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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.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about the
Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance
Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical treatment may
become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information
or methods they should be mindful of their own safety and the safety of others, including parties for whom
they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any
liability for any injury and/or damage to persons or property as a matter of products liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the
material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

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A catalogue record for this book is available from the British Library

ISBN: 978-0-08-101999-3

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visit our website at https://www.elsevier.com/books-and-journals

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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
[email protected]
Guosong Lai
Chutian Young Scholar in Hubei Province
Professor of Chemistry, Hubei Normal University
Huangshi, P. R. China
[email protected]
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
[email protected]
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.
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