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Immunosensing for Detection of Protein
Biomarkers
Immunosensing for
Detection of Protein
Biomarkers

Huangxian Ju
Guosong Lai
Feng Yan
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

Copyright © 2017 Elsevier Ltd. All rights reserved.

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(other than as may be noted herein).

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About the Authors

Huangxian Ju
Changjiang Scholar, Professor of Chemistry, and Director of the State Key
Laboratory of Analytical Chemistry for Life Science, Nanjing University,
Nanjing, P. R. China
hxju@nju.edu.cn
Guosong Lai
Chutian Young Scholar in Hubei Province
Professor of Chemistry, Hubei Normal University
Huangshi, P. R. China
gslai@hbnu.edu.cn
Feng Yan
Leading Talent in Jiangsu medicine.
Deputy Director, Department of Clinical Laboratory and the Province Key
Laboratory of Cancer Molecular Biology and Translational Medicine, Jiangsu
Cancer Hospital; Senior Researcher, Jiangsu Institute of Cancer Prevention and Cure
Professor, Nanjing Medical University
Nanjing, P. R. China
yanfeng2007@sohu.com
Preface

Immunosensing involves the coupling of immunoreactions to appropriate ­transducers

for producing analytical signals, which leads to excellent specificity and high
­sensitivity for selective detection of protein biomarkers in real samples. In recent
­decades, rapid development has seen made in immunosensing and immunoassay
methods with various detection formats and wide applications in different fields, such
as clinical, industrial, environmental, food, and agricultural analyses. In the medical
field, considerable multidisciplinary efforts have been devoted to the development of
precise, rapid, sensitive, and selective immunosensing of disease biomarkers and/or
biomarkers panel for meeting emerging needs of early screening and diagnosis of dis-
eases, monitoring of curative effect, and reliable point-of-care diagnostics in precision
medicine, although there are still problems concerning the assay of analytes in clinical
application due to the stability of immunosensing devices and nonspecific adsorption
from complex sample matrix. A large number of research works in immunosensing
methodology have been reported (more than 78,000 since 2000). These methods are
generally simple to utilize and easy to realize with automation, digitization, and min-
iaturization. Therefore, immunosensing is now becoming one of the most widely used
analytical technologies in protein biomarker detection.
Although a large number of academic papers in immunosensing and immunoassay
have been published in different journals recently, it is still difficult or time-­consuming
for researchers, especially the beginners, to have a good understanding of the princi-
ples, methods, and research progress of immunosensing in a wide scope. The goal of
this book is to not only offer a survey of the principles and methods of immunoassay
and immunosensing, but also to present the latest achievements and detection strate-
gies in different aspects such as electrochemical immunosensors, n­ anoprobe-based
immunoassay, chemiluminescence immunoassay, electrochemiluminescent immuno-
assay, multianalyte immunoassay, optical imaging immunoassay, signal amplification
for immunoassay, and so on. More importantly, based on the experience of these au-
thors, the aim is also to bridge the common gap between research literature and new
research ideas in order to develop immunosensing methodology.
This material is presented in nine chapters, covering all the authors’ study topics
of immunosensing methodology. Some experts who received the PhD degree from
Ju’s group participated in the writing of some chapters, including Dr. Wei Cheng
(Chapter 2), Dr. Zhanjun Yang (Chapter 5), Dr. Dajie Lin (Chapter 6), Dr. Zhifeng Fu
(Chapter 7), Dr. Jie Wu (Chapter 8), and Dr. Kewei Ren (Chapter 9). This book also
contains the previously published works of Dr. Zong Dai, Dr. Jiehua Lin, Dr. Dan Du,
Dr. Lina Wu, Dr. Xuan Liu, Dr. Wenwen Tu, Dr. Shengyuan Deng, Dr. Chen Zong,
Dr. Hong Liu, Dr. Chuan Leng, Miss Hua Yu, Miss Fang Tan, Miss Qiunan Xu, Miss
xPreface

Lisong Wang, Miss Jie Xu, and Mr. Jie Li during the time they studied in this group.
We are very grateful to all these members for their contribution.
This book is one of the monograph series published by the first author (Huangxian
Ju) or with the coauthors, including Electroanalytical Chemistry and Biosensing
Technologies (Science Press, 2006, Chinese), Bioanalytical Chemistry (Science
Press, 2007, Chinese), Electrochemical Sensors, Biosensors and Their Biomedical
Applications (Academic Press, Elsevier, 2007, English, and Chemical Industry
Press, 2009, Chinese), NanoBiosensing—Principles, Development and Application
(Springer, 2011, English, and Science Press, 2012, Chinese), and Nucleic Acid
Detection: Methods for Analysis of DNA and microRNA (Intellectual Property Press,
2015, Chinese). This book offers a good reference for a broad audience, including peer
researchers and graduate students who have similar research interests. It can provide
readers with new research ideas to develop immunosensing methodology. The book
can also be used as a graduate-level textbook for those studying for the master degree
in analytical chemistry and clinical laboratory.
We are fortunate to have the opportunity to undertake this project. We warmly
acknowledge the gracious support of our families. Finally, we also thank Elsevier’s
editors for doing a remarkable job to publish this book.
Huangxian Ju
Nanjing, PR China
Guosong Lai
Huangshi, PR China
Feng Yan
Nanjing, PR China
Introduction
1
Millions of people throughout the world face the risk of malignancies, which have
been one of the leading causes of mortality. In cancer, as tumors develop, the cells or
the organs can release specific proteins into the circulation system. The levels of these
proteins as tumor-related antigens in serum are associated with the stages of tumors
and can therefore be used as tumor biomarkers for screening and clinical diagnosis of
cancer [1]. Hence, reliable, sensitive testing for these tumor biomarkers is crucial in
early clinical diagnosis and in the evaluation of the recovery of patients with certain
tumor-associated diseases. Compared with the conventional biochemistry-, immu-
nology-, and molecular biology–based methods, immunosensors, which combine the
unique advantages of immunoassay and biosensor, have been recognized as significant
and received rapid development in the last decades [2,3].
This chapter briefly introduces the main principles of immunoassay and immu-
nosensor as well as signal labels and the immobilization method of immunoreagents
during the immunoassay of protein analytes. Future perspectives on immunosensors
in the field of protein analysis are also evaluated.

1.1 Immunoassay
Immunoassay is a highly selective bioanalytical method that measures the presence or
concentration of analytes ranging from small molecules to macromolecules in a solution
through the use of an antibody or an antigen as a biorecognition agent. The theoretical
basis of immunoassay is the antibody-antigen immunoreaction as well as its coupling
to appropriate transducers for producing an analytical signal. Thus high specificity is
the unique advantage of immunoassay methods, which results from the use of purified
­antibodies and antigens as analytical reagents. An antibody is a protein (immunoglobulin)
produced by b-lymphocytes (immune cells) in response to stimulation by an anti­
gen. Immunoassays measure the formation of antibody-antigen complexes by labeling or
­labeling-free format [4]. Due to the signal transduction and amplification by using a label-
ing system (e.g., enzyme label), high sensitivity can be also achieved for immunoassays.

1.1.1 Antigen and antibody

The science of immunology is based on an organism’s ability to generate the bio-
logical effect known as the immune response. In higher forms of life, particularly in
mammals, the immune system is a complex mechanism in which identification and
communication take place in the blood and lymph. When a foreign substance (antigen)
enters the body of an advanced animal, certain proteins (antibodies) are synthesized
to identify the invader and to prohibit its harmful effects. Antibodies show very high
specificity and binding constants toward their corresponding antigens.
Immunosensing for Detection of Protein Biomarkers. http://dx.doi.org/10.1016/B978-0-08-101999-3.00001-3
Copyright © 2017 Elsevier Ltd. All rights reserved.
2 Immunosensing for Detection of Protein Biomarkers

Antibodies are divided into five classes (immunoglobulin IgG, IgA, IgE, IgM,
and IgD) based on their structures and biological functions. Of the five classes of
antibodies, IgG is the class used the most frequently for immunoassays because it
exists at the highest level and is readily available. Generally, the structure of IgG is
represented by a Y-shaped figure consisting of four polypeptide units (Fig. 1.1). Two
of them are identified and known as the heavy chains with a molecular weight of
55,000–60,000 Da. The other two sequences are light chains with a molecular weight
of 20,000–24,000 Da. The two double-ended segments of the Y are denoted as Fab
fragments and are the sites at which antibody binds with antigen. The variable and
hypervariable regions of Fab create an active portion that recognizes a specific area of
the antigen. The singular segment at the other end of the Y shape is known as the Fc
fragment, which cannot bind with antigen but has the ability to affix to the cell surface
and to pass through the placenta [5].
The antigen molecule detected by immunoassay is often referred to as an “analyte.”
It may be the natural antigens including such macromolecule substances as proteins
and nucleic acids, or the haptens as some substances with low molecular weight, typi-
cally <1000 Da, as long as proper antibodies that have the adequate properties for the
assay can be developed.
Protein biomarkers for tumor diseases are generally produced by cancer or by other
cells of the body in response to cancer or certain benign (noncancerous) conditions.
Most tumor markers can be made by both normal cells and cancer cells; however, they
are produced at much higher levels in cancerous conditions. These substances can be
found in the blood, urine, stool, tumor tissue, or other tissues or bodily fluids of some
patients with cancer [6]. Therefore, the development of immunoassay methods for se-
lective and accurate measurement of protein biomarkers has shown great importance
for clinical cancer diagnosis.

Complementarity
determining
regions Variable
Antigen
binding site
Constant
Fab region

Light chain
Disulfide
linkages Heavy chain
Hinge
region
Fc region

Fig. 1.1 Schematic representation of an immunoglobulin G (IgG).

Introduction3

1.1.2 Immunoassay format
Immunoassays come in many different formats and variations [7], including labeling
and labeling-free formats. The labeling-free format is based on the immunoreaction
to directly produce the observable detection signal, and the labeling format needs to
use some signal molecules to label the immunological reagents such as antigen or an-
tibody for producing detectable analytical signals on the immunoreaction. The latter
can be divided into homogeneous and heterogeneous immunoassays. In homogeneous
immunoassays, the assay strategies do not require the separation of the immunocom-
plexes from unbound immune reagents. This approach includes agglutination [8], cap-
illary electrophoresis [9], fluorescence polarization [10], and fluorescence resonance
energy transfer-based immunoassays [11]. The other formats described as heteroge-
neous immunoassays impose the initial separation of the immunocomplexes from the
unbound immune reagents. In heterogeneous immunoassays, the immunocomplexes
are bound to a solid substrate such as microplate or immunosensor’s surface, allowing
the retention of the molecules of interest while the unbound ones are washed out of
the system. Heterogeneous assays, although requiring a longer run time and more
complex manipulations, are more versatile, more sensitive, and more specific. Thus,
heterogeneous immunoassays are inevitably more popular than homogeneous ones.
Competitive and sandwich methods are the two most popular heterogeneous immu-
noassay strategies. In both strategies, the “label” or “tag” is utilized as the signal probe
for quantifying the antigen-antibody reaction. In a typical competitive immunoassay,
shown schematically in Fig. 1.2A, the mixture of sample antigens (Ag) and labeled
antigens (Ag*) is added to the surface of the substrate immobilized with antibodies
(Ab). A competitive binding to the immobilized antibodies occurs between the sample

∗
∗
∗ ∗
∗ ∗
(A)
∗
∗ ∗ ∗
∗
∗ ∗
∗
(B)
: Ab1 : Ag ∗ : Labels : Ab2

Fig. 1.2 Schematic illustration of competitive immunoassay (A) and sandwich immunoassay (B).
4 Immunosensing for Detection of Protein Biomarkers

antigens and the labeled antigens. After an antigen-antibody binding equilibrium is

reached, the solid substrate surface is rinsed with buffer to remove unbound antigens,
and the bound label’s signal is detected. Therefore, the measured signal is inversely
proportional to the concentration of antigens in the sample for competitive assay. In a
sandwich immunoassay (Fig. 1.2B), the target analyte (antigen) is exposed to the sub-
strate and captured by the immobilized primary antibodies (Ab1). Then the captured
antigens bind the labeled secondary antibodies (Ab2*) used as the tracer and are rinsed
to remove extra tracer. These tracer antibodies provide a signal that is directly pro-
portional to the concentration of analytes. The use of two different antibodies for the
sandwich immunoassay often imparts greater selectivity since cross-reacting species
rarely bind both the capture and the tracer antibodies. In general, sandwich immuno-
assays are used for macromolecule analytes rather than analytes with a low molecular
weight.

1.2 Immunosensing
As a type of biosensor, the immunosensor is defined as a compact analytical device
incorporating immunological recognition elements either intimately connected to or
integrated within the signal transducer [12]. The general immunosensor design and
its signal transduction strategy are illustrated in Fig. 1.3. Like other types of bio-
sensors, an immunosensor contains three basic components: biorecognition element,
transducer, and detector. Either antigens or antibodies are immobilized on the surface
of a solid substrate and participate in the specific binding, allowing recognition of the
target analyte.

Surface-attached antibodies Transducer

Electrochemical active
substance → Electrode

Heat → Thermistor
Electrical
signal
Light → Photon count

Mass change → Piezo-

electric device

Fig. 1.3 Schematic illustration of the general immunosensor design and the signal
transduction strategy at a solid substrate.
Introduction5

The transducer monitors physicochemical changes resulting from the immunoreac-

tion between immunological recognition elements and target molecules and converts
them to a detectable physical signal. Then the signal is collected and amplified by the
detector to indicate the identification and quantity of the target analyte. Therefore, al-
most all immunosensors reported are based on a heterogeneous immunoassay format
up to now.
Because immunosensors make use of specific interactions between an antibody
and an antigen, they can be used to detect many different analytes (not necessarily
of biological nature as long as they interact specifically with the biorecognition ele-
ment, e.g., antibodies or antigens immobilized on the solid substrate). Meanwhile, as
a biosensing device, they can greatly simplify immunoassay, decrease the analytical
cost, and also miniaturize and automate the detection process. Among immunosens-
ing, the sensitive signal transduction is a key process for quantitative measurements.
Signal transduction in immunosensors can be carried out by different means, taking
advantage of different changes of properties or signal generation, which occurs fol-
lowing the formation of antigen-antibody complex. Based on the difference of the
signal transduction modes, immunosensors can be commonly classified into optical,
electrochemical, mass, or thermal-sensitive immunosensors [13].

1.2.1 Optical immunosensors
In this case, an optical signal that is based on the change in the phase, amplitude,
polarization, or frequency of the input light in response to the antigen-antibody
biorecognition processes is generated. Optical immunosensors can be classified as
colorimetric, fluorescence, chemiluminescent, electrochemiluminescent, and surface
plasma resonance (SPR)–based biosensors.
In colorimetric and fluorescence-based detection, either target or biorecognition
molecules are labeled with chromogenic/fluorescent tag, such as dyes. The change
in the intensity of the color/fluorescence signal indicates the presence of the target
molecules, which is extremely sensitive, with the detection limit down to a single
molecule. For example, horseradish peroxidase (HRP) labels are popularly used for
signal tracing in the enzyme-linked immunosorbent assay (ELISA) systems [14,15].
Based on the HRP-catalytic reaction and spectroscopic detection of its colored prod-
uct, this method is commonly used by molecular biologists for biomarker detection
and quantification. When using various fluorescent labels in immunoassay, several
types (e.g., homogeneous fluorescence, fluorescent excitation transfer immunoas­
say, fluorescence polarization, fluorescence enhancement and fluorescence quench-
ing, fluorescence energy transfer, and time-resolved fluorimetry) of immunoassays
could be developed to detect protein biomarkers with the advantage of high sensi-
tivity, specificity, simplicity, and wide dynamic range [16,17]. Nakamura et al. [18]
fabricated a human chorionic gonadotrophin (hCG) immunosensor coupled with a
flow-immunoassay system and a cation exchange, resin-packed, capillary column.
The hCG and fluorescein isothiocyanate (FITC)-conjugated IgG antibody complex
was separated from free FITC-conjugated IgG on the basis of difference in isoelectric
points. The fluorescence intensity correlated linearly with the concentration of hCG
6 Immunosensing for Detection of Protein Biomarkers

in the range of 25–1500 mIU/mL. For the purpose of minimal reagent consumption,
rapid separations, and automation, Yan et al. [19] developed a time-resolved fluoro-
metric immunosensor for carcinoembryonic antigen (CEA) determination coupled
with a flow injection system. The prepared immunoaffinity column was inserted in
a flow system for immunoreactions. After the enhancement solution was injected to
cleave the Eu-labels from the immunocomplex, the cleaved solution was collected and
detected by time-resolved fluorescence.
Chemiluminescent immunosensors combining antibody-antigen interaction with
sensitive chemiluminescent measurements are popular optical immunosensors. There
are three major chemiluminescent analytical techniques, using direct chemilumines-
cent labels, chemiluminescent substrate labels, and enzyme labels [2]. The former la-
bels chemiluminescent reagents, such as acridinium ester and acridinium sulfonamide
ester, with antibody or antigen. After immunoreaction the labeled reagents can react
with peroxide without addition of enzyme to produce a light emission. When chemi-
luminescent substrates, such as luminol, isoluminol, and dioxetane phosphate ester, or
enzymes, such as HRP and alkaline phosphatase (ALP), are used to label with anti-
body or antigen, the enzymatic reactions to convert substrates to luminescent products
produce detectable signals after immunoreaction. These signals are related to the con-
centration of antigen or antibody. Most of the chemiluminescent immunosensors are
based on the use of enzymes for amplifying the detection signal. By combination with
the enzymatic signal amplification and the automatization of detection procedures by
flow injection system, Ju’s group [20–28] has made great efforts on the development
of chemiluminescent immunosensing strategies for the convenient measurement of
protein biomarkers, which will be introduced in detail in Chapter 5.
Electrochemiluminescence (ECL) is a means to emit measurable luminescent sig-
nals by converting electrochemical energy into radiative energy via an electrochem-
ical reaction. Based on electrochemiluminescent of tris(2,2′-bipyridyl) ruthenium,
quantum dots (QDs), and other miscellaneous nanomaterials including metallic
nanoclusters, carbon nanodots, metallic oxide semiconductors, and even organic
­nano-aggregates, various sensitive immunosensing strategies have been constructed
by studying the enhancement or inhibition mechanisms of the ECL emitters in recent
years [29,30]. Ju’s group [31–35] also worked hard on the development of sensitive
ECL immunosensing methods for measurement of protein biomarkers, which will be
introduced in detail in Chapter 6.
SPR immunosensors often offer label-free, real-time methods to detect the target
molecules in their native forms. This type of detection is relatively easy and cheap to
perform quantitative/kinetic measurement of molecular interactions with a high degree
of accuracy, precision, information content, and sensitivity [36]. SPR is a phenome-
non that occurs when light propagates from a high-refractive-index medium (Fig. 1.4,
prism or grating) toward an interface containing a low refractive-index material (sam-
ple solution). When a thin Au or Ag film is placed at the glass-liquid interface, incident
photons in the evanescent wave generated at the interface by rear illumination couple
with the electrons in the metal (plasmons). This coupling occurs only when the condi-
tions of resonance are established. When biomolecules bind to the external surface of
the metal film, the resulting change in the dielectric properties of the medium adjacent
Introduction7

5 × 5 Au array

Source Prism CCD camera

hv detector
Fig. 1.4 Schematic illustration of SPR arrays on gold films that can be used for detecting
multiple protein biomarkers shown as a side view of an array that is coupled to a charge-
coupled device (CCD) camera for imaging. On the right is the top view of a 5 × 5 array.

to the metal film results in changes in the resonance conditions, and hence modulates
the intensity of the reflected light. Thus SPR has become an effective method for the
detection of virtually any biomarker and has even been extended to array format for
detecting a number of analytes simultaneously [37].
Chou’s group [38] developed an SPR immunosensor for human ferritin, a nonspe-
cific tumor marker, based on the immobilization of antihuman ferritin monoclonal
antibody on an SPR-sensing gold surface. Campagnolo et al. [39] reconfigured a re-
fractometer instrument to detect tumor molecular interactions through changes in SPR
signal. The SPR immunosensors have also been successfully commercialized. Huang
et al. [40] introduced an immunosensing method for the detection of prostate-specific
antigen (PSA) in human serum using a commercially available SPR biosensor based
on mixed self-assembled monolayers. In addition, many efforts have been made to
overcome disadvantages such as inherent low sensitivity. Besselink et al. [41] ampli-
fied the assay sensitivity for PSA in SPR with colloidal gold nanoparticles (Au NPs,
10-nm diameter) and latex microspheres (120-nm diameter) on planar- and gel-type
sensor surfaces. The corresponding detection limit for PSA was ~0.15 ng/mL, which
was sufficient for measuring clinically relevant PSA levels (>4 ng/mL). Yu and Knoll
[42] proposed an immunosensor for hCG based on the diffraction of surface plasmon.
The inherent self-referencing mechanism of surface diffraction was found to be very
effective for compensating fluctuations of the bulk.
Although optical biosensors are highly sensitive, they are bulky, expensive, and
require dedicated personnel to perform the tests. Additionally, colorimetric, fluores-
cence, and luminometric type of sensors require difficult labeling procedures that de-
pend on indirect indicator-based signal schemes.

1.2.2 Electrochemical immunosensors
In this case, an electrical signal in terms of change in currents and/or voltages is mea-
sured, which shows significant differences in magnitude if antigen-antibody complexes
are formed. Based on their operating principle, electrochemical immunosensors can
employ potentiometric (measuring of electrode potential or voltage differences), am-
perometric (measuring of current), capacitative (measuring of reciprocal capacitance),
8 Immunosensing for Detection of Protein Biomarkers

and conductimetric (measuring of conductivity or electrochemical resistance) trans-

ducers converting the chemical information into a measurable electrical signal. The
single analyte enzyme-linked electrochemical immunosensor, developed in the 1990s,
stands as a perfect example [43,44]. Most approaches feature antibodies attached to
the sensor surface, so that antigen capture, enzyme-labeled secondary antibody bind-
ing, and detection are all done on the same surface. The most selective and sensitive
electrochemical immunoassays employ the sandwich format.
Thus, electrochemical immunosensors exclude biosensors, which require light,
mechanical motion, or use of magnetic particles. Due to their low cost, low power,
and ease of miniaturization, electrochemical immunosensors provide great promise
for applications such as point-of-care diagnosis and biological warfare agent detection
[45]. In Chapter 3, detailed samples will be presented to introduce the applications of
electrochemical immunosensors.

1.2.3 Mass-sensitive immunosensors
The study of mass-based immunosensors mainly focuses on microbalances of piezo-
electric crystals [46]. The piezoelectric quartz-crystal device comprises a quartz-­
crystal wafer with different thicknesses sandwiched between two metal electrodes,
which connect the device to an external oscillator circuit. The resonant frequency of
quartz-crystal microbalance (QCM) depends on the mass of the crystal surface as well
as the mass of any layer confined to the electrode area of the crystal. The change of
crystal frequency can reflect a tiny change in mass on the electrode surface. Currently
the piezoelectric immunosensors are used more and more for the determination of
tumor markers in clinical diagnosis due to the advantages of label-free and real-time
detection. Chou et al. [47] proposed a piezoelectric immunosensor for human ferri-
tin by immobilizing antihuman ferritin antibody on a gold disk of a QCM. Human
ferritin could be determined in a linear range from 0.1 to 100 ng/mL. Zhang’s group
[48] presented a multichannel 2 × 5 model of piezoelectric quartz microarray immu-
nosensor for quantitative detection of hCG in serum or urine samples. Compared with
a one-channel immunosensor, this 2 × 5 model of microarray immunosensor could
provide eight times higher detection speed for hCG assay.
In recent years, microcantilever sensors, another kind of mass-based sensor, have
attracted much attention for improved performance of QCM measurements [49].
Microcantilever sensors can sensitively monitor the molecular adsorption, which
bends the microcantilever and changes its resonant frequency. If two surfaces of the
cantilever are chemically different, different molecular adsorption will produce dif-
ferent surface stress between the top and bottom surfaces of the cantilever. When
specific binding of biomolecules occurs on the surface of microcantilever, intermo-
lecular nanomechanical force induces the cantilever to bend, and this can be observed
as changes in cantilever deflection. Microcantilever sensors do not require any label
or reporter molecule to signal the presence of a molecule on a biosensor surface. In
particular, nanomechanical sensors have the advantage of high sensitivity with small
area compared with other label-free biosensors (e.g., the quartz-crystal device). Wee
et al. [50] prepared a label-free PSA immunosensor using self-sensing piezoresistive
Introduction9

microcantilevers. Electrical detection of antigen-antibody specific binding was ac-

complished through a direct nanomechanical response of microfabricated self-sensing
microcantilevers. This cantilever immunosensor was used for the detection of PSA
and C-reactive proteins and shown to be effective for clinic application. Wu and co-
workers [51] developed a label-free immunosensor for PSA with microcantilevers
using a polyclonal antiprostate antibody as a covalent linker. In this system, the PSA
could be detected in the concentration range from 0.2 ng/mL to 60 μg/mL.

1.3 Immobilization method of immunoreagents

As most immunoassays are performed in heterogeneous format, a key feature of im-
munosensors is the use of a solid substrate to couple either the antibody or antigen. To
fabricate a sensitive and reliable immunosensor, a large variety of rigid materials such
as chemical electrodes, polymer membranes or beads, microtiter plates, and magnetic
bead can be applied as a proper matrix for binding the recognition biomolecules. The
immobilization method of immunoreagents will decide their amount, distribution, ori-
entation, and even bioactivity on substrates, thus directly affecting the performance
of the immunosensor such as repeatability, stability, specificity, and detection limit.
Commonly, the methods of physical adsorption, polymer entrapment, covalent bind-
ing, and several oriented immobilization approaches can be used for the immobili-
zation of immunoreagents by combination with the characters of the solid substrate.

1.3.1 Physical adsorption
The physical adsorption method adsorbs proteins on the solid substrate via noncovalent
interactions, mainly electrostatic force, ionic bond, hydrogen bond, and hydrophobic
interaction. The method is simple and generates little effect on bioactivity. However,
physical immobilization often results in random orientation and weak attachment due
to the weak attachment between biomolecules and the substrates. Generally, optical
ELISA involves the nonspecific adsorption of the recognition elements on the bottom
of polystyrene wells [52]. All the immunoassay steps (the series of reaction, washing,
enzymatic reaction, and colorimetric measurement) are performed on this solution/
well interface. For electrochemical immunosensor preparation, capture antibodies
can be directly adsorbed onto rough carbon electrode surfaces by passive adsorption
[53,54]. The coating of a layer of polyethylene glycol on the electrode surface assists
in the antibody immobilization by adsorption [55], which, obviously, can improve the
performance of the electrochemical immunosensor. To enhance the attachment of pro-
teins with electrode surface, Wilson and Rauh designed an electrochemically grown
iridium oxide (IrOx) thin film matrices for antibodies adsorption [56]. The three-­
dimensional porous and hydrous matrix offered better antibody stability and higher
antibody loading, thus improving the recognition efficiency of the immunosensor.
Recently, nanoparticles have attracted growing attention regarding immobilization
of immunoreagents due to their large specific surface area, strong adsorption capacity,
and excellent biocompatibility [57]. Compared to the naked surfaces of bulk materials,
10 Immunosensing for Detection of Protein Biomarkers

the adsorption of protein biomolecules onto the surfaces of nanoparticles can generally
retain their bioactivity. Since most of the nanoparticles carry charges, they can electro-
statically adsorb biomolecules with different charges. Besides the common electrostatic
interaction, some nanoparticles can also immobilize biomolecules by other interactions.
For example, Au NPs can immobilize proteins through the inherent interaction between
the gold atoms and the amine groups and cysteine residues of proteins [58,59]. So Au
NPs are popularly applied to the electrode surface for immobilizing capture antibody
during the immunosensor preparation [60–62].

1.3.2 Polymer entrapment
To ensure high stability and bioactivity of immunosensors, various polymers with a
three-dimensional structural network are often used for the entrapment of immunore-
agents. One typical polymer for the encapsulation of biomolecules is gel matrices with
porous surface and high capacity. Ju’s group [63,64] prepared a novel, titania sol-gel,
thin film by a vapor deposition method. This porous matrix with excellent biocompati-
bility provided an excellent environment to immobilize the antigen for the competitive
electrochemical immunoassay of carbohydrate antigen 19-9 (CA 19-9) [63] and carci-
noma antigen-125 (CA 125) [64], respectively. They also used an organically modified
silicate sol-gel [65] to immobilize the HRP-labeled antibody to achieve the direct
electrochemistry of HRP. Based on the direct immunoreaction at the immunosensor
to inhibit the direct electrochemical signal, novel reagentless immunosensors were
constructed for electrochemical measurement of CEA [66] and hCG [67].
Hydrogels are another widely used polymer for the protein encapsulation during
immunosensor preparation. Commonly used hydrogels are chitosan, dextran, and
commercial carboxymethyl cellulose. Ju’s group prepared an Au NPs-doped cellulose
acetate membrane [68] and an Au NPs-doped chitosan membrane [69] for immobiliz-
ing antigens, thus developing two electrochemical immunosensors for the measure-
ment of CA 125 and CEA, respectively.
The conducting polymers with high electroconductivity and aqueous compatibility
also served as excellent gel materials for the encapsulation of antigens or antibodies
for immunosensor preparation. Sadik et al. [70] reported a simple method for immu-
nosensor construction by means of electrochemical polymerization of pyrrole with
antibodies at a platinum-disk working. Cyclic voltammetry and electrochemical im-
pedance spectroscopy were used to characterize the interaction between human serum
albumin and the antibody-immobilized polypyrrole immunosensor successfully.

1.3.3 Covalent binding
Covalent bonds are mostly formed between side-chain-exposed functional groups of pro-
teins and suitably modified transducer surface, resulting in an irreversible binding and
producing a high surface coverage [71,72]. One of the most commonly used methods for
covalent immobilization is to couple the antibody randomly via their free amino groups
to an activated sensor surface (Fig. 1.5). Chemical coupling agents, such as carbodiim-
ides and succinimidyl esters, may be used to activate carboxylic acids on sensor surfaces.
Introduction11

COOH NH2 OH
+ –NH2

Antibody
with free
a b c amino
group

CONH NHCO

Fig. 1.5 Schematic illustration of covalent immobilization of antibodies to sensor surfaces

via their free amino groups. Reaction (a) involves activation of carboxylic acids (COOH),
achieved with carbodiimides and succinimyl esters. Reaction (b) shows amine surfaces (NH2),
which can be activated using isothiocyanates, epoxides, or aldehydes. Reaction (c) shows
alcohol surfaces that can be activated using periodate oxidation, isothiocyanates, epoxides,
aldehydes, and cyanogen bromide.

Amines or alcohols can be activated by isothiocyanates, epoxides, glutaraldehyde (GA),

or other aldehydes. Oxidation of alcohols is achieved with periodate to yield aldehydes,
which react readily with amines. In addition, conversion of alcohols to a highly reactive
ester by cyanogen bromide allows for further reaction with amines of antibody. For exam-
ple, the ultrasonication treatment of carbon nanotubes (CNTs) in concentrated acid con-
dition can produce abundant carboxyl groups on their surface. When CNTs are modified
on the electrode surface, the carbodiimide/N-hydroxysuccinmide system is commonly
applied to link antibodies with the activated carboxyl groups [60].
Another approach is to use bifunctional cross-linking reagents [73]. The cross-linking
reagents contain two different reactive groups, thereby providing a means of covalently
linking two dissimilar target groups on the sensor surface and protein biomolecules. A
wide variety of these linkers such as (3-aminopropyl)triethoxysilane (APTES) [74],
(3-glycidoxypropyl)-trimethoxysilane (GPTMS) [75], 3-mercaptopropyltrimethoxysilane
(MPTMS) [76], diazonium cation [77], and various thiol derivatives [78–80] are com-
mercially available to cover a broad range of functional groups necessary. For example,
the silanization reaction of APTES at the hydroxyl group-containing substrate (e.g., glass,
electrode, and microwell plate) can provide amino groups at the surface. Then antibodies
can be coupled to the substrate via the cross-linking of GA (Fig. 1.6).

OH O O O O
OH O Si NH2 O Si N=C CH O Si N=C C=N
APTES GA H –NH2 H H
OH O O O
OH O Si NH2 O Si N=C CH O Si N=C C=N
OH O O H O H H
O

Fig. 1.6 Schematic illustration of immobilization of antibody by the bifunctional cross-linking

reagents of APTES and GA.
Introduction23

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Title: And it was good

Author: A. Earley

Illustrator: Dan Adkins

Release date: November 11, 2023 [eBook #72092]

Language: English

Original publication: New York, NY: Ziff-Davis Publishing Company,

1962

Credits: Greg Weeks, Mary Meehan and the Online Distributed

Proofreading Team at http://www.pgdp.net

*** START OF THE PROJECT GUTENBERG EBOOK AND IT WAS

GOOD ***
 And It Was Good

By A. EARLEY

Illustrated by ADKINS

A moving tale of a second chance

for man—and for his maker.

[Transcriber's Note: This etext was produced from

Amazing Stories February 1962.
Extensive research did not uncover any evidence that
the U.S. copyright on this publication was renewed.]
When she came back he looked at her and put down the piece of
wood which he had been carving.
He always carved in anxious moments. Many years before he had
been apprenticed to a carpenter. He still loved the smooth, creamy
feel and the warm tang of a good piece of wood. Usually he whittled
away at it until it suggested a design to work on. More often than not
it turned out to be a face, rugged peasant features with the simple
wisdom of age engraved on them, or the chubby whorls of a child
dimpled with delight. Today, he thought, it might make a tree heavy
with fruit and the crown of leaves.
"He's decided to do it, then"? he said, and she nodded without
looking at him. She did not want to see the pain in her son's eyes.
He got up and stood beside her and put his arm round her
shoulders.
"When"? he asked her softly, patiently.
"Right away".
"Did you ask him if he would let me go again instead"?
"I couldn't"! she said and pulled him to her. "I couldn't bear it again
after what they did to you last time".
"Am I any the worse for it"? he smiled at her. "Besides, it was a long
time ago and people have changed".
"You'd suffer and you'd be away for years", she said. "I couldn't go
through that. Not again".
"Is he very sad about it"? he asked.
"You know how he is when he has to do a thing like that", she said.
"He said you weren't to worry too much. I was to tell you he'd like to
talk to you about it later. He might want you to go there for a short
visit while it's on".
He went back to his whittling, but his mind was busy with other
things and the tree would not take shape.

Spring had been late before. As the Times pointed out, there had
been snow as late as mid-May in 1569 and at the end of April in
1782, yet the chronicles recorded bumper crops for both years.
Agricultural experts advised closer pruning of fruit trees to speed
budding, and an American firm of Artificial Fertilizer Manufacturers
brought out a new product called 'Shoot-boost'. But the
correspondence columns of the newspapers carried letters pointing
out that, while spring might have been late before, this time the
weather was entirely spring-like, yet still there was no sign of shoot,
blossom or bud. Excessive radiation resulting from nuclear tests was
blamed.
It was mid-May before the people and their governments became
seriously alarmed. Trees still stood bare as in the depth of winter,
lawns bore the bruising of last season's mowing but no new growth,
flower beds showed the unbroken rills of after-seed raking. Farmers
walked their fields day after day and crouched down to silhouette the
furrows against the sky, the better to see the green whiskers when
they sprouted. They prodded their heifers and ewes and went down
to the villages to consult the vet. Their wives searched the hen-
houses and put down extra grain and bricks of chalk.
The Pope's call to world-wide prayer and the British Government's
announcement of the introduction of rationing fell on the same day.
In most countries, the Pope's call found little response because the
people were too busy lining up at food stores trying to lay in stocks.
There were bread riots in Teheran.

Rumors of a cattle disease began to circulate several days before

official news of the full extent of the additional catastrophe was
released. That night, the British Prime Minister spoke on the BBC.
"With Her Majesty's consent," he said after reviewing the 'grave and
disquieting situation', "I have given instructions for all available ships
of the Royal Navy to put to sea immediately as an emergency fishing
fleet." Meanwhile, he continued, divers and frogmen were asked to
place their services at the disposal of the Ministry of Agriculture and
Fisheries. They would be required to "glean nourishment for the
nation from the laden larders of the deep." "Human ingenuity, skill
and tenacity will conquer yet," he concluded. The Prime Minister's
broadcast was followed by the announcement of emergency
regulations for the disposal of dead cattle.
On 16th June, the President of the United States informed an
Emergency Meeting of the General Assembly of the United Nations
that Professor Braunweiler of Columbia University had perfected a
method of extracting carbon sugar from wood. All suitable industrial
plants throughout America were to be geared to the mass-production
of the necessary equipment. The United States was prepared to
supply the whole world with this equipment and with power-operated
tree-felling implements on a lend-lease basis. Teams of instructors in
the use of the equipment would be available to proceed to all parts of
the world by the end of the month. The offer, which became known
as USASAW, (USA SUGAR AID TO THE WORLD), was accepted
with gratitude by all but the Soviet delegation.
Shortly after Sugar-Aid started, a Frenchman named Dr. Muller
discovered, (in desperation, vineyards stood barren), that tree-sugar
caused a fermentation in the still-plentiful needles of coniferous trees
which, when distilled, resulted in a drink rich in alcohol and vitamins.
He gave the drink the name 'BOIGNAC' in melancholy memory of
happier days. Within six weeks, France had a surplus in the World
Bank, and a French admiral was appointed to command the NATO
Mediterranean fleet. Undoubtedly, boignac helped; yet, by the end of
August, even that could not arrest the death rate.

On 3rd September, a Soviet Task Force landed troops and armor at

sixteen places along the East-African coast. Moscow Radio informed
the world that 'the glorious forces of the USSR have taken this step
under the personal command of Mr. Khrushchev to safeguard
Africa's rich resources in animal life against the depredations of the
Capitalist Warmongers'. Thus, the world was told, all peaceloving
peoples would be assured an equitable and adequate supply of meat
in the hard months to come.
At an Emergency Meeting of the NATO Council immediate counter-
measures were agreed upon, but it was decided to confine retaliation
to Africa and not to use nuclear weapons unless Russia did so first.
The 'British Left', which had come into being after the Labor Party
had split, withdrew from the House of Commons in protest, and the
workers of the largest motor works in Italy assembled outside their
long-closed factory to call for strike action.
By mid-December, the war in Africa had settled down to a stalemate.
There was a good deal of patrolling; the opposing armies 'lived off
the land', in other words on what game they could bag before the
other side got it. Food-finding became more important than fighting,
and hunger closed the eyes of higher command to the proximity of
the enemy, except of course when the enemy was engaged in
tracking the same game. Reports from the front recorded these
'patrol skirmishes', and gave account of the really violent artillery
duels. Loading and firing guns required less waning energy than
infantry slogging in the heavy country. The fact that the wide no-
man's-land between the opposing armies formed the main hunting-
ground exposed friend and foe to the same gunfire. Casualties were
consequently high. The Neutral Investigating Commission appointed
after much vetoing by the United Nations—it consisted of delegates
from Costa Rica, Kashmir and Monaco—found the situation rather
confusing and withdrew to Cannes to consider its findings.
Early in January, a British scientist invented a Very-High-Frequency
Lamp, regular exposure to which substituted a certain amount of the
energy normally absorbed in food. The equipment was fantastically
expensive to produce and was therefore available to very few
people. A portable, cheaper and far less efficient model was mass-
produced for the armed forces and essential workers. The dashing
victories in Africa, forecast by enthusiastic politicians as a certain
result of the new machine, did not however materialize. The new
energy induced in picked units was expended in a redoubled quest
for food. The papers reported increased patrol activity.
An agent planted by the Communists in the Ministry of Defense in
London succeeded in photographing the plans of the ray-lamp.
Within six weeks, a Russian version of the equipment reached the
Red forces in Africa. As a result, the stalemate became staler still.
Both sides began to lose control of their troops, which scattered over
wide areas of Africa well outside the zone of battle; game had
become scarce, and pursuit led both sides further and further afield.
On a swampy peninsula, formed by a hairpin bend of a crocodile-
infested river, a British and a French soldier had established their
laager. They had joined forces to hunt for edible snakes, and a few
hundred yards up-river one of them had trodden on a carelessly
buried anti-personnel mine. The soggy ground had prevented the
contraption from jumping as high as the designer had intended, and
the dense, though leafless undergrowth had screened them from the
worst of the blast.
They took it in turns to fetch water in their hats from the river and to
bathe each other's wounds. Starving and feverish, neither of them
knew for certain when the stranger joined them. He was not in
uniform; he spoke English and French so well that they both claimed
him for a fellow-countryman. He did not enlighten them, and they did
not persist in their questions. He insisted on nursing them and
waiting on them. He fetched water for them from the river, and he put
clay from the river bank on their septic wounds; he said it would heal
them. The Englishman was embarrassed to see that the stranger
had tears in his eyes while he did it. To pretend that he had not
noticed, the Tommy talked about the flipping bastards who strew
flipping mines all over the flipping place. The stranger smiled at that
and said he would try to get them some fish from the river. He was
away a long time, and when the Englishman crawled down to the
river to see what had happened, he saw the stranger on his knees
on the river bank. He wanted to shout that one could not catch
flipping fish that flipping way, but then he changed his mind and
crawled back to the Frenchman. The stranger turned up a little later
with his hat full of fine fish. He wanted to light a fire to cook them, but
the Frenchman pointed up to where shells from both sides were
hissing over them, and they ate the fish raw. It tasted wonderful.
The stranger settled down to stay with them and brought fish and
water as often as they felt hungry or thirsty. When he was not
otherwise engaged, he used one of their bayonets to whittle away at
pieces of wood. Their wounds were clearing up fast and did not hurt
any more. The Frenchman insisted on giving the stranger his
gascape to sleep in because he had nothing else, and the Tommy
pulled out his only spare pair of socks because the stranger's were
walked to shreds.
Sometimes the stranger left them for a few days, but he always
made sure that they had enough water and fish before he left. He
came back dusty and dirty and tired out, but he did not seem to need
much sleep. Once, when the Tommy woke in the middle of the night
and wanted a drink, he saw the stranger kneeling under a nearby
tree. Flipping shell-shock, probably. Poor bastard.

The Russian soldier stumbled into their laager one evening just as
they were getting ready for sleep. He dropped his rifle in his surprise
and then held his hands up high because the Frenchman was
groping for his bayonet. They stood for a while looking at each other
until the Frenchman put his weapon down and the Russian's arms
fell slowly to his sides. He watched them for a few minutes, then he
saw a fishtail lying on the ground and picked it up and began to
gnaw it. The Tommy glanced at his companions and crawled to the
hole in the rocks behind them where they kept their supplies and
gave the Russian a whole fish. The Russian grinned and took it, and
while he was eating it he sat down and gradually wriggled his way
closer to them. They showed him another fish and he said 'da' and
they gave it to him. "First time I knew a flipping Ivan could say yes
too," the Tommy said.
To their amazement, the stranger spoke to the Russian with the
same ease with which he spoke English and French.
The Russian spent the night with them, and in the morning, after
more fish, he wandered off. He came back dragging mounds of
branches with which he built a shelter for the wounded men under
one tree, and another one for the stranger. He grinned all over his
broad face, pointed to the fish, to them, to himself and to the
shelters. Then he shook hands all round.
That afternoon a Russian fighting patrol passed close by. The officer
heard their voices, crept up behind them and threw a hand grenade
among them. The stranger threw himself on top of it just as it went
off. The Englishman shot the officer through the head before the dust
and smoke had cleared, and the remainder of the patrol withdrew.

When they turned the stranger over, the ants were already swarming
in his blood. At first they tried to brush them off with twigs, but more
and more ants came. The Russian pointed to the river and gestured
that it would be kindest to throw the body in. The Frenchman shook
his head, and the Englishman started to drag the body to the hole in
the rocks. They laid the stranger inside and rolled a rock against the
entrance and sealed the gaps with clay.
They missed him a great deal. Not only because of the fish and
water.
Next day the Russian left them. Before going, he banged them on
the back and shook hands with them several times and tears left
streaks on his dirty face.

She was overjoyed to have her son back with her. She could not
stop looking at him for the sheer joy of it.
"Was it very terrible"? she asked.
"No", he smiled at her. "In a way it was wonderful".
"But the suffering and the killing", she said.
"I saw more than that", he said.
"Did you tell him all of it"? she asked.
"All of it". He picked up his knife and whittled away at the wood.
"And"? she insisted.
"He's angry, and sad. And at the same time he's pleased", he said,
and that was all he would tell her. But she felt comforted and she
knew it was going to be all right.
He shaved the last of the bark of the wood and looked at the grain
and set to work. This time it would be a child, with fat round cheeks
and the dimples of laughter in them.

THE END
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