising
their original Provincial code in accordance with requirements which
they were able to discover only through personal investigation, and
at the same time they took up the grave business of establishing a
sound judiciary.
There was always something new to be talked over at our family
table, or during the long evening hours on the verandah overlooking
the Bay and, in spite of the fact that much of our “news” presented
itself in the form of fresh delays and exasperating difficulties, life was
very entertaining.
Not long after we returned from our trip through the South Mrs. J.
Franklin Bell invited my sister Maria and me to go with her on an
expedition, on which she expected to accompany her husband,
through the mountains of northern Luzon which are inhabited by
non-Christian tribes only. General Bell was commander of troops in
the North and this was to be an inspection trip. It meant several
weeks on horseback, over dangerous trails where, in parts at least, no
white woman had ever been, but we were most anxious to go. The
trouble was that I had never ridden in my life, so I looked with
considerable trepidation to the prospect of a long and necessarily
intimate association with a horse. I brought the proposition up in
family council and my husband advised me, by all means, to go. I
should probably have gone without this advice, but it was comforting
to have it because if anything happened I could “blame it all on him.”
In fact, I began to do this even before I left. When my courage
dwindled a little I promptly told him that it was all his fault; that if
he hadn’t urged me to go I never should have thought of such a
thing; but that as long as I had promised I should have to see the
adventure through, though I knew I should never survive it. He only
laughed and assured me that we would have a glorious time and that
the trip would do us “all the good in the world.”
Major Stevens, who expected to accompany us, brought me an
American horse, of formidable dimensions, and volunteered to
superintend my first encounter with him. He was as gentle as a lamb.
I wouldn’t let him go faster than a walk the first evening and the fact
that I was pretty stiff at the end of my ride made me almost hopeless.
The second evening I let him out a little, and began, much to my
surprise to enjoy the exhilaration of the exercise. By the third
evening I had progressed so far that I decided for myself that the
poor old beast had no speed in him at all.
We took a Spanish steamer, the Salvadora, from Manila up to
Vigan, where General Bell was stationed, and, though I am glad to
have had the experience, I shouldn’t care to repeat it. When we got
on board we were shown at once to a most promising-looking
stateroom, quite spacious, and with four berths in it. The trip to
Vigan was to take from Thursday to Saturday and we were glad to
note that we were going to be quite comfortable. But our self-
congratulations came to a sudden end. Upon inspection we found the
room was indescribably dirty, the beds were without sheets, the
pillows were like rocks, there were insects galore, and the
thermometer stood at 110 degrees. Ventilation was out of the
question because the room opened into a sort of public saloon where
innumerable Filipinos, in various states of undress, slept, stretched
out on the floor, on the tables, on chairs, on anything that could
serve as a resting place. The second night I got the Captain’s
permission to sleep on the bridge, since the decks, too, were covered
with a miscellaneous crowd and were rendered additionally
uncomfortable by odoriferous strings of cabbages and other
vegetables which hung from the awnings.
The food on the ship was all Spanish; indeed, I might say, terribly
Spanish; still, I was rather used to it and didn’t mind much as long as
I could get into a wind-swept corner of the deck to eat it. But there
were some American women on board who had just come out from
the United States and they complained violently.
We were put ashore early Saturday morning; much earlier, in fact
than we had been expected to arrive. We had to drive three miles
before we reached the Bells’ house, and when we did get there we
were delighted to find that they were just having breakfast. They
were eating real, human food and, however heroically we had
adapted ourselves to the peculiarities of Spanish cookery, there was
nothing we stood so much in need of. They were a most homelike
and comfortable-looking party. Besides the General and Mrs. Bell
there were two young officers, Mr. Wilcox and Mr. Nolan, and a
young lady whose name was Miss Bubb, a daughter of General Bubb,
and whose general characteristics had won for her the nickname of
“Bubbles.”
The first thing we learned was that Mrs. Bell would, after all, be
unable to go with us on the trip through the mountains. She was not
at all well and the doctor had forbidden it. We were greatly
disappointed. Mrs. Bell is so jolly and full of fun that she is an
addition to any party, and on such an expedition as we were
contemplating we were sure to miss her tremendously. But, the party
was all made up. General Bell was to take command; “Bubbles” was
going; then, in addition to my sister and me, there were Major Rice,
Major Stevens, Captain Shearer and Captain Haight—eight in all.
First let me say that the northern part of the island of Luzon bears
just about as much resemblance to the rest of the archipelago as the
Alps bear to the plains of Nebraska. We began to notice the
difference even at Vigan, though Vigan is at sea-level and is as hot as
a sea-level town is supposed to be in that latitude. But it feels and
looks like a little foreign city; foreign, that is, to the Philippines. Its
houses are well built of ancient-looking stone, with heavy red-tiled
roofs; its streets are narrow and crooked and it has a fine plaza filled
with fire-trees which, when I saw them first, were in full bloom.
There is no way to describe the magnificence of a grove or avenue of
fire-trees. They make a veritable cloud of flame which, seen against a
background of blue hills, or overhanging the mouldy, old-world grace
of a Spanish church and convent, fairly “takes one’s breath.” The
world-famed cherry trees of Japan, wonderful as they are, seem pale
and soulless in comparison. I wonder the Spaniards didn’t line the
streets of Manila with fire-trees and make for themselves the
reputation of having created the most amazing city in the world.
While we were at Vigan, and before starting on the long trip, we
made an excursion to Bangued, in the province of Abra. Mrs. Bell
went with us. This town, a short time before, could be reached only
by raft up the Abra River, no launch ever having been built that could
go through the rapids, but the establishment of an Army post made
necessary the building of a piece of road which shortened the journey
at the Bangued end. The first part of the trip, however, had still to be
made by water and all the supplies for the soldiers were sent up on a
fleet of twenty or more rafts which started out together every
morning. When there was a breeze each of them would run up a sail
of bright, striped Igorrote cloth.
We had a grand raft with a bamboo awning. And there were
comfortable rattan chairs, to say nothing of a picnic luncheon and a
carefully wrapped and jealously guarded box of ice. Ice was the rarest
of all luxuries in the provincial towns of the Philippines in those
days.
ON THE LONG TRAIL IN NORTHERN
LUZON. MRS. TAFT SECOND FROM
THE LEFT

MR. TAFT AND CHARLIE ENJOYING

THEIR FAVOURITE EXERCISE

We moved very slowly against the current of the swift-flowing

river, but we had no desire to hurry. It was really enchanting. From
narrow, pebbly beaches on either bank rose rugged cliffs which
seemed to tower mountain high, throwing deep shadows into the
canyon and leaving only a narrow ribbon of sky above us. And these
cliffs were hung with a tangled undergrowth through which small,
white waterfalls rushed and rustled. Where the river broadened, here
and there, we came upon groups of bright-clad natives who regarded
us with great curiosity, and at one place we saw half a dozen women
starting up the steep bank with graceful brown water-jars balanced
on their heads. Each one was carrying at least six, one on top of
another, and all of them full. How they managed it was beyond
comprehension. We watched them until they were out of sight and
not one of them ever raised a hand to her head. As a matter of fact,
they were nonchalantly smoking and chattering away as if they were
quite unconscious of their burdens, though the slightest
unrhythmical motion would have spelt disaster for them all.
At eleven o’clock we reached the village where the road begins and
the whole population gathered around in curious groups and gazed
at us. White women were still a novelty in that region and I’m sure
we looked much more peculiar to them than they looked to us. There
were crowds of school children from the new American school, and
one very much embarrassed little girl, who had had her English book
only about four months, read some English for me very nicely. Likely
as not that same little girl has by this time won a normal school
certificate and is herself teaching English in an “American” school.
Such is the history of many of her generation.
When we reached Bangued the young men in the command of
Major Bowen, who was our host, gave up their house to the ladies,
and we had three comfortable beds, with mosquito nets, in a large,
airy room. It was a fine afternoon for a siesta because it rained in
torrents for the rest of the day and the patter of rain on nipa thatch is
a soothing sound. The young men’s house was just across from the
Major’s and by evening the street was such a river that we had to be
carried over for dinner. But nobody minded; and we enjoyed even
the music of the native band which stationed itself down under our
windows and enlivened the occasion with a wonderful medley of
sound. When the band-men came upstairs for refreshments Mrs. Bell
and two of the young officers ran down and tried their powers on the
instruments, and I can only say that the result was joyful
pandemonium.
 The next morning we left our hospitable hosts and, escorted by
Lieutenant Ingram, made the return trip all the way down the river.
The water was high and, though it had taken us an interminable time
to go up, it took only three hours and a half to go down; and some of
the rapids were most exciting. We took our lunch basket and chairs
ashore on a lovely, green, shaded knoll and dallied there for several
hours. Only a month before nobody, who was not compelled to, ever
went over this route on account of the danger of being shot, but the
last of the Abra insurgents had surrendered, and so safe did we feel
that we were absolutely unarmed.
If I should try to write a detailed account of this expedition I am
afraid I could not avoid conveying the idea that we encountered
nothing but a continuous downpour. It was the “rainy season” and
we were wet most of the time, but Mr. Taft was right when he
promised that we would have a glorious time and that the trip would
do us a “world of good.” Down in the heat and the political turmoil of
Manila I was taking things much too seriously, while up in the far-
away north there was nothing to do but dismiss all worry and accept
things as they came along. After we left Vigan on the long trail the
only way we could get even a letter through was by messenger who
had to travel hundreds of miles through a most difficult country. So I
enjoyed myself thoroughly, as did every one else in the party,
hardships and physical discomforts seeming only to add to our
gaiety.
At first I thought that my riding lessons in Manila were not going
to do me much good. We had had a most luxuriously easy time in the
beginning. We left General Bell’s house in an Army ambulance,
instead of on horses, for our first day’s journey on the “long trail.”
General Bell was in command and he knew what he was doing. All he
had to do was to issue orders; we obeyed. That is what it is to go
camping with a soldier. One learns what discipline means.
We were permitted to take with us only such things as were
absolutely necessary. Even then, the “absolute necessities” which we
eventually discarded as useless impedimenta would have made a
long list. Everything was done up in waterproof bundles and when
we started out these were stuffed so full that they would hardly
fasten, but they grew slimmer as time went on. The most important
articles, we found, were our slickers and wraps. It was wet and cold
and we had to have them, but all our toilet appurtenances together
went easily into Miss Bubb’s saddle-bags.
The first day we forded a river—the same river—several times, and,
finally, we had to cross it on a raft which was so small that it could
carry only one thing, or one person at a time. My sister, Miss Bubo
and I sat on the bank above the ford for more than two hours waiting
for all our things to get across. While we waited many natives came
along driving carabaos, and it was amusing to see the two-wheeled,
awkward carts hustled onto the swaying raft—one thing after another
falling into the river—while each poor old carabao was forced to
swim, dragged along by his master who held fast to a string attached
to a ring in the animal’s nose. If I had been able to speak the dialect I
would have said: “Your friend the Carabao, being a water-buffalo,
could probably swim the river much more easily without your
assistance.” I have had to look on and suffer at many things in the
Philippine Islands merely because I was unable to speak a dozen-odd
different dialects. In the provinces Spanish was seldom of any use
because the common tao knows little or nothing of it, and it is with
the common tao that one wishes there to communicate.
On our first day’s journey we did thirty-seven miles in a jolting
Army wagon, but the air was so invigorating, and we were having
such a good time, that we were not exhausted. We didn’t even
murmur when we were told to be ready to start at four the next
morning.
This was at Candon and we were joined there by Major Stevens,
which made our party complete. The next evening, at Concepción, we
camped in a lovely, new nipa-thatched house which had been built by
a man who was known generally as “Windy” Wilson, an Army
captain. We were extremely thankful for the shelter, because it was
raining as it can rain only in northern Luzon and we had every
reason to believe that this would be the last house we would be
permitted to occupy for many a day. We were striking straight into
the mountains and our shelter-to-be was a small field tent slung on
the cargo saddle of a commissary mule.
Captain Wilson’s house was quite spacious. It had two rooms; one
small and one large one. The ladies slept in the smaller room on
Army cots, while the four stalwart officers of our military escort
stretched themselves out on blankets and slickers on the split
bamboo floor of the larger room. The walls and partitions were of
woven nipa palm leaves, known locally as suali, while the two
windows were made of braided bamboo and were set in grooves so,
when we wanted to open them, all we had to do was to give them a
gentle shove. There were no “trappings of civilisation,” but we
managed to be perfectly comfortable.

AN IGORROTE HEAD DANCE, AND A

COMPANY OF CARGODORES WITH
THEIR DOGS, WHICH ARE TO BE
KILLED FOR FOOD

The next day, before the sun was very high, we found ourselves in
the midst of mountain-tops, on a trail which rose in great upward
sweeps around the densely wooded slopes, to an altitude of 5600
feet. By this time we were all on horseback with eight Igorrote boys
behind us carrying a sedan chair to be used in case of accident or a
dangerous washout on the trail. I wish I could describe the
magnificence of the scene which lay all about us when we reached
that amazing summit. General Bell, who had been all through the
Rocky Mountains, the Yellowstone, and the Yosemite Valley, said
there was nothing that he had ever seen which could compare with it.
And its grandeur is accentuated by vivid colouring. The Igorrotes
have, for hundreds of years, been building extraordinary rice terraces
and these have gradually climbed the mountains until, in some
places, only the rugged crests are left uncultivated. The terraces are
as symmetrical as honeycomb and are built in solid walls of finely
laid masonry out of which grow ferns and tangled vines. The brilliant
colour of the young rice fairly glows against the dark greens of pine
trees, of spreading mangoes, and of tropic forest giants whose names
I do not know. And wherever one looks there are peaks, jagged sunlit
peaks which rise from sombre valleys upward into a strange light
whose every ray seems to shine in its own individual hue. In the far
distance we could see the ocean, with white breakers dashing against
the cliffs; while in the valley below the Santa Cruz River, though
actually foaming and dashing through its winding, rocky bed,
seemed to us to be lying still, without motion of any kind, or sound.
In my diary, which I kept on that trip, I find that at each stopping
place I have solemnly set down the observation that: “the scenery to-
day was the finest we have yet found”; and when we reached Sagada I
took the trouble to record for my own future reference that: “I shall
not rest until Will has seen it.” He never has.
At Sagada we found ourselves quite far up in the Igorrote country,
where Filipinos as a rule, do not go. We had come from Cervantes
over a trail where the horses cautiously kept to the inside, and where
we were told to let go of our inner stirrups so, in case a horse went
over the edge of the precipice its rider would have a chance of falling
clear on the terra-firma side instead of being hurled out into open
space. There are a great many people who have to be taken over such
trails blind-folded, but there were no dizzy-heads among us, and as
each turn of the way revealed to us different and more wonderful
views, we filled the day with exclamation points.
Here and there we met bands of Igorrotes, marching “Indian file,”
carrying great bundles of rice up short-cut mountain trails, which
wound through the rice terraces and were “as steep as the side of a
house.” All the men had long, murderous-looking spears, while the
women were evidently the burden-bearers. Along the main trail we
came, now and then, upon a company of men leading home a
whimpering and pitiful little pack of very thin dogs. We knew these
were to be killed and eaten and, naturally, the thought was sickening,
but in the Igorrote country the dog-loving white man has to get used
to this. Some day, perhaps, it will be different, but not until herds
and flocks have been substituted and entirely new ideas have
patiently been instilled into the minds of these people. For the time
being dog flesh is their most cherished article of diet.
I wish it were known just where these curious wild tribes came
from; just what their race history is. They are as unlike Filipinos as
American Indians are unlike Englishmen. They have but one thing in
common with the Filipinos, and that is their colour, which is a soft,
dark brown. There is hardly an American who has ever lived among
them for any length of time who has not a real admiration and
affection for them and yet, to all intents and purposes, they are
naked savages. They are most amenable to civilising influences. They
take to education eagerly. They are, in their physical development,
beautiful to look upon—when they are cleaned up—perfectly formed,
straight and muscular, with features strongly marked and with wide,
clear eyes which inspire confidence. They are entirely fearless; and
they are loyal to the “last ditch.” Also, it is these same
incomprehensible “naked savages” who have built the thousands of
acres of rice terraces which are a marvel and a mystery to every
irrigation expert or technical engineer who has ever seen them.
 Bureau of Science, Manila.

VIEWS OF THE EXTRAORDINARY IGORROTE RICE

TERRACES. PRACTICALLY ALL OF THE NORTH CENTRAL
LUZON IS CULTIVATED IN THIS MANNER

Bontoc, which we reached after a day’s weary, wet riding over

slippery trails from Sagada, is the capital of what is now known as
the Mountain province. For the first time in their known history the
Igorrotes are united under one central government, each tribe having
its lieutenant governor—an American always. There are the
Benguets, the Bontocs, the Ifugaos, the Ilongots, the Kalingas and
others, and they have been engaged in inter-tribal warfare since time
began, their chief pleasure being derived from the taking of each
other’s heads. When I went into the Igorrote country head-hunting
was still in full force and houses were still decorated with festoons of
human skulls, while no man ever ventured forth, even to his rice
fields, without his spear and shield and head-axe. They all carry
spears even yet, but head-hunting, having been made by the
American government a capital offence, is not so popular. Mr. Dean
C. Worcester, as Secretary of the Interior, in direct charge of all wild
tribes, actually succeeded in introducing substitutes for the sport in
the form of baseball and other inter-tribal athletic contests and
peaceful, though rough and strenuous pastimes. For fourteen years
Mr. Worcester was to these children of the hills a most highly
respected Apo-apo,—chief of chiefs.
Miss Bubb, my sister Maria and I were the first white women who
ever set foot in Bontoc and to say that we created a sensation is to
describe our reception too mildly. We were the guests of three
American miners who had a comfortable house and who, having
lived among the Igorrotes for a long time—one of them for more than
a year without visiting civilisation—could give us much interesting
first-hand information. The people gathered around us in hordes,
but they kept at a respectful, not to say a reverential, distance. I think
they were afraid of us; especially the women, not one of whom would
let us look at her baby. But we were used to that. Many Christian
Filipinos believe firmly in the “evil eye.” There was one little dwarf
who was bolder than the rest and who followed us everywhere we
went. He was like a little, brown, toy-child, beautifully formed, and
looking not more than one year old, but we were told that he was at
least fourteen.
Everybody wanted to give us things. The evening I arrived I
received a present from one of the headmen, of three live chickens,
and the next day, as we were picking our way through the native
village, another man ran after me and, very graciously and gracefully,
presented me with two fresh eggs. We learned to say “mapud,” which
means “good,” and, in connection with smiles and gestures, found it
served us famously for all purposes of social intercourse.
Bontoc is in a deep valley, on the bank of a wide, swift river and
surrounded by close sheltering hills, so it is not as cold as it is in
Sagada and some other places we visited; but it is cold enough, and I
failed to understand how the natives could live in a state of almost
complete nakedness. But they do and, in fact, all these people do,
even in the coldest regions. The Bontoc Igorrote wears a very bright-
coloured clout called a “G-string” with a heavy, brass chain around
his waist, while his long, black hair is tucked into a little, flat, straw
hat which is fastened, in some mysterious way, on the back of his
head. They nearly all wear heavy, brass earrings which make their
ears unsightly, and the Bontoc “dandy” usually has a long, black,
homemade and half-smoked cigar tucked behind one ear for all the
world like the pencil of an absentminded bookkeeper.