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Methods in
Molecular Biology 2504

Maurizio Federico
Barbara Ridolfi Editors

Extracellular
Vesicles in Diagnosis
and Therapy
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

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 Extracellular Vesicles
in Diagnosis and Therapy

Edited by

Maurizio Federico and Barbara Ridolfi

National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy
Editors
Maurizio Federico Barbara Ridolfi
National Center for Global Health National Center for Global Health
Istituto Superiore di Sanità Istituto Superiore di Sanità
Rome, Italy Rome, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2340-4 ISBN 978-1-0716-2341-1 (eBook)
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Dedication

In memory of Elisabetta Federico, Lisa, too young a victim.

For more on Lisa’s story, Dr. Maurizio Federico has written an

essay, “Dedicated to Lisa. An Italian Story that Should Never
Repeat” available online.

v
Preface

Transport of macromolecules among cytoplasmic structures is mediated by vesicles moving

in a densely populated microenvironment. Part of these vesicles can be released into the
extracellular milieu, thereby being identified as extracellular vesicles (EVs). They are part of
the mechanism of intercellular communication, also at long distance. The discovery of
nano/micro-vesicular structures released into the extracellular space, containing a multitude
of factors including signaling molecules, proteins, and nucleic acids, has added a new layer of
complexity to our understanding of cell-to-cell communication.
All EV subtypes are limited by a lipid bilayer membrane surrounding a specific cargo of
molecules, and having different sizes and buoyant densities. Current research mainly con-
siders two types of EVs according to their biogenesis, i.e., ectosomes/microvesicles and
exosomes. Ectosomes/microvesicles are of 150–1000 nm in diameter and bud directly from
plasma membrane. Exosomes refer to vesicles of 30–150 nm in diameter generated intracel-
lularly by inward invagination of endosome membranes leading to the formation of intra-
luminal vesicles. They became part of multivesicular bodies which are released in the
extracellular space upon fusion with plasma membrane
Body fluids (e.g., blood, urine, saliva, amniotic fluid, bronchoalveolar lavage fluid,
synovial fluid, breast milk) contain various types of membrane-enclosed vesicles recognizing
diverse pathways of biogenesis. These vesicles possess different biophysical features and
functions in health, e.g., protein clearance, immune regulation, cell signaling, as well as in
disease, such as in infections and cancer. The presence in many biological fluids of EVs
prompted many research groups to investigate their possible use as disease biomarkers as
well as tools for the development of new therapies.
The first part of the present volume is opened by an introductive chapter by Tan and
colleagues overviewing current methods to detect cancer exosomes through aptasensors,
i.e., biosensors using aptamers (single-stranded DNA or RNA molecules) as recognition
element. Al-Jamal et al. then provide a detailed application of the aptasensor-based tech-
nique. This section also comprises a couple of papers from Takov and colleagues and
McDonnell and colleagues, describing methods to isolate EVs from both human and
mouse plasma. In the last chapter of the section, van der Pol et al. report a detailed protocol
for evaluating concentrations of EVs in human plasma by flow cytometry.
The second part of this book is dedicated to the description of different methods to
isolate and quantify EVs from specialized tissues/organs and body fluids, i.e., from placenta
by Salomon and colleagues, cardiovascular stem cells by Yang and colleagues, saliva by
Punyadeera and colleagues, and urine by Reithmair and colleagues. Together, these chapters
offer to readers the ability to deal with effective analysis of EVs isolated from a wide range of
districts.
In the third section, methods devoted to analyzing EV components are presented. In
particular, Jin and colleagues furnish a protocol for the miRNA detection in EVs, whereas
Iliuk presents a method for the detection and analysis of EV phosphoproteins. This section is
then completed by the contribution of Saito et al., who describe a protocol for the analysis of
EV lipids.
The last frontier in terms of macromolecule delivery by natural nanovesicles is repre-
sented by engineered EVs. In the last section of this book, a number of methods to engineer

vii
viii Preface

EVs are provided. This section is opened by a review from Chiozzini and colleagues offering
a comparative analysis of EV-based versus other nanovesicle-based methods in terms of
antigen delivery to induce immunity. Dieterich and colleagues then describe a protocol for
fluorescently labeling the membrane of tumor EVs, whereas Manfredi and colleagues
propose a way to incorporate fluorescent proteins into the EV lumen. Finally, two different
methods to upload miRNAs and other nucleic acids are provided by Pomatto and colleagues
and Jeyaram and colleagues, respectively.
Overall, this book provides an exhaustive picture of current methods to detect, isolate,
and analyze EVs from diverse sources. In addition, the methods of EV labeling and
macromolecule uploading would be of outmost utility for the study of still incompletely
known EV processes (i.e., cell attachment, cell entry, fate of EV cargo) as well as for the use
of EVs as immunogens/therapeutics.
Clearly, EVs have been proven to have great potentialities as disease biomarkers as well as
delivery tools of therapeutic/immunogenic molecules. Recent advances pave the way for a
wide use of EVs in both diagnosis and therapy. Protocols included in the present volume are
expected to be of great utility to further enlarge the number of scientists interested in EV
research.

Rome, Italy Maurizio Federico

Barbara Ridolfi
Contents

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I EXTRACELLULAR VESICLES DETECTION AND ISOLATION

1 Aptasensors for Cancerous Exosome Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Jin Li, Sitao Xie, Fengli Qu, and Weihong Tan
2 Detection of Cancer-Derived Exosomes Using a Sensitive
Colorimetric Aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Lizhou Xu and Khuloud T. Al-Jamal
3 Isolation of Circulating Extracellular Vesicles by High-Performance
Size-Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Kaloyan Takov, I-Jou Teng, and Manuel Mayr
4 Isolation and Proteomic Analysis of Mouse Serum Small Extracellular
Vesicles for Individual Subject Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Federica Anastasi, Marialaura Dilillo, Davide Pellegrini,
and Liam A. McDonnell
5 Protocol for Measuring Concentrations of Extracellular Vesicles
in Human Blood Plasma with Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Najat Hajji, Chi M. Hau, Rienk Nieuwland,
and Edwin van der Pol

PART II ISOLATION AND CHARACTERIZATION OF TISSUE

AND BIOFLUID-SPECIFIC EVS

6 Targeted Mass Spectrometry-Based Proteomics Method

to Quantify Placental Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Andrew Lai, Carlos Palma, Alexis Salas, Flavio Carrion,
and Carlos Salomon
7 Isolation and Characterization of Extracellular Vesicles Secreted
from Human Pluripotent Stem Cell-Derived Cardiovascular
Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Qiang Wu, Min-Xia Ke, and Huang-Tian Yang
8 Isolation and Characterization of Salivary Exosomes for Cancer
Biomarker Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Lucas Trevisan França de Lima, Juliana Müller Bark,
Mohammad Rasheduzzaman, Chameera Ekanayake Weeramange,
and Chamindie Punyadeera

ix
x Contents

9 Isolation and Characterization of Urinary Extracellular Vesicles

for MicroRNA Biomarker Signature Development with Reference
to MISEV Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Marlene Reithmair, Anja Lindemann, Veronika Mussack,
and Michael W. Pfaffl

PART III ANALYSIS OF EV COMPONENTS

10 Direct Detection of Extracellular Vesicle miRNAs Using a Single-Step
RT-qPCR Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Ayyanar Sivanantham, Heedoo Lee, and Yang Jin
11 Purification and Phosphoproteomic Analysis of Plasma-Derived
Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Anton B. Iliuk
12 Lipidomic Analysis of Extracellular Vesicles Isolated
from Human Plasma and Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Yuchen Sun, Kosuke Saito, and Yoshiro Saito

PART IV EV ENGINEERING

13 Extracellular Vesicles and Their Use as Vehicles of Immunogens . . . . . . . . . . . . . . 177

Chiara Chiozzini, Barbara Ridolfi, and Maurizio Federico
14 Isolation and Fluorescent Labeling of Extracellular Vesicles
from Cultured Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Noelle Leary, Sarina Walser, and Lothar C. Dieterich
15 Generation, Characterization, and Count of Fluorescent
Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Flavia Ferrantelli, Valentina Tirelli, Valeria Barreca,
and Francesco Manfredi
16 Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading
with Synthetic miRNA Mimic Using Electroporation . . . . . . . . . . . . . . . . . . . . . . . 219
Margherita A. C. Pomatto, Federica Negro,
and Giovanni Camussi
17 Extracellular Vesicle Loading Via pH-Gradient Modification . . . . . . . . . . . . . . . . . 231
Stephanie M. Kronstadt, Steven M. Jay,
and Anjana Jeyaram

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Contributors

KHULOUD T. AL-JAMAL • Institute for Pharmaceutical Science, King’s College London,

London, UK
FEDERICA ANASTASI • NEST Laboratories, Scuola Normale Superiore, Pisa, Italy; Fondazione
Pisana per la Scienza ONLUS, Pisa, Italy
VALERIA BARRECA • National Center for Global Health, Istituto Superiore di Sanità, Rome,
Italy
GIOVANNI CAMUSSI • Department of Medical Sciences, University of Turin, Turin, Italy
FLAVIO CARRION • Departamento de Investigacion, Postgrado y Educacion Continua
(DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba, Santiago, Chile
CHIARA CHIOZZINI • National Center for Global Health, Istituto Superiore di Sanità (ISS),
Rome, Italy
LOTHAR C. DIETERICH • Institute of Pharmaceutical Sciences, Swiss Federal Institute of
Technology (ETH) Zurich, Zurich, Switzerland
MARIALAURA DILILLO • Fondazione Pisana per la Scienza ONLUS, Pisa, Italy
MAURIZIO FEDERICO • National Center for Global Health, Istituto Superiore di Sanità (ISS),
Rome, Italy
FLAVIA FERRANTELLI • National Center for Global Health, Istituto Superiore di Sanità,
Rome, Italy
NAJAT HAJJI • Laboratory Experimental Clinical Chemistry, Amsterdam University Medical
Centers, location AMC, Amsterdam, the Netherlands; Vesicle Observation Center,
Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands
CHI M. HAU • Laboratory Experimental Clinical Chemistry, Amsterdam University
Medical Centers, location AMC, Amsterdam, the Netherlands; Vesicle Observation Center,
Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands
ANTON B. ILIUK • Tymora Analytical Operations, West Lafayette, IN, USA
STEVEN M. JAY • Fischell Department of Bioengineering, University of Maryland, College
Park, MD, USA; Program in Molecular and Cell Biology, University of Maryland, College
Park, MD, USA
ANJANA JEYARAM • Fischell Department of Bioengineering, University of Maryland, College
Park, MD, USA
YANG JIN • Division of Pulmonary and Critical Care Medicine, Department of Medicine,
Boston University, Boston, MA, USA
MIN-XIA KE • CAS Key Laboratory of Tissue Microenvironment and Tumor, Laboratory of
Molecular Cardiology, Shanghai Institute of Nutrition and Health, University of Chinese
Academy of Sciences (CAS), Shanghai, China
STEPHANIE M. KRONSTADT • Fischell Department of Bioengineering, University of Maryland,
College Park, MD, USA
ANDREW LAI • Exosome Biology Laboratory, University of Queensland Centre for Clinical
Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane,
QLD, Australia
NOELLE LEARY • Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology
(ETH) Zurich, Zurich, Switzerland

xi
xii Contributors

HEEDOO LEE • Department of Biology and Chemistry, Changwon National University,

Changwon, Korea
JIN LI • The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang
Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of
Sciences, Hangzhou, Zhejiang, China
ANJA LINDEMANN • Institute of Human Genetics, University Hospital Munich, Ludwig-
Maximilians-University Munich, Munich, Germany
FRANCESCO MANFREDI • National Center for Global Health, Istituto Superiore di Sanità,
Rome, Italy
MANUEL MAYR • King’s College London British Heart Foundation Centre, School of
Cardiovascular Medicine and Sciences, London, UK
LIAM A. MCDONNELL • Fondazione Pisana per la Scienza ONLUS, Pisa, Italy
JULIANA MÜLLER BARK • Saliva and Liquid Biopsy Translational Laboratory, The School of
Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD, Australia
VERONIKA MUSSACK • Department of Animal Physiology and Immunology, TUM School of
Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany
FEDERICA NEGRO • Department of Medical Sciences, University of Turin, Turin, Italy
RIENK NIEUWLAND • Laboratory Experimental Clinical Chemistry, Amsterdam University
Medical Centers, location AMC, Amsterdam, the Netherlands; Vesicle Observation Center,
Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands
CARLOS PALMA • Exosome Biology Laboratory, University of Queensland Centre for Clinical
Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane,
QLD, Australia
DAVIDE PELLEGRINI • NEST Laboratories, Scuola Normale Superiore, Pisa, Italy; Fondazione
Pisana per la Scienza ONLUS, Pisa, Italy
MICHAEL W. PFAFFL • Department of Animal Physiology and Immunology, TUM School of
Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany
MARGHERITA A. C. POMATTO • Department of Medical Sciences, University of Turin, Turin,
Italy
CHAMINDIE PUNYADEERA • Saliva and Liquid Biopsy Translational Laboratory, The School of
Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD, Australia;
Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery
(GRIDD) and Menzies Health Institute Queensland (MIHQ), Griffith University,
Brisbane, QLD, Australia
FENGLI QU • The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang
Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of
Sciences, Hangzhou, Zhejiang, China
MOHAMMAD RASHEDUZZAMAN • Saliva and Liquid Biopsy Translational Laboratory, The
School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD,
Australia
MARLENE REITHMAIR • Institute of Human Genetics, University Hospital Munich, Ludwig-
Maximilians-University Munich, Munich, Germany
BARBARA RIDOLFI • National Center for Global Health, Istituto Superiore di Sanità (ISS),
Rome, Italy
KOSUKE SAITO • Division of Medicinal Safety Science, National Institute of Health Sciences,
Kawasaki City, Kanagawa, Japan
YOSHIRO SAITO • Division of Medicinal Safety Science, National Institute of Health Sciences,
Kawasaki City, Kanagawa, Japan
 Contributors xiii

ALEXIS SALAS • Department of Pharmacology, Faculty of Biological Sciences, University of

Concepcion, Concepcion, Chile
CARLOS SALOMON • Exosome Biology Laboratory, University of Queensland Centre for
Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland,
Brisbane, QLD, Australia; Department of Pharmacology, Faculty of Biological Sciences,
University of Concepcion, Concepcion, Chile; Departamento de Investigacion, Postgrado y
Educacion Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba,
Santiago, Chile
AYYANAR SIVANANTHAM • Division of Pulmonary and Critical Care Medicine, Department of
Medicine, Boston University, Boston, MA, USA
YUCHEN SUN • Division of Medicinal Safety Science, National Institute of Health Sciences,
Kawasaki City, Kanagawa, Japan
KALOYAN TAKOV • King’s College London British Heart Foundation Centre, School of
Cardiovascular Medicine and Sciences, London, UK
WEIHONG TAN • The Cancer Hospital of the University of Chinese Academy of Sciences
(Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese
Academy of Sciences, Hangzhou, Zhejiang, China; Molecular Science and Biomedicine
Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics,
College of Chemistry and Chemical Engineering, College of Biology, Collaborative
Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha,
China; Institute of Molecular Medicine (IMM), Renji Hospital, State Key Laboratory of
Oncogenes and Related Genes, Shanghai Jiao Tong University, School of Medicine, and
College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,
Shanghai, China
I-JOU TENG • King’s College London British Heart Foundation Centre, School of
Cardiovascular Medicine and Sciences, London, UK
VALENTINA TIRELLI • Core Facilities, Istituto Superiore di Sanità, Rome, Italy
LUCAS TREVISAN FRANÇA DE LIMA • Saliva and Liquid Biopsy Translational Laboratory, The
School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD,
Australia
EDWIN VAN DER POL • Laboratory Experimental Clinical Chemistry, Amsterdam University
Medical Centers, location AMC, Amsterdam, the Netherlands; Vesicle Observation Center,
Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands;
Biomedical Engineering and Physics, Amsterdam University Medical Centers, location
AMC, Amsterdam, the Netherlands
SARINA WALSER • Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology
(ETH) Zurich, Zurich, Switzerland
CHAMEERA EKANAYAKE WEERAMANGE • Saliva and Liquid Biopsy Translational Laboratory,
The School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove,
QLD, Australia
QIANG WU • CAS Key Laboratory of Tissue Microenvironment and Tumor, Laboratory of
Molecular Cardiology, Shanghai Institute of Nutrition and Health, University of Chinese
Academy of Sciences (CAS), CAS, Shanghai, China
SITAO XIE • The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang
Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of
Sciences, Hangzhou, Zhejiang, China
xiv Contributors

LIZHOU XU • Hangzhou Global Scientific and Technological Innovation Center, Zhejiang

University, Hangzhou, China; Institute for Pharmaceutical Science, King’s College
London, London, UK
HUANG-TIAN YANG • CAS Key Laboratory of Tissue Microenvironment and Tumor,
Laboratory of Molecular Cardiology, Shanghai Institute of Nutrition and Health,
University of Chinese Academy of Sciences (CAS), CAS, Shanghai, China; Translational
Medical Center for Stem Cell Therapy and Institute for Heart Failure and Regenerative
Medicine, Shanghai East Hospital, Tongji University School of Medicine and Shanghai
Institute of Stem Cell Research and Clinical Translation, Shanghai, China; Institute for
Stem Cell and Regeneration, CAS, Beijing, China
 Part I

Extracellular Vesicles Detection and Isolation

 Chapter 1

Aptasensors for Cancerous Exosome Detection

Jin Li, Sitao Xie, Fengli Qu, and Weihong Tan

Abstract
Cancerous exosomes that carry multiple biomarkers are attractive targets for the early diagnosis and therapy
of cancer. As one of the powerful molecular recognition tools, aptamers with excellent binding affinity and
specificity toward biomarkers have been exploited to construct various aptamer-based biosensors (aptasen-
sors) for exosome detection. Here, we review recent advances in aptasensors for the detection of cancerous
exosomes. We first discuss the importance and potential of cancerous exosomes in cancer diagnosis and then
summarize some conventional aptasensors from the perspective of biomarker recognition and signal
collection strategies. Finally, we comment on the outlook for aptasensor research and new directions for
cancerous exosome detection.

Key words Cancerous exosome, Aptamer, Early diagnosis, Aptasensor, Exosome detection

1 Cancerous Exosome Detection for Diagnosis and Therapy

The increasingly high incidence of cancers is marked by equally

high worldwide mortality [1]. Clinical and basic scientists have
agreed that the survival rate of patients depends on early diagnosis
and therapeutic intervention and that this goal can be improved by
the detection of cancer biomarkers. Different types of detection
tools have been developed to accurately identify various types of
cancer [2–4]. In particular, single-factor biomarker identification
has achieved some success, but the simultaneous detection and
classification of multiple biomarkers is more likely to lead to
improved prognoses in the future [5–9].
One such ideal biomarker is the exosome which is a class of
extracellular vesicle-like structures released by cells. Ranging in size
from 30 to 120 nm, the exosome contains DNAs, RNAs, proteins,
and lipids. It participates in the regulation of normal physiological
processes, but it is also suspected of regulating many cancerous
processes [10]. Specifically, the formation and production of exo-
somes in source cells, whether normal or cancerous, involves the
process of the membrane invagination of late endosomes and

Maurizio Federico and Barbara Ridolfi (eds.), Extracellular Vesicles in Diagnosis and Therapy,
Methods in Molecular Biology, vol. 2504, https://doi.org/10.1007/978-1-0716-2341-1_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

3
4 Jin Li et al.

multivesicular body (MVB) fusion with the plasma membrane.

Thus, exosomes share some features in common, such as nucleic
acids (mRNA, miRNA, and long non-coding RNAs), lipids (cho-
lesterol and ceramide), and proteins (tetraspanins, heat shock pro-
teins, membrane transport and fusion proteins) [11–14]. Owing to
the presence and stability of exosomes in most bodily fluids, cancer-
derived exosomes can carry cargoes reflective of changes in genetic
material or signals in cancer cells, thus acting as an early warning
system for the presence of cancer and allowing the collection and
analysis of exosomes in bodily fluids for diagnostic purposes. As the
role of exosomes begins to emerge in cancer diagnosis, remarkable
progress has been achieved by researchers in advancing the devel-
opment of exosome detection methods using nanomaterials [15],
optics [16], electrochemistry [17, 18], and so on [11].

2 Aptamers for Cancerous Exosome Recognition

Since exosomes can carry multiple biomarkers from sourced cancer

cells, they may allow clinicians to determine the type and progress
of cancers. However, in order to simultaneously and accurately
detect and analyze the species and abundance of biomarkers carried
by exosomes, powerful molecular recognition tools are required.
Aptamers, known as “chemical antibodies,” are short single-
stranded nucleic acids evolved by the process termed systematic
evolution of ligands by exponential enrichment (SELEX)
[19, 20]. The essence of aptamers is the three-dimensional struc-
ture produced by the folded nucleic acid. Aptamers can fold into
specific secondary or tertiary structures and bind to their targets by
forming stable complexes with their targets. With high binding
affinity and specificity to targets, such as metal ions [21], proteins
[22], whole cells [23], and even tissues [24], and the advantages of
easy chemical modification and programmability, aptamers are
attractive alternatives to antibodies for molecular recognition,
making them a powerful tool for the accurate recognition and
identification of multiple biomarkers on the exosome plasma
membrane [25].
Table 1 summarizes some cancerous protein targets commonly
used for exosome detection. Related cancer types and some
corresponding aptamers are listed. In the next section, we review
recent advances in aptasensor development for the detection of
cancerous exosomes. In particular, fluorescent-, electrochemical-,
and colorimetric-based methods are discussed. Our concluding
remarks include some perspectives on the research directions and
opportunities in developing aptasensors for cancerous exosome
detection.
 Aptasensors for Cancerous Exosome Detection 5

Table 1
Summary of cancerous protein target and corresponding aptamers

Protein
target Aptamer Cell type References
CD24 CD24A_2 HT29 [26]
CD63 LL4A, CD63-1 Melanoma cells, MDA-MB-231, HT29, [27–30]
MCF-7, HepG2
PD-L1 aptPD-L1, XQ-P3 A549, MDA-MB-231 [31]
EGFR CL4, GR200, TuTu22 A549, A431, U87, U251 [32, 33]
HER2 Aptamers 2-2, Heraptamer N87, SKOV3 [34, 35]
EpCAM 19-nt RNA aptamer, Ep1 Breast, colorectal, gastric cancer cells [36]
aptamer
PSMA xPSM aptamer PSMA-expressing LNCaP cells [37]
PTK7 sgc8 CCRF-CEM cells [23]
CEA YJ-1 CEA-positive cell lines [38]
MUC1 S1.3, 5TR1, GalNAc3 MUC1-positive cell lines [39]
aptamers
AFP AP273 HepG2 [40]
HSP70 A8 Breast, lung, ovarian cancer cells [41]

3 Aptasensors for Exosome Detection

Aptamers are oligonucleotides that can be easily synthesized and

modified with desired functional groups [42, 43]. Based on these
characteristics, aptamer-based sensors (aptasensors) have been
developed to detect exosomes through different signal transduc-
tion mechanisms, e.g., fluorescent [44, 45], electrochemical
[46, 47], and colorimetric [48, 49]. In this section, aptasensor-
based exosome detection strategies are introduced.

3.1 Fluorescent Fluorescence imaging has the advantages of high sensitivity and
Aptasensor-Based reliability, noninvasiveness, and short-term analysis and is, there-
Methods fore, the most commonly used method in exosome detection
[50, 51]. Fluorescent aptasensors can be easily constructed by
integrating aptamers with fluorescent dyes, quantum dots, upcon-
version nanoparticles, or other fluorescent materials. For example,
Zhang et al. designed an “on-off”-type aptasensor for detecting
cancer exosomes (Fig. 1a) [52]. A TAMRA fluorophore and a
Dabcyl quencher were labeled at the two ends of the hairpin-like
aptamer, respectively. In the absence of tumor exosomes (TEX), the
fluorescence generated by TAMRA was quenched by the
6 Jin Li et al.

Fig. 1 Aptasensor-based fluorescent platform for exosome detection. (a) Fluorescence resonance energy
transfer (FRET)-based aptasensor for exosome detection, reproduced from Zhang 2018 with permission from
Elsevier [52]. (b) HCR-based signal amplification and detection of exosomes [53]. (c) Exosome detection based
on nuclease-assisted signal amplification, reproduced from Wang 2018 with permission from Elsevier [54]. (d)
Identifying exosomes with a FP-based aptasensor, reproduced from Zhang 2019 with permission from Royal
Society of Chemistry [55]

neighboring Dabcyl, and the aptasensor was in the “off” state.

However, upon aptamer binding to its cognate target, Mucin
1 (MUC1), on the exosome plasma membrane, TAMRA and Dab-
cyl separated, turning on the aptasensor for exosome detection by
measuring the fluorescence intensity of TAMRA. This simple fluo-
rescent aptasensor achieved the detection of MCF-7 cell-derived
exosomes with a low detection limit (3
105 particles μL1). In
another work, Zhang et al. constructed another turn-on fluores-
cent aptasensor for exosome detection [56]. This time,
Cy3-modified CD63 aptamer was first adsorbed onto Ti3C2
MXene nanosheets, and the fluorescence was quenched by the
nanosheets. When exosomes were added to the nanocomposites,
CD63 aptamers were released from the nanosheets owing to spe-
cific binding between CD63 aptamers and the CD63 proteins
present on exosomes. The fluorescence of aptasensors was then
recovered and the exosomes were identified according to the recov-
ered fluorescence. The detection limit of this fluorescent aptasensor
was reported to be 1.4
103 particles mL1. Meanwhile, by
integrating other specific aptamers with Ti3C2 MXene nanosheets,
 Aptasensors for Cancerous Exosome Detection 7

multiple proteins on different exosomes were identified. Thus, this

Ti3C2 MXene nanosheet-based fluorescent aptasensor could profile
multiple biomarkers on exosomes.
Aptamers can also be easily combined with multiple cyclic
amplification reactions, including hybridization chain reaction
(HCR), nuclease-assisted signal amplification (NSA), and rolling-
circle amplification (RCA), to construct aptasensors for accurate
detection of exosomes. For example, Shi et al. developed an
HCR-based fluorometric assay for highly sensitive detection of
HepG2 cell-secreted exosomes (Fig. 1b) [53]. In this design, exo-
somes could be captured by anti-CD63 antibody-modified mag-
netic nanoparticles (MNPs), and epithelial cell adhesion molecule
(EpCAM) aptamers could specifically bind to the captured exo-
somes. The bound EpCAM aptamers then initiated a hybridization
chain reaction between two FAM-labeled probes to obtain a strong
fluorescence signal. Finally, exosomes were detected by measuring
the fluorescence intensity of FAM, and the detection limit of this
HCR-based aptasensor was reported to be 100 particles·mL1.
As oligonucleotides, aptamers are easily degraded by nucleases.
Several NSA-based aptasensors have been constructed for exosome
detection. By constructing an NSA platform, Wang et al. achieved
detection of colorectal cancer exosomes with a limit of detection
(LOD) of 2.1
104 particles μL1 (Fig. 1c) [54]. In this work,
fluorophore-labeled aptamers were adsorbed onto graphene oxide
(GO), and the fluorescence was quenched by GO. Once exosomes
were added, aptamers could specifically bind to the exosomes, and
the fluorescence was recovered. Subsequently, the aptamer-bound
exosomes were degraded by DNase I, and more fluorophore-
labeled aptamers that had adsorbed to GO could bind to the
“free” exosomes, resulting in cyclic digestion of aptamers and
amplification of the fluorescent signal. Importantly, this
NSA-based aptasensor has been used to distinguish healthy and
cancer patients by detecting their blood serum samples, showing
its potential in cancer diagnosis. Using a similar strategy, Jin et al.
identified cancer cell-secreted exosomes with a LOD of 1.6
102
particles μL1 and profiled seven biomarkers on exosomes that
derived from five types of cells [57].
Rolling-circle amplification-based aptasensors are also widely
used to detect exosomes by generating a large number of repeating
units to achieve satisfactory signal amplification. Huang et al., for
instance, constructed a branched RCA (BRCA)-based aptasensor
for highly sensitive detection of exosomes derived from gastric
cancer cells [50]. In this design, the MUC1-binding aptamer was
sequentially used to identify exosome, trigger circularization of a
padlock probe, and initiate the BRCA reaction. SYBR Green I
bound to BRCA products and was used as a fluorescent dye to
identify exosomes. The LOD of this aptasensor was reported to be
4.27
104 particles·mL1. This aptasensor has also been used to
8 Jin Li et al.

test plasma samples, and it showed promising clinical potential. In

another work, Huang et al. constructed a dual-signal amplification-
based aptasensor to detect leukemia cell-secreted exosomes
[51]. RCA and NSA reaction were used for primary signal amplifi-
cation and secondary signal amplification, respectively. Using this
dual-signal amplification strategy, the detection of exosomes was
down to 102 particles μL1.
The fluorescence polarization (FP) assay, which does not rely
on quenchers or donor-acceptor pairs, is another good choice for
exosome detection. Zhang et al. developed an FP-based assay that
does not require amplification of fluorescence or separation of
exosomes from human plasma for detection (Fig. 1d) [55]. When
low molecular weight dye-labeled aptamers bound to the high
molecular weight exosomes, the FP value increased, and exosomes
could be identified according to the change in FP value. The LOD
of this FP-based aptasensor was reported to be 5
102 particles
μL1. Importantly, this simple (one step) and fast (within 30 min)
assay has been used to directly quantify exosomes in clinical samples
of healthy and lung cancer patients, showing their potential in
cancer diagnosis and therapy monitoring.

3.2 Electrochemical Electrochemical assays have attracted widespread attention in bioa-

Aptasensor-Based nalysis for their high sensitivity, low cost, and fast response. In
Methods recent years, by using aptamers as a recognition element, electro-
chemical aptasensors have been developed for exosome detection
[58, 59]. Zhou et al. developed an electrochemical aptasensor-
based assay to detect exosomes by directly modifying aptamers
and redox reporters onto gold electrodes [60]. In the presence of
exosomes, CD63 aptamers could specifically bind to exosomes,
resulting in the release of their complementary sequences modified
by redox moieties. A decreased electrochemical signal was detected
by the release of the redox moieties, and exosomes could then be
identified according to the changed redox signal. The LOD of this
electrochemical-based aptasensor was reported to be 1
103 par-
ticles μL1. Single-stranded DNA probes immobilized on electro-
des tend to become entangled, thwarting highly sensitive detection
of exosomes. To solve this problem, Wang et al. constructed a
nanotetrahedron (NTH)-assisted aptasensor to improve the detec-
tion performance of aptamers on gold electrodes (Fig. 2a) [61]. In
this design, DNA tetrahedrons contain an aptamer at the top
vertex, while three thiols at bottom vertices were constructed and
immobilized onto gold electrodes through the thiols. Compared
with single-stranded aptasensors, this NTH-assisted aptasensor
achieved exosome detection with 100-fold higher sensitivity.
To improve the sensitivity of exosome detection, amplification
strategies, such as HCR, RCA, NSA, and DNA walker, were used in
developing electrochemical-based aptasensors. An et al. con-
structed an HCR-based electrochemical aptasensor to detect
 Aptasensors for Cancerous Exosome Detection 9

Fig. 2 Aptasensor-based electrochemical platform for exosome detection. (a) NTH-assisted aptasensor for
exosome detection, reproduced from Wang 2017 with permission from American Chemical Society [61]. (b)
HCR-based signal amplification and detection of exosomes, reproduced from An 2019 with permission from
Elsevier [62]. (c) RCA-based signal amplification and detection of exosomes [63]. (d) Dual-amplified-based
aptasensor for exosome detection, reproduced from Zhao 2019 with permission from American Chemical
Society [64]

exosomes derived from MCF-7 cells (Fig. 2b) [62]. In this design,
CD63 aptamers were immobilized onto glassy carbon electrodes to
capture exosomes. Then, a DNA initiator was anchored to the
captured exosomes to initiate HCR between two biotin-labeled
DNA monomers. Finally, horseradish peroxidase (HRP) was com-
bined with the HCR products through interactions between biotin
and streptavidin. HRP could catalyze the oxidation of
o-phenylenediamine (OPD) to obtain detectable electrical signals,
thus identifying exosomes. By using this HCR-based amplification
strategy, exosomes could be detected with a LOD of 9.6
101
particles μL1. The ability of this HCR-based electrochemical apta-
sensor to detect exosomes in serum samples has also been proved.
In another work, Huang et al. constructed an RCA-based
electrochemical aptasensor for detecting gastric cancer exosomes
(Fig. 2c) [63]. In this work, anti-CD63 antibodies were immobi-
lized onto gold electrodes to capture exosomes. MUC1 aptamers
bound to the captured exosomes and subsequently triggered RCA
to generate a range of G-quadruplex-hemin complexes. The G-
quadruplex-hemin complexes catalyzed the reduction of H2O2 to
10 Jin Li et al.

obtain electrical signals for exosome detection. The LOD of this

RCA-based electrochemical aptasensor was reported to be
9.54
102 particles mL1. This economical and simple aptasensor
was also used to analyze plasma samples, revealing its potential
application in the diagnosis of gastric cancer.
The DNA walker has also been used to construct a highly
sensitive electrochemical aptasensor for exosome detection
(Fig. 2d) [64]. Zhao et al. developed a dual-amplified system to
detect exosomes derived from MCF-7 cells. The system contains
two parts. In the first part, CD63 aptamers and single-stranded
DNAs that contain a ribonucleobase (rA) cleavage point were
labeled to magnetic beads (MB). When exosomes were added,
exosomes could be captured by the CD63 aptamer and another
aptamer (EpCAM) linked to a DNAzyme could then bind to the
captured exosomes. The DNAzyme acted as a DNA walker to
induce the cleavage of the multiple single-stranded DNAs on the
MB, leading to the first signal amplification of exosomes and releas-
ing an oligonucleotide fragment (namely, P1) to the second part. In
the second part, methylene blue-labeled, hairpin-like DNAs
(MB-DNA) were immobilized on the gold electrode to capture
P1. MB-DNA hybridized with P1 to form a double-stranded DNA,
followed by Exo III digestion of MB-DNA in the double-stranded
DNA. This resulted in the release of P1 from the ds-DNA for
hybridization with another MB-DNA, leading to cyclic digestion
of MB-DNA and the second signal amplification of exosomes.
Finally, a large number of short DNAs were left on the electrode
to capture ferrocene-modified DNAs to generate detectable elec-
trical signals for exosome detection. Based on this dual-amplified
strategy, exosomes could be detected with a LOD of 1.3
104
particles mL1. This dual-amplified aptasensor was also used to
analyze complex biological samples and differentiate plasma sam-
ples between healthy and breast cancer patients, indicating that it is
a promising tool for exosome-based cancer diagnosis.

3.3 Colorimetric Colorimetric sensors have been widely used in bioanalysis for their
Aptasensor-Based simplicity and ability to convert sample data into a signal readable
Methods by the naked eye. Thus far, several colorimetric aptasensors have
been constructed for exosome detection [49, 65]. Xia et al. con-
structed a single-walled carbon nanotubes (s-SWCNTs)-based col-
orimetric aptasensor to detect exosomes derived from MCF-7 cells
(Fig. 3a) [66]. In this design, CD63 aptamers able to improve the
peroxidase activity of s-SWCNTs were first absorbed onto
s-SWCNTs. The absorbed s-SWCNTs could then efficiently cata-
lyze the oxidation of 3,30 ,5,50 -tetramethylbenzidine (TMB) to
generate a large number of products with blue color. In the pres-
ence of exosomes, CD63 aptamers specifically bound to exosomes,
and their release from s-SWCNTs led to a decrease in peroxidase
activity of s-SWCNTs. As a consequence, fewer products with blue
 Aptasensors for Cancerous Exosome Detection 11

Fig. 3 Aptasensor-based colorimetric platform for exosome detection. (a) s-SWCNTs-based aptasensor for
exosome detection, reproduced from Xia 2017 with permission from Elsevier [66]. (b) Multicolor detection
platform for visual detection of exosomes, reproduced from Zhang 2019 with permission from American
Chemical Society [67]. (c) Profiling the membrane proteins of exosomes with Aptamer/AuNP sensing platform,
reproduced from Jiang 2017 with permission from Wiley [68]

color were generated, and exosomes could be determined by the

solution’s color change. Using this colorimetric aptasensor, exo-
somes were detected with a LOD of 5.2
105 particles μL1. This
s-SWCNTs-based aptasensor has the advantages of fast response,
low cost, freedom from labeling, and visual analysis, holding poten-
tial in the construction of point-of-care testing devices for the
detection of exosomes.
In another work, Zhang et al. developed a multicolor detection
platform for visual detection of exosomes (Fig. 3b) [67]. Exosomes
were first captured by CD63 aptamer-modified magnetic beads
(MB). Cholesterol-labeled DNAs containing a DNA initiator was
anchored to the captured exosomes through hydrophobic interac-
tion between cholesterol and lipid membrane of exosomes. Then,
12 Jin Li et al.

the DNAs initiated HCR to load alkaline phosphatases (ALP),

thereby promoting the generation of ascorbic acid. Subsequently,
silver ions were reduced by ascorbic acid, and the obtained silver
was deposited on Au nanorods to generate multicolor Au@Ag
nanorods. Thus, exosomes could be identified according to variable
colors in the solution with LOD down to 9
103 particles μL1 as
detectable by the naked eye. Compared with single-color-based
visual analysis, this multicolor detection platform is novel and
useful as multicolor variation is easier to discern by the naked eye.
In addition to detecting exosomes, aptasensors have also been
used to profile the membrane proteins of exosomes. By developing
a colorimetric aptasensor platform (namely, Aptamer/AuNP),
Jiang et al. successfully profiled five proteins on four different
exosomes (Fig. 3c) [68]. In this work, aptamers were first absorbed
to gold nanoparticles (AuNP) to prevent their aggregation. Once
exosomes were added, aptamers could bind to exosomes through
the specific binding between aptamers and their target proteins on
exosomes, resulting in AuNP aggregation and changing of the
solution’s color. In this way, the proteins present on exosomes
could be identified by observing the color change of the solution
by the naked eye and monitored by UV-vis spectrometry. Impor-
tantly, the developed Aptamer/AuNP sensor was able to profile
membrane proteins of exosomes within a few minutes, making it
suitable for high-throughput analysis of clinical samples.

3.4 Other Surface plasmon resonance (SPR)-based [69], surface-enhanced

Aptasensor-Based Raman scattering (SERS)-based [70], or electrochemilumines-
Methods cence (ECL)-based [71] aptasensors have also been developed for
exosome detection. SPR has been widely used in bioanalysis
because it is a label-free and real-time detection assay. Wang et al.
developed an SPR-based, dual-amplification aptasensor for detect-
ing cancerous exosomes (Fig. 4a) [27]. In this work, exosomes
were first captured by CD63 aptamers immobilized on the Au
film and detected by measuring the SPR signal. Then, CD63
aptamer-modified A30-gold nanoparticles (A30-AuNP) could spe-
cifically bind to the captured exosomes via interactions between
CD63 aptamers and CD63 proteins on exosomes, resulting in the
first amplification of an SPR signal. Subsequently, T30-AuNPs
bound to the A30-AuNPs through hybridization between the
A30 sequence and the T30 sequence to achieve the dual-signal
amplification. This dual-amplification aptasensor could be used to
detect exosomes with a LOD of 5 particles μL1 and to distinguish
exosomes derived from MCF-7 breast cancer cells from MCF-10A
normal breast cells.
SERS-based aptasensors are also powerful tools for exosome
detection because they can be used to screen different exosomes
with single-molecule sensitivity. Wang et al. constructed a SERS-
based aptasensor for simultaneous detection of multiple exosomes
 Aptasensors for Cancerous Exosome Detection 13

Fig. 4 (a) SPR-based, dual-amplification aptasensor for exosome detection, reproduced from Wang 2019 with
permission from Elsevier [27]. (b) Identifying exosomes with a SERS-based aptasensor, reproduced from
Wang 2018 with permission from Royal Society of Chemistry [72]. (c) Exosome detection based on AuNPs-
MXenes-Apt nanoprobes, reproduced from Zhang 2020 with permission from American Chemical Society [73]

(Fig. 4b) [72]. In this design, magnetic nanobeads were modified

by CD63 aptamers to capture and separate exosomes. Raman
reporter and a specific aptamer (HER2 aptamer, PSMA aptamer
or CEA aptamer) were labeled to gold nanoparticles for specific
recognition of target exosomes and provide a SERS signal. Once
exosomes were added, the CD63 aptamer-modified magnetic
nanobeads and the specific aptamer-labeled gold nanoparticles
bound to the target exosomes to form a ternary complex, leading
to a decreased intensity of SERS signal in the supernatant. Thus,
exosomes could be determined by the changed SERS signal. Using
14 Jin Li et al.

this SERS-based aptasensor, three kinds of exosomes (derived from

SKBR3, T84, and LNCaP cells) could be detected with a LOD of
32, 73, and 203 particles μL1, respectively. Furthermore, this
SERS-based aptasensor could be used to determine exosomes in
blood samples, revealing its potential applications in exosome-
based cancer screening.
Owing to its low background signal and high sensitivity, the
ECL assay has also been used for exosome detection. Zhang et al.
constructed an ECL-based aptasensor for highly sensitive detection
of tumor exosomes (Fig. 4c) [73]. In this design, CD63 aptamers
were immobilized on glassy carbon electrodes to effectively capture
exosomes. Then, the CD63 aptamer-modified Ti3C2 MXenes
nanosheets bound to the captured exosomes through the specific
binding between CD63 aptamers and their target proteins present
on exosomes. The Ti3C2 MXenes nanosheets reduced HAuCl4 to
generate gold nanoparticles in situ, thereby forming AuNPs-
MXenes-Apt nanoprobes for exosome detection. This ECL-based
aptasensor could be used to detect exosomes (and their surface
proteins) secreted by various cancer cells in serum, indicating that
it is a promising tool for clinical exosomes detection and cancer
diagnosis.

3.5 Aptasensor- Cancerous exosomes carrying abundant cancer biomarkers, e.g.,

Based Exosome cancer-related proteins, are attractive targets for early diagnosis of
Detection for Cancer cancer. Due to their high specificity and affinity, flexible design and
Diagnosis the ability to simultaneously detect multi-parameter targets, apta-
mers have become promising tools for exosome-based cancer diag-
nosis [54, 55, 57, 62]. Huang et al. developed a thermophoresis-
based aptasensor to detect programmed death-ligand 1 (PD-L1)
on circulating exosomes for cancer diagnosis [74]. In this study,
fluorescent dye-labeled anti-PD-L1 aptamers were first mixed with
isolated circulating exosomes to achieve the specific binding
between PD-L1 and the aptamers (Fig. 5a). Then, the mixture
was exposed to an infrared laser to generate a temperature gradient
that could induce the accumulation of aptamer-bound exosomes,
and thus result in an amplified signal for the quantification of
exosomes. By using this thermophoresis-based aptasensor, exoso-
mal PD-L1 could be detected with a LOD of 17.6 pg mL1.
Importantly, the authors found that the level of exosomal PD-L1
can not only distinguish cancer patients from healthy controls, but
also has a positive correlation with tumor metastasis. These results
indicate that this thermophoretic-based aptasensor has great poten-
tial in the early diagnosis of cancer.
In another work, Li et al. developed an aptasensor-based nano-
platform for diagnosing prostate cancer [75]. PSMA aptamer
which can specifically bind to the prostate specific membrane anti-
gen was first modified to Fe3O4 nanoparticles and then hybridized
with two single-stranded DNAs (Fig. 5b). In the presence of
 Aptasensors for Cancerous Exosome Detection 15

Fig. 5 (a) Thermophoresis-based aptasensor for cancer diagnosis [74]. (b) Aptasensor-based nanoplatform for
cancer diagnosis, reproduced from Li 2019 with permission from American Chemical Society [75]. (c)
Thermophoretic aptasensor for cancer screening and classification, reproduced from Liu 2019 with permis-
sion from Springer Nature [76]

PSMA-positive exosomes, PSMA aptamers could bind to the exo-

somes and cause the release of the ssDNAs. The released ssDNAs
could then cyclically initiate the hybridization of two hairpin mono-
mers to obtain fluorescence signal for exosome detection. By using
this strategy, 100 exosomes could be detected in 1 μL urine sample.
The level of PSMA-positive exosomes in the urine of prostate
cancer patients is higher than that in healthy persons. This
aptasensor-based nanoplatform has the ability to distinguish pros-
tate cancer patients from healthy controls revealing its potential
applications in diagnosing prostate cancer.
Compared with the detection of single protein biomarker on
exosomes for cancer detection, multi-protein biomarkers profiling
on exosomes is more attractive because it can be used to screen and
classify cancer. In a recent work, by developing a thermophoretic
aptasensor (Fig. 5c), Liu et al. could profile the surface proteins of
extracellular vesicles that derived from 6 types of cancers with
7 aptamers [76]. Fluorescent aptamers were first incubated with
16 Jin Li et al.

the diluted serum sample and the mixture was then locally heated
with an infrared laser to accumulate extracellular vesicles and
obtained a detectable fluorescence signal. Then, the surface pro-
teins of extracellular vesicles could be profiled by detecting the
fluorescence signal. Importantly, this isolation-free, small-volume
(<1 μL serum sample), low-cost, and noninvasive extracellular
vesicle detection strategy had been used to detect and classify
6 types of cancers, and to distinguish prostate cancer from benign
disease. These results indicate that this thermophoretic aptasensor
has great potential in the early screening and classification of
cancers.

4 Conclusion and Prospective

Exosomes have emerged as promising cancer biomarkers for non-

invasive cancer diagnosis. In the past few years, especially from
2019, aptasensors with the ability to specifically recognize and
bind to their targets have been widely employed to detect cancer
exosomes based on multiple signal transduction mechanisms, such
as fluorescence, electrochemistry, and colorimetry. These aptasen-
sors have proved to be powerful tools for detecting exosomes or
profiling exosomal surface proteins because they can analyze exo-
somes with high sensitivity, fast response, and easy-to-read signals.
However, most aptasensors can only be used to identify exosomes
in buffers or diluted biological fluids, indicating the challenges
ahead to extend aptasensor-based exosome detection from labora-
tory testing to clinical analysis. (1) More exosomal biomarkers
against specific cancers need to be identified for accurate diagnosis
of specific cancers. (2) Currently, only a limited number of exo-
somes can be determined by aptasensors by the lack of sufficient
variety of aptamers. Therefore, to achieve exosome-based cancer
screening, more aptamers must be selected against specific biomar-
kers on the plasma membrane surface of exosomes. Meanwhile, to
directly detect exosomes in clinical samples, the stability and affinity
of aptamers in biological fluids need to be improved. Selecting
aptamers in complex biological matrices, rather than in simple
buffer, may help to achieve this goal. (3) In most published work,
highly sensitive exosome detection requires complicated experi-
mental procedures and special equipment, which may limit its
clinical applications. To detect exosomes with aptasensors in the
clinic, the simplicity and usability of the sensing system should be
considered. (4) Compared with identifying exosomes based on a
single biomarker, simultaneous identification of multiple biomar-
kers, such as proteomics analysis of exosomes [77], will make it
easier to achieve accurate exosome detection and cancer diagnosis.
We believe aptasensors can serve as powerful tools for exosome-
based cancer screening and exosome-based prognosis monitoring
once these challenges are overcome.
 Aptasensors for Cancerous Exosome Detection
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 Chapter 2

Detection of Cancer-Derived Exosomes Using a Sensitive

Colorimetric Aptasensor
Lizhou Xu and Khuloud T. Al-Jamal

Abstract
Exosomes are a type of extracellular vesicles that contain constituents including proteins, DNAs, and RNAs
of the cells that secrete them. Cancerous exosomes are potential biomarkers for cancer diagnosis. Biosensors
are useful analytical tools for quantification of biomarkers and targeted molecules. An aptasensor uses the
aptamer as the biorecognition element to bind to the target and is one main type of biosensors that is
promising for exosomes analysis and clinical cancer detection. The assay described in this chapter allows for
reliable, sensitive, and specific detection of cancer-derived exosomes using a colorimetric aptasensor that is
promising for point-of-care testing.

Key words Exosomes, Aptamer, Biosensor, Cancer detection, Biomarker, Aptasensor

1 Introduction

Exosomes are extracellular, membrane-enclosed vesicles, of

50–150 nm in diameter and are derived from most, if not all,
eukaryotic cells through an endosomal pathway and circulate in
body fluids [1, 2]. Exosomes contain constituents on the surface or
inside the lumen including proteins, DNAs, and RNAs of the cells
that secrete them. Exosomes shed from tumorous tissues may
exhibit clinical significance and can be identified as potential non-
invasive cancer biomarkers, as they reflect the genetics or proteins
originating from parent tumors [3]. In the context of a disease,
early diagnosis generally renders the best chances of a cure and
based on their reliability, exosomes biomarkers can provide an
understanding of disease diagnosis, progression, and response to
treatment [4]. For example, cancer cell derived human epidermal
growth factor receptor 2 (HER2) positive exosomes are considered
to be promising tumor biomarkers for clinical diagnosis [5, 6]. Exo-
somes are also advantageous in that they are often very stable due to

Maurizio Federico and Barbara Ridolfi (eds.), Extracellular Vesicles in Diagnosis and Therapy,
Methods in Molecular Biology, vol. 2504, https://doi.org/10.1007/978-1-0716-2341-1_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

21
22 Lizhou Xu and Khuloud T. Al-Jamal

the double-layer membrane and can be stored for extended periods

of time without degradation [7]. Therefore, there is a significant
clinical potential for the use of exosomes as biomarker tools and it is
increasingly needed for robust and accurate cancerous exosomes
detection toward potential noninvasive cancer diagnosis.
Biosensors are smart analytical devices combining specific rec-
ognition of target and sensitive readout of signals [8]. They are fast,
reliable, and precise analytical methods and have been widely used
in medical and biological markets worldwide [9]. Novel biosensors
have recently been studied for detecting and quantifying a variety of
exosomes from cell culture and/or patient samples, facilitating
rapid, facile, or cost-effective detection of the exosomal biomarkers
at a point-of-care setting [10, 11]. Recently, there are many
emerging biosensing methods reported for exosomes detection
using aptamers in the modality of optical, electrochemical, and
electrical biosensors for a variety of applications in cancers as well
as other diseases [10, 12, 13].
Aptamers are single-stranded DNA or RNA molecules that
bind target molecules with high affinity and specificity [14]. Apta-
mers are increasingly used as alternatives of antibodies for the
designing of a sensitive and specific biosensor [15]. Aptasensors
are biosensors that utilize the aptamer as the recognition element in
a biosensor for the detection of specific analytes such as protein
markers, DNA sequences, cells, or small molecules [16]. Aptamers
are generally more resistant to pH, temperature, or other environ-
mental changes than antibodies, and can be synthesized with a high
grade of purity and reproducibility, enabling easier fabrication pro-
cesses of biosensors. Aptasensor can be easily adapted to electro-
chemical, optical, and other sensing principles in a label-free or
labeled manner [17]. Therefore, the aptasensor holds great poten-
tial for exosomes detection with future development of point-of-
care detection kits for cancer diagnosis in a clinical setting [18].
This chapter describes the detailed protocols for the surface
marker protein detection on exosomes using a sensitive and specific
colorimetric aptasensor [12]. The detection principle of this apta-
sensor is shown in Fig. 1. In this work, a specific CD63 aptamer was
used for selective binding/recognition to the target protein CD63,
a common exosomal marker, on exosomes surface. The aptamer
was modified with biotin on one end of the sequence. Target
exosomes are firstly captured by latex beads via aldimine condensa-
tion, followed by specific binding with the aptamer. The aptamer is
then conjugated to streptavidin conjugated horseradish peroxidase
(HRP) through biotin-streptavidin binding. Colorimetric detec-
tion can be achieved in 10 min via enzymatic catalysis to produce
dark colored polydopamine (PDA) from colorless substrate dopa-
mine (DA) in especially prepared H2O2 reaction solution. Colori-
metric signals are significantly visualizable by naked eye.
 Colorimetric Aptasensor for Sensitive Exosome Detection 23

Fig. 1 Schematic illustration of the principle of a colorimetric aptasensor for the sensitive detection of cancer-
derived exosomes

Alternatively, the quantification of the signal can be simply carried

out by absorbance measurement. The detection result reflects the
surface marker expression level for the target exosomes.

2 Materials

Prepare all solutions used in this work using the deionized water
(18 MΩ-cm at 25
C) and analytical grade reagents. Store all
reagents and as-prepared solutions in the fridge at 4
C (unless
indicated otherwise). When disposing off waste materials, please
follow all waste disposal regulations.

2.1 Preparation of Prepare exosomes samples from tissue culture according to Xu et al.
Exosomes Stock [12, 19, 20] or Faruqu et al. [21], or use a commercial exosomes
Solution isolation kit. Prepare the exosomes samples in PBS solution (see
below) at a certain concentration as the stock (see Note 1). Store
the exosomes stock at 80
C for long term or 4
C for short term.

2.2 Bio-Capture of 1. 1 Phosphate-buffered saline (PBS) buffer: transfer 100 mL of

Exosomes onto Latex 10 PBS stock solution (Sigma-Aldrich, UK) to 900 mL of
Beads deionized H2O. Pass the solution through a filter with 0.22 μm
membrane. Autoclave the solution at 120
C for 20 min
before use.
2. Latex beads: aldehyde/sulfate latex beads, 4% w/v, 4 μm,
Sigma-Aldrich.
3. Bovine serum albumin (BSA) blocking buffer: weigh 3 g BSA
(Sigma-Aldrich, UK) and add to 100 mL of deionized H2O to
prepare 3% BSA/PBS solution. Mix well before use (see Note
2). Store at 4
C.
24 Lizhou Xu and Khuloud T. Al-Jamal

4. Glycine buffer: add 0.75 g glycine (Sigma-Aldrich, UK) into

100 mL of PBS (1) to prepare 100 mM glycine buffer.
5. Eppendorf tube: 1.5 mL microcentrifuge tubes.
6. 5 mL Falcon® Round-Bottom Polystyrene Tubes.
7. Centrifuge.

2.3 Color 1. Aptamer solution: acquire appropriate aptamer sequence from a

Development by the commercial supplier (e.g., Eurogentec, Integrated DNA Tech-
Aptasensor nologies). The sequence information is as below: CD63 apta-
mer: 50 /CAC-CCC-ACC-TCG-CTC-CCG-TGA-CAC-TAA-
TGC-TA/30 . Order the aptamer with the form of biotin-
labeled on one end. Prepare the stock aptamer solution accord-
ing to the product information (see Note 3). Store the stock
solution at 80 or 20
C. Then prepare the 0.1 μM biotiny-
lated aptamer working solution with 3% BSA/PBS solution.
Store the working solution at 4
C or on ice before use and use
it in same day.
2. Streptavidin-HRP working solution: prepare the streptavidin-
HRP conjugate using VECTASTAIN® Elite® ABC HRP Kit
(Vector Laboratories, UK) by mixing one drop of reagent A
and one drop of B in 0.5 mL of 3% BSA/PBS (according to
supplier’s protocol). Store the working solution at 4
C and use
it in same day (see Note 4).
3. Dopamine (DA)-H2O2 reaction solution: DA-H2O2 solution
should be freshly prepared by rapidly dissolving 10 mg of DA
hydrochloride powder (Sigma-Aldrich, UK) in 2 mL of tris
buffer (10 mM, pH 8.5), followed by quick addition of
40 μL of H2O2 (1 M). Quickly mix well and use immediately
(see Note 5).

2.4 Quantification of 1. Transparent 96-well plate, Costar, USA.

Color Intensity 2. Plate reader, Microplate spectrophotometer (FLUOstar
Omega, BMG LABTECH, UK) (see Note 6).

3 Methods

3.1 Bio-Capture of 1. Add 40 μL of exosomes sample (in PBS) with 10 μL of latex

Exosomes onto Latex beads solution in a 1.5 mL Eppendorf tube. Mix gently using
Beads the pipette and incubate for 15 min at room temperature. For
the negative control sample (blank sample), add equal volume
of PBS instead and run the sample protocol as below (see
Note 7).
2. Add 5 μL of 100 μM BSA (in PBS) to the exosomes-beads
mixture. Mix well and gently using the pipette and incubate for
15 min at room temperature.
 Colorimetric Aptasensor for Sensitive Exosome Detection 25

3. Add sterile PBS to top up to 1 mL. Mix gently using the pipette
and incubate for 60 min at room temperature with mild
agitation.
3. Gently transfer the solution to a 5 mL Falcon® Round-Bottom
Polystyrene Tubes using an appropriate pipette, e.g., p1000
(see Note 8).
4. Pellet the beads with centrifugation at 4
C for 5 min at
580  g.
5. Carefully remove the supernatant using an appropriate pipette,
e.g., p200 (see Note 9).
6. Add 1 mL of 100 mM glycine buffer in PBS to the sample tube,
mix gently using the pipette, and incubate for 30 min at room
temperature with mild agitation.

3.2 Color 1. After incubation, pellet the beads with centrifugation at 4
C

Development by the for 5 min at 580  g.
Aptasensor 2. Remove the supernatant using an appropriate pipette, e.g.,
p200 or p1000, with care.
3. Add 200 μL of prepared 0.1 μM biotinylated CD63 aptamer
solution. Mix gently using the pipette and incubate for 2 h at
room temperature with mild agitation.
4. Wash the mixture with 1 mL of PBS solution by first pelleting
the beads with centrifugation at 4
C for 5 min at 580  g, and
then adding fresh PBS solution.
5. Centrifuge the mixture again at 4
C for 5 min at 580  g.
6. Carefully remove the supernatant using an appropriate pipette.
7. Add 100 μL of streptavidin-HRP conjugate working solution.
Mix gently using the pipette and incubate for 30 min at room
temperature with mild agitation.
8. Pellet down the mixture with centrifugation at 4
C for 5 min
at 580  g. At the same time, prepare the fresh DA-H2O2
reaction solution (to be used immediately) (see Note 10).
9. Quickly add 100 μL of fresh DA-H2O2 reaction solution to the
pellet after pipetting out the supernatant.
10. Mix gently using the pipette and allow incubation for 10 min at
room temperature to develop the colorimetric signal (see
Note 11).

3.3 Quantification of 1. When incubation period finishes, observe the final color of the
Color Intensity samples directly by naked eye and make reasonable comparison
to estimate the CD63 expression level on exosomes (see
Note 12).
2. To obtain the absorbance spectra and quantitative data, transfer
100 μL of the sample using an appropriate pipette, e.g., p200,
26 Lizhou Xu and Khuloud T. Al-Jamal

to a clear-bottomed 96-well plate and scann from 300 to

600 nm in a Microplate spectrophotometer (see Note 13).
Transfer the same volume of the DA-H2O2 reaction solution
only and record the signal as well which is treated as the back-
ground noise.
3. Record the absorbance values at 400 nm for all the samples.
4. Export the data and analyze the data using a software, e.g.,
Microsoft Excel.

3.4 Data Analysis The final signal intensity for a sample should be the absorbance
reading subtracted by the background noise. This resulting (rela-
3.4.1 Analyze the Final
tive) signal intensity represents the exosomes marker (CD63 in this
Result of a Sample
case) expression level in the correlating sample tested (Fig. 2). The
final detection result can also be visualized qualitatively by naked
eye for each sample (Fig. 3).

3.4.2 Establish a 1. Detect a serial samples with different exosomes stock concen-
Calibration Curve trations (which means different surface marker amount) using
the proposed aptasensor method.
2. Record the final absorbance signal for all the samples (see
Subheading 3.4.1).
3. Then plot the data between the log value of known exosomes
concentration (X-axis) and the final absorbance signal intensity
of the correlating exosomes samples (Y-axis). Build a calibra-
tion curve using the fitting function in the software.

Fig. 2 Detection of CD63 amount on exosomes from P04.03 cancer cells using
the aptasensor. The calibration curve was obtained by plotting the color signal
intensity over a serial concentrations of exosomes samples tested (4.3  103,
4.3  105, 4.3  107, 4.3  109, 4.3  1011 particles/mL). The limit of
detection (LOD, dashed line) was established at 1.583, which were expressed
as mean  3  SD (n ¼ 3) of the blank control sample (without exosomes) (see
Note 14)
 Colorimetric Aptasensor for Sensitive Exosome Detection 27

Fig. 3 Images showing the representative colorimetric signals as observed by

naked eye for the samples tested and the blank control sample (first on the left)
using the aptasensor

3.4.3 Detect an Unknown 1. Test an unknown sample by using the sample protocol.
Sample 2. Analyze the final absorbance signal results of the unknown
testing sample as described in Subheading 3.4.1.
3. Fit the final absorbance signal into the calibration curve
(Y-axis) and obtain the corresponding exosomes concentration
of the tested samples. This result suggests the surface CD63
expression level in this tested sample.
4. In the case of clinical testing, first measure the exosomes
marker expression level by using this aptasensor assay. Then
obtain the final result for each sample. At last, compare the
results between healthy control samples and patient samples.

4 Notes

1. Always try to keep the exosomes in a relatively high concentra-

tion, such as in the order of magnitude of 1011 particles/mL or
above.
2. Always prepare fresh BSA/PBS buffer prior to use. Keep in the
fridge while not in use during the operating steps on the
same day.
3. Use DNase-free H2O to prepare the aptamer stock to minimize
the degradation of the nucleic acids.
4. Prepare the streptavidin-HRP working solution fresh the day
use. After mixing the drop A and B to the buffer in the same
container, mix immediately, and allow the reagent to stand for
15–30 min before use.
28 Lizhou Xu and Khuloud T. Al-Jamal

5. Always prepare the DA-H2O2 reaction solution right before

use. Use it immediately once prepared; otherwise the color will
start to develop and this may cause interference in signal
measurement.
6. Any types of plate reader should be working, as long as there is
the function of measuring absorbance signals. However, it is
suggested to use the same machine for one set of experiments
in order to make accurate comparison between samples.
7. Use one tube for one sample only. Prepare more if more than
one samples are tested in parallel. Always check the maximum
capacity of the centrifuge and do not operate too many samples
at the sample time.
8. This purpose to transfer the liquid to a 5 mL round-bottom
Falcon tube is to make the removal of liquid easier using a
pipette after centrifugation.
9. This step must be operated with special care to avoid any extra
beads (mixture) being sucked into the sharp pipette tip and
then thrown away.
10. The DA-H2O2 reaction solution needs to be freshly prepared
right before use. Always estimate the time needed for this
preparation so once the bead-exosome-aptamer/biotin-strep-
tavidin/HRP conjugate has formed (Subheading 3.2, step 7),
the color is ready to be developed with this DA-H2O2 reaction
solution.
11. The color in the tube becomes pink-orange and gradually turns
to (dark) brown during the incubation time. To ensure equal
comparison of the results between different samples, always
allow the same incubation time for all the samples tested.
12. To estimate the exosomes marker expression level of an
unknown sample by naked eye, it is better to conduct the
detection of positive samples with known exosomes and
marker concentration in advance. Record the color intensity
using digital image or color strip/paper which is very conve-
nient to be used as reference.
13. In this demonstration, the Microplate spectrophotometer
(FLUOstar Omega, BMG LABTECH, UK) was used. How-
ever, a plate reader with the function of absorbance measure-
ment should be working for this purpose. Please follow the
user manual of the product before operating the instrument for
correct measurement.
14. In this work, exosomes from P04.03 cancer cells were used as
an example for the demonstration of cancer-exosomes detec-
tion using the aptasensor. However, the biosensing application
is not limited to this type of cancer exosomes or this surface
marker. The exosomes were characterized with their size,
 Colorimetric Aptasensor for Sensitive Exosome Detection
surface charge, surface common marker, morphology informa-
tion, and so on. The readers are suggested to conduct proper
characterization in advance to identify the exosomes popula-
tion before running the aptasensor method for specific marker
detection.
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 Chapter 3

Isolation of Circulating Extracellular Vesicles by

High-Performance Size-Exclusion Chromatography
Kaloyan Takov, I-Jou Teng, and Manuel Mayr

Abstract
Circulating extracellular vesicles (EVs) are gaining increased attention as carriers of proteins, nucleic acids,
and lipids. Blood contains EVs from different cell sources that constitute an attractive target for biomarker
studies. However, there is no consensus on the best approach to isolate EVs from blood. Non-EV proteins
and lipoproteins in plasma/serum tend to contaminate isolated EVs and confound functional experiments.
Here we describe a single-step, high-performance size-exclusion chromatography procedure for separation
of EVs from most lipoproteins and non-EV proteins, and compare it to ultracentrifugation, still the most
commonly used method for EV isolation.

Key words Extracellular vesicles, Exosomes, Size-exclusion chromatography, HPLC, Plasma, Lipo-
proteins, Proteomics

1 Introduction

Extracellular vesicles (EVs) are secreted nano- and microsized

membrane-encapsulated particles that carry lipids, proteins, and
RNAs. They are formed intracellularly in multivesicular body orga-
nelles and subsequently released in the extracellular milieu (i.e.,
exosomes), or by cell membrane budding (e.g., microvesicles, apo-
ptotic bodies) [1]. EVs have been implicated in various intercellular
signaling processes. The EV content of biological fluids, including
blood, is altered in disease [2]. Hence, blood-derived EVs have
potential as biomarkers to inform diagnosis and prognosis of dis-
ease [3]. Yet, the isolation of pure EV populations from blood has
proven exceptionally difficult, especially from plasma and serum.
Blood plasma contains the most complex proteome of the human
body and the concentration of plasma proteins spans a very high
dynamic range. Abundant plasma proteins, for example, albumin,
fibrinogens, and immunoglobulins, tend to mask low-abundant
soluble proteins and EVs. To add to this complexity, lipoprotein

Maurizio Federico and Barbara Ridolfi (eds.), Extracellular Vesicles in Diagnosis and Therapy,
Methods in Molecular Biology, vol. 2504, https://doi.org/10.1007/978-1-0716-2341-1_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

31
32 Kaloyan Takov et al.

particles, including APOB-containing lipoproteins with lower den-

sity [e.g., chylomicrons, very-low density lipoproteins (VLDL),
intermediate-density lipoproteins (IDL), low-density lipoproteins
(LDL)] as well as APOA1-containing high-density lipoproteins
(HDL), are also present in high abundance. It is estimated that
there are ~1016 HDL particles per mL blood, ~1015 non-HDL
lipoproteins per mL blood, while EVs are 5–6 orders of magnitude
lower in abundance with ~1010 per mL [4, 5]. In fact, due to the
lipoprotein contamination, this number is likely to be an overesti-
mate for EVs. Therefore, even an EV isolation method that
removes 99.99% of lipoproteins will still have 100 times more
lipoproteins than EVs.
Ultracentrifugation (UC) is by far the most widely used
method for isolation of plasma EVs while other techniques such
as size-exclusion chromatography (SEC) are increasingly applied
[6]. In comparison to UC, SEC fractionation relies only on the
hydrodynamic radius of the particles/protein complexes, it induces
less damage and aggregation of EVs/particles, minimizes the loss
of binding partners, and it is also faster and more flexible. On the
other hand, methods relying only on size separation are prone to
co-isolation of lipoproteins (e.g., chylomicrons, VLDL, LDL) with
EVs [7–10]. To address these methodological constraints, it has
been suggested that using a combination of methods may be
required to obtain sufficient EV purity [11–13]. However,
approaches with multiple steps tend to result in a considerable
loss of EVs, while only marginal improvements in purity are
achieved. Moreover, a combinatorial approach is time-consuming
and laborious, and collection of EV-depleted fractions for down-
stream control experiments is complicated. SEC, on the other
hand, has potential as a single-step isolation technique for EVs. It
provides: (1) flexibility: vast choice of columns, methods, and
fraction collection settings; (2) automatization: for example,
using a high-performance liquid chromatography (HPLC) system;
(3) easy collection of EV-rich and EV-poor fractions; and (4) pres-
ervation of endogenous protein complexes.
Here we present a high-performance SEC method for plasma
fractionation which separates EVs from most APOB, APOA1, and
albumin in a single step. Notably, rather than investigating only the
EV isolates, which are also not completely devoid of lipoproteins or
abundant plasma protein, the method provides EV-, protein-, and
lipoprotein-rich fractions that can be studied in parallel in down-
stream experiments. In comparison to the currently available SEC
methods, the one presented here uses an HPLC system and there-
fore can be automated, it is of higher resolution due to use of
multiple, long SEC columns, can process minimal volumes of
plasma (20 μL or less), and it offers a flexibility for an accurate
fraction collection that cannot be achieved using gravity-driven,
manual SEC fractionation platforms [14]. Due to the automation,
the method also minimizes any operator-induced variability.
Another random document with
no related content on Scribd:
 “1. Rapid penetration of the substance into the
cell by virtue of its high lipoid solubility.
2. Hydrolysis by the water within the cell, to form
hydrochloric acid and dihydroxyethylsulphide.
3. The destructive effect of hydrochloric acid upon
some part or mechanism of the cell.
“Although hydrochloric acid does not penetrate
cells readily and is easily neutralized by the buffer
action of the fluids of the body, we might expect by
flooding the body with large quantities of acid to
produce some of the characteristic effects of mustard
gas. Stimulation of the respiratory center is a well
known effect of acid. Convulsions and salivation may
be produced by injection of hydrochloric acid and we
have been able to produce slowing of the heart by
rapid injection of this acid.
“The delayed action of mustard gas might be
explained by the formation of some compound with
some constituent of the blood. However, blood taken
from dogs which had been poisoned with mustard
gas and were exhibiting typical symptoms at the time,
injected into normal dogs produced no effect. Serum
treated in vitro with mustard gas and allowed to stand
and then injected into a dog, produced no effect. The
fluid which is formed in the vesicle and blebs
produced by the application of mustard gas to the
skin produces no mustard gas effects.”
In studying the toxicity of mustard gas for dogs, it was observed
that a concentration of 0.01 mg. per liter could be tolerated indefinitely.
If this value is considered as a threshold value, and subtracted from
the toxicity values for varying periods of time, it is found that there is a
definite relation between the toxic concentration and the time of
exposure. This is expressed by the formula

(C - 0.01)t = K
 where C is the concentration observed for a given time
t
. K has the approximate value of 1.7, where
t
varies between 7.5 and 480 minutes.

Vesicant Action
In addition to its toxicity mustard gas is highly important because of
its peculiar irritating effect upon the skin. Its value is seen when we
realize that one part in 14,000,000 is capable of causing conjunctivitis
of the eye and that one part in 3,000,000 and possibly one part in
5,000,000 will cause a skin burn in a sensitive person on prolonged
exposure. According to Warthin, the lesions produced by mustard gas
are those of a chemical, not unlike hydrochloric acid, but of much
greater intensity. The pathology of these lesions has been carefully
studied and fully described by Warthin and Weller in their book on The
Pathology of Mustard Gas. Our observations will therefore be confined
to certain striking features of the vesicant action of this substance.

Variation in Susceptibility of the Skin

Every worker who has worked with mustard gas has noticed that
some individuals are much more susceptible to skin burns from this
substance than are others. Marshall made a study of 1282 men at
Edgewood Arsenal, using a 1 per cent and a 0.01 per cent solution of
mustard gas in paraffin oil. A small drop of these solutions was applied
to the skin of the forearm of the subject and the arm allowed to remain
uncovered for about 10 minutes. The presence or absence of a
positive reaction is indicated by the appearance or absence of
erythema 24 hours later. The results were as follows:
1% 0.01% % of Total
Positive Positive 3.3
Positive Negative 55.3
 1% 0.01% % of Total
Negative Negative 41.4
The test made on 84 negroes gave the following results:
1% 0.01% % of Total
Positive Positive 0.0
Positive Negative 15.0
Negative Negative 78.0
Questionable Negative 7.0
“It is seen from the above tables that negroes as a
race, have a much more resistant skin than white
men. No negro of the 84 examined reacted to the 0.1
per cent solution, and of course none would react to
a more dilute one. About 10 per cent of white men
react to the 0.1 per cent solution, while 2 to 3 per
cent react to the 0.01 per cent solution or are
hypersensitive. About 78 per cent of the negroes fail
to react to the 1 per cent solution, while only 20 to 40
per cent of the white race do not show a reaction.”
 Fig. 37.

The same individual may also show variations in susceptibility and

this has also been studied by Marshall.
“The effect of exercise and sweating was
investigated. A number of individuals were given
vapor burns (one to five minutes exposure) and then
exercised until in a profuse sweat, and then the same
exposure to vapors made. In all cases the burn
produced after exercising was more severe.
Sweating produced by having the subjects place their
feet in hot water, produced the same increase in
susceptibility. That the moisture on the skin produced
by sweating is at least partly, if not entirely,
responsible for the increased susceptibility, was
shown in the following way: An area of the forearm
 was kept moist for a few minutes with wet cotton. The
sponge was then removed and two vapor tests made,
one over the moist area and one over normal, dry
skin. In all cases the moist burn was the more
severe, in one, producing a blister where the control
did not.
“The skin of different areas of the body is
undoubtedly somewhat different in its susceptibility.
All our tests have been applied to the forearm. The
hands are considerably more resistant than the
forearm. Tests made by the oil method on the
forearm, chest, and back, however, indicate very little
difference in susceptibility of these areas. The skin in
the neighborhood of old burns has been shown to be
more susceptible.
“In general, the same individual does not become
more susceptible to skin burns from continued
exposure to the vapor. The great number of tests
which have been made on the same individual at
different times and under the same conditions,
indicate a remarkable constancy in reaction. A series
of men who were tested at various times during a
period of four months, revealed slight changes from
time to time in some of the men. No man who
originally reacted to only the 1 per cent solution ever
reacted to the 0.01, and likewise, no man who
originally reacted to the 0.01 ever failed to react to
the 0.1 per cent.
“Susceptibility of skin of animals. The paraffin oil
test was used on a number of animals and indicated
that differences in susceptibility exist in different
species and in different individuals of the same
species.”
Species Number Percentage Positive to
Tested
 Species Number 1Percentage
Per 0.1 Per
Positive
0.01 Per
to
Tested Cent Cent Cent
Horse 1 100 100 100
Dog 91 1 Per
83 0.135
Per 0.01 0 Per
Goat 11 Cent
55 Cent
36 Cent
0
Rat 10 30 20 0
Mouse 7 70 14 0
Rabbit 2 100 0 0
Guinea-pig 12 33 0 0
Monkey 9 22 0 0
The horse appears to be the most sensitive and the monkey and
guinea-pig the most resistant species, while the dog would seem to
have a sensitivity as near man as any of the species studied. No
animal has yet been found which will give a blister from the application
of mustard gas.
Smith, Clowes and Marshall[22] have studied the mechanism of
absorption by the skin. They find that it is quite evident that the
mustard gas is at first rapidly taken up by some element on, or
adjacent to, the surface of the skin and for two to three minutes it may
be completely removed, and for ten to fifteen minutes partially
removed by prolonged washing With an organic solvent, and to a
lesser extent with soap and water.
An interesting phenomenon is observed when the untreated
normal skin of one subject is impressed for five minutes upon an area
of skin of another subject, which has been exposed previously to the
vapors of mustard gas. Under these circumstances both donor and
recipient may develop burns (due to the transposition of the poison
from one skin to another), the intensity of which will vary according to
the circumstances and the respective sensitiveness of the participants.
The degree of transposition is most strikingly observed in the intensity
of the burn on the donor’s arm. If two similar exposures are made on
the arm of a sensitive man, and one of these burns is treated, so to
speak, by contact for five minutes, with the skin of a resistant man, the
treated burn will be markedly less severe than the control, in some
cases being entirely prevented. If, however, the recipient is equally
sensitive to or more sensitive than the donor, the burns on the latter
will exhibit far less difference. Both treatments may be effected at
once, using two recipients, one more, and the other less, resistant
than the donor. In such a case the burn brought into contact with the
more resistant skin will be the less severe.
Similarly, if a sensitive individual impresses his arm alternately
against burns of the same concentration and exposure on a resistant
and sensitive man, the recipient receives a more severe burn from the
sensitive than from the resistant man.
This indicates that the skin of a resistant individual exhibits a
greater affinity or capacity for mustard gas than that of a sensitive one.
There is an actual partition of the gas between the two skins, with an
evident tendency to establish an equilibrium in which the larger portion
of the gas will remain in that skin which possesses the greater
capacity for it.
“A tentative explanation of this phenomenon can
be made as follows. A three phase system is involved
—the air over the skin surface constitutes the outer
phase; some fatty or keratinous elements of the skin,
the central phase; and a cellular portion of the skin
the inner phase. The central phase is rich in lipoids
and poor in water, while the inner phase is rich in
water and poor in lipoids. After exposure to the
vapors of dichloroethylsulphide the central phase is
the absorbing agent and tends to establish
equilibrium with the other two phases. On account of
the lipoid nature of the central phase no damage is
produced here because the compound is not
hydrolyzed. On its passage from the central to the
inner phase hydrolysis takes place within the cell and
damage results when a sufficient concentration of
hydrochloric acid is attained. The outer phase is
constantly being freed from vapor by diffusion and
convection currents, so more and more can
evaporate from the central phase. The susceptibility
 of an individual depends on the relative power of the
central phase to hold the poison in an inactive form
(not hydrolyzed) and prevent its entry into the inner
phase at a sufficient velocity to result in the formation
of a toxic concentration. We do not attempt to localize
the central or inner phases with any definite structure
of the skin. As mustard is known to penetrate the
sebaceous ducts the fat here might form one phase
and the epithelial lining another.”

Tactical Use of Mustard Gas

As before stated, mustard gas, like most other materials used in
war, was discovered in peace. Indeed, Victor Meyer in 1886 worked
out fairly completely its dangerous characteristics. Like phosgene and
chlorine used before it, the materials for its production were available
in considerable quantities through the manufacture of components
either for dyes or photographic chemicals.
Mustard gas, besides being highly poisonous, has so many other
important qualities as to have given it the designation during the war of
the “king of gases.” That broad distinction it still holds. Its introduction
at Ypres, on the night of July 12, 1917, changed completely the whole
aspect of gas warfare and to a considerable extent the whole aspect
of warfare of every kind. It is highly poisonous, being in that respect
one of the most useful of all war gases. It produces no immediate
discomfort. It has a considerable delay action. It burns the body inside
or out, wherever there is moisture. Eyes, lungs and soft parts of the
body are readily attacked. It lingers for two or three days in the
warmest weather, while in cold, damp weather it is dangerous for a
week or ten days, and in still colder weather may be dangerous for a
month or longer whenever the weather warms up sufficient to volatilize
the liquid. It is only slowly destroyed in the earth, making digging
around shell holes dangerous for weeks and months and in some
cases possibly a year or more.
The Germans first used it simply to get casualties and interfere
with or break up the threatened heavy attacks by the British on the
Ypres salient. While not stopping the inauguration of these attacks in
the fall of 1917, the German use of mustard gas was so effective as to
delay the beginning of those attacks for at least two weeks and thus
gain valuable time for the Germans, besides causing serious
casualties with consequent partial break up of companies, regiments
and divisions in the English Army.
The German used his mustard gas throughout the fall of 1917 and
the winter of 1917 and 1918, as above stated, to produce casualties,
to destroy morale, to break up units, and to interfere with operations
generally. During that time, however, he developed a more scientific
use and when he started his big offensives in March, April, May and
June, 1918, he used mustard gas before the battles to cause losses,
break up units and destroy morale, and also during the progress of
battles to completely neutralize strong points which he felt he did not
want to attempt to take by direct assault. Perhaps the most noted case
of this was at Armentières in April, when he deluged the city to such
an extent that mustard gas is said to have actually run in the streets.
So effective was this gassing that not only did the British have to
withdraw from the city but the Germans could not enter it for more
than two weeks. It, however, enabled the Germans to take the city with
practically no loss of life. There were numerous other cases on a
smaller scale where mustard gas was used in the same way.
On account of its persistence it has been generally referred to as a
defensive gas and for that purpose it is incomparable. The use of
sufficient quantities of mustard gas will almost certainly stop the
occupation of areas by the enemy and probably even stop his crossing
them. It also enables strong points which it is not desired to attack to
be completely neutralized,—that is, made so unhabitable that the area
must be evacuated.
A use that was proposed toward the end of the war, and that will
undoubtedly be made of the gas in the future, is to have it planted in
drums in the ground and exploded when an enemy is attempting to
advance. This would be a highly economic way to distribute great
quantities of the material at the moment and in the place most heeded.
It has even been proposed, and this would seem entirely feasible, to
sprinkle certain of these areas with mustard gas by means of
sprinklers attached to drums or even tanks mounted on trucks.
 Just before the Armistice the German made another development
in the use of mustard gas. Instead of the ordinary amount of explosive,
which only fairly opened up the shell and allowed the liquid to escape,
he filled nearly 30 per cent of the total space of the shell with high
explosive. This completely broke up the shell and distributed the
greater part of the liquid mustard gas in the form of a fine spray. This
spray, when breathed, proved extremely deadly, as might be expected
from the fact that when in the form of minute particles one can draw
into the lungs in a single breath one hundred times or more the
amount that he would get of pure gas.
Since mustard gas has such a delay action and is effective in such
small concentrations it can be used very effectively in small calibre
guns, as the 75 mm. or 3-inch. Furthermore, since it lasts for two or
three days at the very least, a small number of guns can keep a very
large area neutralized with the gas. With phosgene and similar non-
persistent gases that volatilize almost completely upon the burst of the
shell it is necessary to build up a high concentration immediately. The
exact opposite is true of mustard gas. Mustard gas can be fired very
slowly with the certain knowledge that all shells fired at one moment
will be effective when the next is fired, though twelve hours or more
may intervene between the first and last firing. Thus, while with
phosgene a large number of guns are needed for a gas attack, with
mustard gas the number can be reduced to one-tenth or even less.
Mustard gas may be in the future and has been in the past used safely
in hand grenades because of its very low vapor tension, whereby the
pressure at ordinary temperatures is exceedingly low. This has an
important bearing on cylinders and other containers for shipping
mustard gas, that is, they need be only strong enough to be safe
against handling and not to withstand the high pressure encountered
with phosgene or chlorine cylinders.
In the future, mustard gas will be used in all the ways above stated
and undoubtedly in many more. It can be fired in large quantities upon
strong points to force their evacuation. It can be fired on the flank of
attacking armies for protection against counter-attacks. It can be fired
against the enemy artillery at all times to silence them and stop their
firing. It was thus used by the Americans in the Argonne against the
enemy on the east bank of the Meuse River, this river separating the
American and German armies. It was extremely effective in stopping
the enemy’s artillery. The high explosive mustard gas shell, not only
because of its persistency but because of its quick deadliness, can be
fired singly and be depended upon to do its work wherever there be
men or animals. One of the greatest uses will be by simple sprinkling
from aeroplanes.
The future will see mustard gas used at nearly all times with a
certain quantity of a powerful lachrymator or tear gas. This is for the
reasons, as stated in the beginning, that mustard gas causes no
immediate discomfort and has no objectionable smell. Accordingly, if
the battle be critical, men may continue to fight from four to eight hours
in a mustard gas atmosphere without masks. It is true the casualties
will be high with a high death rate. Nevertheless, this period of time
might enable the artillery to do such effective work as to completely
stop an attack. If, however, at the instant mustard gas firing is begun a
number of powerful lachrymatory shells are sent over, the immediate
wearing of the mask is forced. The enemy is then subject to all the
burning effects of mustard gas as well as the discomfort of long
wearing of the mask.
It may confidently be expected that further developments in the use
of mustard gas will be made, as well as further developments in
methods of throwing it upon the enemy or of bursting shell containing
it in his midst.
 CHAPTER X
ARSENIC DERIVATIVES

Since arsenic is well known as an insecticide in the form of lead

arsenate, arsenic acid etc., and in pharmacy, specially in the form of
salvarsan and neosalvarsan, it is not surprising that the Germans
should have endeavored to discover an arsenic derivative which
would be of value from the point of view of chemical warfare. Very
early in the war persistent rumors were circulated that the Germans
were to use arsine. These rumors led to the use of sodium
permanganate in the canister, but as far as is known, no arsine was
actually used. Another suggestion which received considerable
attention from American workers was the use of arsenides, which
might decompose under the influence of the atmospheric moisture
with the liberation of arsine. Calculation of the amount of arsenide
necessary to establish a lethal concentration of arsine showed,
however, that there was no possibility of using the material on the
field.
Because of the use of arsenic trichloride in the manufacture of
organic arsenic compounds, a method of preparation was developed
from arsenic trioxide and sulfur chloride or hydrogen chloride. It was
also shown experimentally that the phosgene of the tail gas of
phosgene plants might be converted into arsenic trichloride by
reaction with arsenic trioxide. Charcoal is the catalyzer of this
reaction.
Arsenic trichloride is also of interest because it was one of the
constituents of the mixture vincennite, early used by the French. This
was a mixture of hydrocyanic acid, stannic chloride, arsenic
trichloride and chloroform. While extensively used at first, it was
gradually replaced by phosgene.
 Arsenic triflouride was also prepared by the action of sulfuric acid
upon a mixture of calcium flouride and arsenic trioxide. The
compound is very easily decomposed by the moisture of the air, and
furthermore is not very toxic.
Organic arsenic derivatives are the most important compounds
from the military point of view. The first substance used was
diphylchloroarsine, a white solid, which readily penetrated the
canister and caused sneezing. This was used alone, and in solution
in phenyl dichloroarsine. Later methyl and ethyl dichloroarsines were
introduced.

Fig. 38.—Apparatus for the Manufacture of

Methyldichloroarsine.
 Methyldichloroarsine
The Germans apparently used ethyldichloroarsine because they
had no suitable method for the preparation of methyl dichloroarsine,
which is a more satisfactory material. The Chemical Warfare Service
developed the following method of preparation of the methyl
derivative. Sodium arsenite (Na₃AsO₃) is prepared by dissolving
arsenic trioxide in sodium hydroxide solution. The action of methyl
sulfate at 850 C. gives disodium methyl arsenite, Na₂CH₃AsO₃.
Sulfur dioxide reduces the arsenite to methyl arsine oxide, CH₃AsO,
which is then reacted with hydrochloric acid to give methyl
dichloroarsine. The final product is distilled from the mixture and
condensed. This material costs from two to two and a half dollars per
pound for chemicals (war prices).
Methyldichloroarsine is a colorless liquid of powerful burning
odor, which boils at 132° C. It is somewhat soluble in water and is
soluble in organic solvents. The specific gravity is 1.838 at 20° C.
The vapor pressure at 25° was found to be 10.83 mm. mercury. Not
only is the material toxic but it has remarkable vesicant properties,
comparing favorably with mustard gas in this respect.
Ethyldichloroarsine, which was used by the Germans, was
prepared by the method given above, using ethyl sulfate, but the
yield was never over 20 per cent. In general this has properties
similar to the methyl derivative.

Diphenylchloroarsine
The best known of the arsenicals, however, is
diphenylchloroarsine or sneezing gas. Although this is an old
compound (having been prepared by German chemists in 1885),
there was no method for its preparation on a large scale when, first
introduced into chemical warfare. It was finally discovered that the
interaction of triphenyl arsine with arsenic trichloride was fairly
satisfactory and a plant was erected for its manufacture.
 When pure, diphenylchloroarsine is a colorless solid, melting at
44°. Because of this, it was always used in solution in a toxic gas or
in a shell which contained a large amount of explosive so that on the
opening of the shell the material would be finely divided and
scattered over a wide territory.
Its value lay in the fact that the fine particles readily penetrated
the ordinary mask and caused the irritation of the nose and throat,
which resulted in sneezing. This necessitated the perfection of
special smoke filters to remove the particles, after which the other
toxic materials were removed by the absorbent in the canister.
It causes sneezing and severe burning sensations in the nose,
throat and lungs in concentrations as slight as 1 part in 10 million. In
higher concentrations, say 1 in 200 to 500 thousand it causes severe
vomiting. While neither of these effects are dangerous or very
lasting, still higher concentrations are serious, as in equal
concentrations diphenylchloroarsine is more poisonous than
phosgene.
Various other arsenical chemicals were developed in the
laboratory, but with one or two exceptions they were not as valuable
as diphenylchloroarsine and methyldichloroarsine and were
therefore discarded.

German Methods for Manufacturing

Arsenicals[23]

Diphenylchloroarsine

“This substance (Blue Cross) was a famous gas

of the Germans and was made in large quantities.
The method used by the Germans was different
from the one worked out by the Allies, and on
account of the fact that the German method could
be carried out without specially designed apparatus
and required as raw materials substances readily
obtainable, it was probably preferable. It is doubtful,
however, whether the Allies would have made this
gas, for as the result of its use no fatalities were
reported. The German process consisted in
preparing phenylarsenic acid by condensing
benzene diazonium chloride with sodium arsenite.
The acid was next reduced by sulfur dioxide to
phenylarsenous acid, which was, in turn, condensed
with the diazonium compound to form
diphenylarsenic acid. This acid was reduced to
diphenylarsenous oxide, which with hydrochloric
acid yielded diphenylchloroarsine. The chemical
equations for the reactions will make clearer the
steps involved.

C₆H₅N₂Cl + C₆H₅AsO₃Na₂ +
=
Na₃AsO₃ NaCl + N₂
C₆H₅AsO₃Na₂ + C₆H₅AsO₃H₂ +
=
2HCl 2NaCl
C₆H₅AsO₃H₂ + C₆H₅AsO₂H₂ +
=
SO₂+H₂O H₂SO₄
C₆H₅N₂Cl + (C₆H₅)₂AsO₂Na +
=
C₆H₅AsO₂Na₂ NaCl + N₂
(C₆H₅)₂AsO₂Na + (C₆H₅)₂AsO₂H +
=
HCl NaCl
2(C₆H₅)₂AsO₂H + [(C₆H₅)₂As]₂O +
=
2SO₂ + H₂O 2H₂SO₄
[(C₆H₅)₂As]₂O + 2(C₆H₅)₂AsCl +
=
2HCl H₂O.
“The entire process was carried out at Höchst.
The method used at Höchst was as follows: In
preparing the diazonium solution, 3 kg.-mols of
aniline were dissolved in 3000 liters of water and the
theoretical quantity of hydrochloric acid. The
temperature of the solution was reduced to between
0° and 5° and the theoretical amount of sodium
nitrite added. The reaction was carried out in a
wooden tank of the usual form for the preparation of
diazonium compounds. A solution of sodium
arsenite was prepared which contained 20 per cent
excess of oxide over that required to react with the
aniline used. The arsenous oxide was dissolved in
sodium carbonate, care being taken to have enough
of the alkali present to neutralize all of the acid
present in the solution of the diazonium salt. To the
solution of the sodium arsenite were added 20 kg. of
copper sulfate dissolved in water, this being the
amount required when 3 kg.-mols of aniline are
used. The solution of the diazonium compound was
allowed to flow slowly into the solution of the
arsenite while the temperature was maintained at
15°. The mixture was constantly stirred during the
addition which requires about 3 hrs. After the
reaction was complete, the material was passed
through a filter press in order to remove the coupling
agent and the tar which had been formed.
Hydrochloric acid was next added to the clear
solution to precipitate phenylarsenic acid, the last
portions of which were removed by the addition of
salt.
“The phenylarsenic acid was next reduced to
phenylarsenous acid by means of a solution of
sodium bisulfite, about 20 per cent excess of the
latter over the theoretical amount being used. For
100 parts of arsenic acid, 400 parts of solution were
used. The reaction was carried out in a wooden
vessel and the mixture stirred during the entire
operation. A temperature of 80° was maintained by
means of a steam coil. Phenylarsenous acid
separated as an oil. The aqueous solution was
decanted from the oil, which was dissolved in a
solution of sodium hydroxide, 40° Bé. The solution
of the sodium salt of phenylarsenous acid was
treated with water so that the resulting solution had
a volume of 6 cu. m. when 3 kg.-mols of the salt
were present. Ice was next added to reduce the
temperature to 15° and a solution of benzene
diazonium chloride, prepared in the manner
described for the first operation, was slowly added.
After the coupling, diphenylarsenic acid was
precipitated by means of hydrochloric acid. The acid
was removed by means of a filter press and
dissolved in hydrochloric acid, 20° Bé. For one part
of diphenylarsenic acid, 3 parts of hydrochloric acid
were used. Into this solution was passed 5 per cent
excess of sulfur dioxide over that required for the
reduction. The sulfur dioxide used was obtained
from cylinders which contained it in liquid condition.
“The reduction was carried out in an iron tank
lined with tiles and a temperature of 80° was
maintained. About 8 hrs. were required for the
reaction. The diphenylarsenic acid on reduction by
the sulfur dioxide was converted into
diphenylarsenous oxide which, in the presence of
the hydrochloric acid, was converted into
diphenylchloroarsine, which separated as an oil.
The oil was next removed and heated in the best
vacuum obtainable until it was dry and free from
hydrochloric acid. The compound melted at 34°. It
was placed in iron tanks for shipment. The yield of
diphenylchloroarsine calculated from the aniline
used was from 25 to 30 per cent of the theoretical.
No marked trouble was observed in handling the
materials and no serious poisoning cases were
reported.
 Diphenylcyanoarsine

“This compound was prepared by treating diphenylchloroarsine

with a saturated aqueous solution of potassium or sodium cyanide.

(C₆H₅)₂AsCl + NaCN = (C₆H₅)₂AsCN + NaCl.

Five per cent excess of the alkaline cyanide was used. The
reaction was carried out at 60° with vigorous stirring. The yield was
nearly theoretical.

Ethyldichloroarsine

“This compound was prepared at Höchst from ethylarsenous

oxide which was obtained from the Badische Anilin und Soda Fabrik.
“Preparation of Ethylarsenous Oxide—The compound was
prepared by treating sodium arsenite with ethyl chloride under
pressure. The resulting sodium salt of ethylarsenic acid was
converted into the free acid and reduced by sulfur dioxide. The
ethylarsenous acid formed in this way lost water and was thereby
transformed into ethylarsenous oxide. The reactions involved are as
follows:
=
C₂H₅Cl + Na₃AsO₃ C₂H₅AsO₃Na₂ + NaCl
C₂H₅AsO₃H₂ + 2
C₂H₅AsO₃Na₂ + 2 HCl =
NaCl
C₂H₅AsO₃H₂ + SO₂ +
= C₂H₅AsO₂H₂ + H₂SO₄
H₂O
2C₂H₅AsO₂H₂ = (C₂H₅As)₂O + H₂O.
“The ethyl chloride used in the preparation was in part made in
this factory, and in part received from other sources. As ethyl
chloride is an important product used in peace time, it is not,
therefore, essentially a war product and its preparation was not
described.
“In preparing the solution of sodium arsenite, one molecular
weight of arsenous oxide was dissolved in a solution containing 8
molecular weights of sodium hydroxide. The solution of the base was
prepared from a 50 per cent solution of sodium hydroxide to which
enough solid alkali was added to make the solution a 55 per cent
one. In one operation 660 kg. of arsenous oxide were used. For 100
parts of arsenous oxide, 130 parts of ethyl chloride were used, this
being the theoretical amount of the latter.
“The reaction was carried out in a steel autoclave of about 300
liters capacity. The temperature was maintained at between 90° and
95°. The ethyl chloride was pumped in, in 3 or 4 portions, and the
pressure in the autoclave was kept at from 10 to 15 atmospheres.
The several portions of ethyl chloride were introduced at intervals of
about 1½ hrs. During the entire reaction, the contents of the
autoclave were vigorously stirred. After all the ethyl chloride had
been added, the material was stirred from 12 to 16 hrs., at the end of
which time the pressure had fallen to about 6 atmospheres. The
excess of ethyl chloride and the alcohol formed in the reaction were
next distilled off. At this point a sample of the solution was drawn off
for testing. This was done by determining the amount of arsenite
present in the solution. If not more than 20 per cent of sodium
arsenite had not reacted, the preparation was considered
satisfactory. Water was then added to the contents of the autoclave
in sufficient amount to dissolve the solid material. The product was
next drawn over into a tank and neutralized with sulfuric acid. It was
then treated with sulfur dioxide gas until there was an excess of the
latter present. The mixture was then heated to about 70° when the
ethylarsenous oxide precipitated as a heavy oil. This was readily
separated and shipped without further purification. The yield of
ethylarsenous oxide, from arsenic oxide, was from 80 to 82 per cent
of a product which contained about 93 per cent of pure
ethylarsenous oxide.
“Preparation of Ethyldichloroarsine—The compound was
prepared by treating ethylarsenous oxide with hydrochloric acid. The