Handbook of Immunoassay Technologies: Approaches, Performances, and Applications

Handbook of Immunoassay Technologies

Approaches, Performances, and Applications

Editors

Sandeep K. Vashist

John H.T. Luong

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Chapter 1. Immunoassays: An Overview

1. Overview of Immunoassays

2. Antibody Structure

3. Need for Immunoassays

4. Immunoassay Formats

5. Conclusions and Future Trends

Chapter 2. Antibody Immobilization and Surface Functionalization Chemistries for Immunodiagnostics

1. Introduction

2. Surface Functionalization Chemistries

3. Antibody Immobilization Chemistries

4. Surface Characterization

5. Conclusions, Challenges, and Future Trends

Chapter 3. Monoclonal Antibody Generation by Phage Display: History, State-of-the-Art, and Future

1. Introduction

2. Phage Display Selection

3. Antibody Libraries

4. In Vitro Selection of Antibodies for Specific Applications

5. Conclusion and Outlook

Chapter 4. Bioanalytical Requirements and Regulatory Guidelines for Immunoassays

1. Introduction

2. Bioanalytical Requirements for an Immunoassay

3. Critiques and Outlook

4. Conclusions

Chapter 5. Enzyme-Linked Immunoassays

1. Introduction

2. Conventional Enzyme-Linked Immunoassays

3. Emerging Enzyme-Linked Immunoassays

4. Portable Analyzer–Based Immunoassays

5. Critiques and Outlook

6. Conclusions

Chapter 6. Surface Plasmon Resonance–Based Immunoassays: Approaches, Performance, and Applications

1. Introduction

2. Surface Plasmon Resonance–Based Immunoassays

3. Future Trends in Surface Plasmon Resonance–Based Immunoassays

4. Conclusions

Chapter 7. Lateral Flow Immunoassays

1. Introduction

2. Advances in Lateral Flow Immunoassays

3. Challenges and Future Directions

4. Bibliographic and Commercial Data

5. Conclusions

Chapter 8. Paper-Based Immunoassays

1. Paper-Based Immunoassays: Strategies and Detection Principles

2. Development of the Paper-Based Immunoassays Devices

3. Conclusions

Chapter 9. Acoustic Wave–Based Immunoassays

1. Introduction

2. Clinical Diagnostics

3. Detection of Microbial Pathogens and Toxins

4. Detection of Parasites

5. Detection of Viruses

6. Quartz Crystal Microbalance and Surface Acoustic Wave-Based Electronic Noses

7. Quartz Crystal Microbalance and Surface Acoustic Wave Immunoassays in Environmental Monitoring and Allergens Detection

8. Integrated Acoustic Wave Immunosensors for Point of Care

9. Commercial Acoustic Wave Immunosensors

10. Market Potential and Conclusions

Chapter 10. Interferometry-Based Immunoassays

1. Introduction: General Context

2. Principles of Operation

3. Sensor Surface Functionalization

4. Application of Interferometric Immunosensors

5. Conclusions and Future Perspectives

Chapter 11. Nanomaterial- and Micromaterial-Based Immunoassays

1. Introduction

2. Micromaterial-Based Immunoassay

3. Colorimetric Immunoassay

4. Electrochemical Immunoassay

5. Fluorescent Immunoassay

6. Conclusion

Chapter 12. Microcantilever-Based Sensors

1. Introduction

2. Microcantilevers and Their Modes of Operation

3. Detection Methods

4. Bending Behavior of Microcantilevers

5. Fabrication of Microcantilevers

6. Microcantilever-Based Sensors

7. Electronic Nose

8. Nanocantilevers

9. Commercial Availability

10. Conclusions and Future Trends

Chapter 13. Quartz Crystal Microbalance–Based Sensors

1. Introduction

2. Detection of Biomolecules

3. Detection of Bacteria

4. Detection of Volatile Organic Compounds

5. Detection of Chemical Analytes

6. Detection of Gaseous Analytes

7. Special Analytical Applications

8. Other Analytical Applications

9. Conclusions and Future Trends

Chapter 14. Electrochemical Immunosensors: Fundamentals and Applications in Clinical Diagnostics

1. Introduction

2. Electrochemical Techniques in Immunosensing

3. Advanced Approaches in Electrochemical Immunosensors

4. Biorecognition Elements in Immunosensors

5. Electrochemical Immunosensors

6. Conclusions and Outlook

Chapter 15. Lab-on-a-Chip (LOC) Immunoassays

1. Introduction

2. Lab-on-a-Chip Immunoassays

3. Critiques and Outlook

4. Conclusions

Chapter 16. Smartphone-Based Immunoassays

1. Introduction

2. Signal Detection in Smartphone-Based Immunoassays

3. Applications of Smartphone-Based Immunoassays

4. Conclusions, Challenges, and Future Trends

Chapter 17. Immunoassays: Future Prospects and Possibilities

1. Immunoassays: Trends and Prospects

2. Evolving Healthcare

3. Challenges

4. Future Directions

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1800, San Diego, CA 92101-4495, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2018 Elsevier Inc. All rights reserved.

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

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

Notices

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

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

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

Library of Congress Cataloging-in-Publication Data

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

British Library Cataloguing-in-Publication Data

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

ISBN: 978-0-12-811762-0

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: John Fedor

Acquisition Editor: Linda Versteeg-buschman

Editorial Project Manager: Pat Gonzalez

Production Project Manager: Kiruthika Govindaraju

Cover Designer: Christian Bilbow

Typeset by TNQ Books and Journals

Contributors

Hugo A. Arends,     HAN University of Applied Sciences, Arnhem, The Netherlands

Dan Bai,     Xi'an Jiaotong University, Xi'an, P.R. China

Pranjal Chandra,     Indian Institute of Technology Guwahati, Guwahati, India

Christian Frisch,     Bio-Rad, Puchheim, Germany

Electra Gizeli

University of Crete, Heraklion, Greece

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece

Aristea Grammoustianou

University of Crete, Heraklion, Greece

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece

Christian Hentrich,     Bio-Rad, Puchheim, Germany

Jie Hu,     Xi'an Jiaotong University, Xi'an, P.R. China

Sotirios Kakabakos,     Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece

Achim Knappik,     Bio-Rad, Puchheim, Germany

Marjo Koets,     Wageningen University & Research, Wageningen, The Netherlands

Georgios Koukouvinos,     Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece

Suveen Kumar,     Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea

D. Lemass,     Dublin City University, Dublin, Ireland

Zedong Li,     Xi'an Jiaotong University, Xi'an, P.R. China

Min Lin,     Xi'an Jiaotong University, Xi'an, P.R. China

Zhi Liu,     Xi'an Jiaotong University, Xi'an, P.R. China

John H.T. Luong,     University College Cork, Cork, Ireland

Kuldeep Mahato,     Indian Institute of Technology Guwahati, Guwahati, India

Eleni Makarona,     Institute of Nanoscience and Nanotechnology, Athens, Greece

Pawan K. Maurya,     Universidade Federal de Sao Paulo-UNIFESP, Sao Paulo, Brazil

Konstantinos Misiakos,     Institute of Nanoscience and Nanotechnology, Athens, Greece

K.L.M. Moran,     Dublin City University, Dublin, Ireland

R. O'Kennedy,     Dublin City University, Dublin, Ireland

Panagiota Petrou,     Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece

Zhiguo Qu,     Xi'an Jiaotong University, Xi'an, P.R. China

Ioannis Raptis,     Institute of Nanoscience and Nanotechnology, Athens, Greece

Renu Singh,     University of Minnesota, Saint Paul, MN, United States

Ananya Srivastava,     Indian Institute of Technology Delhi, New Delhi, India

Andre Ten Haaf,     Bio-Rad, Puchheim, Germany

Aart van Amerongen,     Wageningen University & Research, Wageningen, The Netherlands

Sandeep K. Vashist,     IDS Immunodiagnostic Systems Deutschland GmbH, Frankfurt am Main, Germany

Jeroen Veen,     HAN University of Applied Sciences, Arnhem, The Netherlands

Feng Xu,     Xi'an Jiaotong University, Xi'an, P.R. China

Francisco Ylera,     Bio-Rad, Puchheim, Germany

Minli You,     Xi'an Jiaotong University, Xi'an, P.R. China

Penghui Zhang,     Xi'an Jiaotong University, Xi'an, P.R. China

Preface

As the smart eyes of healthcare professionals, immunoassays play a prominent role in clinical diagnostics to confirm suspected diseases, enabling clinical decisions and subsequent treatments. Ideally, immunoassays must have high detection selectivity and specificity together with a high degree of automation, low cost, and fast analysis time. In clinical settings, pertinent treatments based on fast and reliable diagnosis favor the patient's prognosis and prevent the disease spreading. Other important applications of immunoassays include veterinary diagnostics, food analysis, environmental monitoring, defense and security, and other bioanalytical settings. To date, immunoassays are available for a plethora of analytes such as disease biomarkers, pathogens, drug impurities, environmental contaminants, allergens, food adulterants, drugs of abuse, and various biomolecules.

This book unravels the role and technology evolution of immunoassays in bioanalytical sciences. During the last four decades, immunoassays have been transformed from conventional enzyme-linked immunosorbent assays to more advanced formats including smartphone-based point-of-care devices. Such advanced formats stem from rapid bioanalytical procedures, novel biosensing schemes, fully integrated lab-on-a-chip platforms, prolonged biomolecular storage strategies, device miniaturization and interfacing, and the emerging smart system technologies equipped with personalized mobile healthcare tools.

Objectives of the Book

This book aims to provide the comprehensive details of various types of immunoassays, which have been performed in healthcare, industrial, environmental, and other bioanalytical settings. The multifarious aspects of immunoassays are discussed to cover the principle of operation, characteristics, pros and cons, and potential bioanalytical applications.

Scope of the Book

This book outlines various aspects of immunoassays to encompass antibody/protein immobilization, surface functionalization, point-of-care devices, and regulatory requirements. Various types of immunoassays together with their bioanalytical performance and characteristics are discussed in depth.

Target Audience

This book aims to provide a thorough understanding and comprehensive view of immunoassays and in vitro diagnostics (IVD) to healthcare professionals, biomedical engineers/scientists, researchers, healthcare economists, policy makers, investors, professionals in preventive healthcare, and persons interested in personalized healthcare. This book serves as a very useful resource and teaching aid for professionals and researchers in immunoassays, IVD, biosensors, biomedical diagnostics, medicine, bioengineering, and multidisciplinary scientific disciplines.

Book Organization

This book targets the various immunoassay formats developed to date and the multifarious aspects of immunoassays. The first two chapters provide an overview of immunoassays, antibody immobilization, and surface functionalization chemistries. The third chapter provides an in-depth expert insight into monoclonal antibody generation. The bioanalytical requirements and regulatory requirements for immunoassays are discussed in the fourth chapter. The subsequent chapters 5–16 discuss the various types of immunoassays developed to date in comprehensive detail. They provide the principles of operation, advantages, potentials, challenges, limitations, bioanalytical applications, and future trends of each immunoassay. The detection platforms are based on enzyme-linked immunoassay, surface plasmon resonance, lateral flow, paper, acoustic wave, interferometry, nano- and micromaterials, microcantilevers, quartz crystal microbalance, electrochemical, lab-on-a-chip technologies, and smartphone readout. The concluding chapter discusses the future prospects of immunoassays.

We highly appreciate the efforts devoted by the authors in drafting the outstanding book chapters. The knowledge and insight provided by this book on immunoassays will lend considerable support to more effective healthcare management as it provides the most comprehensive and updated information about various types of immunoassays and its varied aspects including the immunoanalytical platforms/systems/devices. Immunoassays play an instrumental role in clinical decision-making by enabling rapid and highly sensitive detection of disease biomarkers and analytes. Moreover, they are critically important in the biopharmaceutical industry for the manufacture of improved drugs with lesser impurities. Additionally, they have a prominent role in environmental monitoring. Therefore, they form an integral part of bioanalytical sciences in healthcare, industry, environmental, and other bioanalytical settings. The information provided in this book will empower the targeted end-users with the desired knowledge, competent skills, and comprehensive resource pertaining to the immunoassays. It will empower the targeted users to effectively manage the bioanalytical applications and lead to critically improved outcomes in industrial, healthcare, research, and other settings.

The guided insight and knowledge of immunoassays and varied aspects of immunoassays provided in this book will act as a potential resource for professionals, researchers, and students, who are working or would like to work in the field of bioanalytical sciences.

Sandeep Kumar Vashist,     Aachen, Germany

John H.T. Luong,     Cork, Ireland

Chapter 1

Immunoassays

An Overview

Sandeep K. Vashist¹, and John H.T. Luong²     ¹IDS Immunodiagnostic Systems Deutschland GmbH, Frankfurt am Main, Germany     ²University College Cork, Cork, Ireland

Abstract

The last two decades have witnessed striking advances in immunoassays (IAs), thanks to innovation and developments in antibody immobilization strategies, rapid diagnostic platforms, novel IA formats, new biosensing concepts, portable detectors, and complementary technologies. These advances have led to the emergence of a wide range of IAs for diversified bioanalytical applications. Most of the initial efforts focused on IAs for central laboratories while the subsequent efforts aimed at decentralized settings. Thereafter, the critical advances in complementary technologies, such as lab-on-a-chip platforms, microfluidics, portable detectors, and novel assay formats, paved the way to several point-of-care IAs. The current trend is strongly inclined toward IA formats for point-of-care testing and personalized mobile healthcare to empower the patients and the general population to monitor and manage their own health. This chapter provides an overview of the critical advances achieved in IAs.

Keywords

Antibody; Bioanalytical applications; Immunoassay formats; Immunoassays

1. Overview of Immunoassays

Immunoassays (IAs) play a critical role in various bioanalytical settings, such as clinical diagnostics, biopharmaceutical analysis, environmental monitoring, security, and food testing. During 1995–2017, a wide range of IAs have been developed to provide the quantitative, semiquantitative, or qualitative detection of analytes. The precise early-stage detection of analytes is an essential requirement for all bioanalytical settings to effectively monitor and manage the quality of the biopharmaceutical drugs, foods, and environment. It is even more critical to effectively diagnose, monitor, and manage the patients' health. Considering the prominent role that IAs play in the clinical decision-making, they are indispensable for healthcare settings.

Tremendous advances in IA formats, bioanalytical platforms, immunoanalytical systems, and complementary technologies have led to various emerging IA formats. Initially, most of the IAs, developed several decades ago, were based on radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Consequently, RIA- and ELISA-based kits for a wide range of analytes became commercially available as the antibodies were generated against such analytes. Owing to extremely high sensitivity, specificity, precision, and throughput of ELISA, this format has served as the gold standard for a plethora of analytes. The last two decades has seen tremendous innovation in ELISA technology. Automated assays are equipped with robotic workstations while the microtiter plate (MTP) readers have also improved drastically in terms of technology, features, and cost-effectiveness. The conventional 96-well MTP formats are transformed into higher-throughput 384- and 1536-well formats. The bioanalytical performance of ELISA has also improved significantly by better antibody immobilization chemistries [23,61,78–80], development of ultrasensitive enzyme substrates, use of micromaterial- or nanomaterial-based signal enhancement strategies [20,71,74], and novel IA concepts [38]. The current trend in ELISA deviates toward wash-free ELISA such as AlphaLISA by Perkin Elmer [8], which has critically reduced the IA duration and complexity.

Advanced bioengineering, microfluidic, and biosensor technologies [24,31,35,55,62] have further led to the development of prominent immunoanalytical systems including surface plasmon resonance (SPR)-based BiaCore instruments for label-free IA from GE Healthcare (previously Pharmacia Biosensor AB, Biacore AB Corporation). These systems have become the global standard for developing rapid IA for the detection of analytes and screening of immunological components based on the determination of biomolecular interactions [27,54]. Although GE Healthcare is still the leader in this domain, other competitors have developed SPR-based immunoanalytical systems.

The advent of microfluidics has started the quest for novel IA platforms and formats, which enable rapid IA using the minimal volume of reagents [77]. An example is the Optimiser ELISA by Siloam Biosciences, USA, which involves the conversion of the conventional 96-well MTP-based ELISA into microfluidic ELISA [29]. The analysis time is only a few minutes as this hybrid system employs significantly reduced number of steps and a microfluidic IA protocol. Similarly, a large number of prospective lab-on-a-chip technologies and formats have been developed toward the development of various microfluidic IA [41]. The most widely used microfluidic IA format is the lateral flow IA (LFIA). Although most of the LFIAs are qualitative, many prospective quantitative LFIAs have been demonstrated in recent years such as those based on smartphone (SP) detection [42]. Another prospective development route has been the development of electrochemical IA [55].

There is also an emerging trend toward the development of multiplexed IA that holds a tremendous potential in in vitro diagnostics and bioanalytical sciences [32] as exemplified by the two most widely used formats: multiplex bead-based Luminex assays by Thermo Fisher Scientific and multiplex IA by Meso Scale Discovery.

Other new biosensing concepts are based on microgravimetry (cantilevers and quartz crystal microbalance) [21,57,60,70], interferometry [39], and acoustic waves [33]. Although such formats have been demonstrated in various laboratories, they could not gather much commercial attention. Of interest is the deployment of mass spectroscopy–based IAs, which have been extensively used as the clinical and industrial IA standard for a large number of analytes. Albeit it requires highly sophisticated and costly instrumentation along with highly skilled analysts, this technique offers unmatched high sensitivity, specificity, and analytical performance. Accordingly, it is being used as the established clinically validated IAs for various analytes, which makes it essential to use these IAs to determine the precision of newly developed IAs. The development of low-cost IA formats for the developing nations has received considerable attention [40] as attested by paper-based IA formats for a number of IAs [20,26]. Despite being low-cost and easy to produce in large numbers, they still lack high analytical sensitivity comparable with that of the established IAs [63].

The most recent trend is the development of SP-based IA (SPIA) formats and devices, which outperform the central laboratory IA and instruments [62,63,66]. SPIA and devices have been developed for LFIA, electrochemical IA, colorimetric IA, chemiluminescent IA, fluorescent IA, SPR-based IA, lab-on-a-chip–based IA, and other IA formats. The use of SPs enhances the capabilities of diagnostics and enables the analysis at the point-of-care settings, such as decentralized and remote locations, and personalized settings. The IA readings together with the time and date stamp and spatiotemporal information can be stored securely on the SP and the centralized server by Cloud computing via wireless or Internet connectivity. The readings can be immediately processed on the SP or the centralized server and the results are transmitted back to the user. SPs have emerged as the ideal point-of-care device for IA as they are extensive outreach and capabilities considering over 7  billion SP users, i.e., ∼96% of the world's population [3].

Other parallel improvements are antibody immobilization chemistries, rapid IA strategies, portable readout devices, microfluidic and lab-on-a-chip technologies, innovative diagnostic platforms, and assisting complementary technologies. These ongoing advances have considerably improved the bioanalytical performance of IA and would further lead to continuous improvements in IA during the coming years.

2. Antibody Structure

The antibody, also known as immunoglobulin, is the most critical component of an IA as it provides the desired high specificity and sensitivity for the analyte of interrogation. The screening and generation of the specific antibody are the preliminary tasks before the development of IA for an analyte. Among five classes of antibodies that are present in humans, the most predominantly used Ab in immunodiagnostics is immunoglobulin G (IgG). IgG accounts for ∼75% of total serum immunoglobulin and protects the newborn during the initial months as it can cross the placenta in humans. An IgG is a Y-shaped glycoprotein consisting of four polypeptide chains with two identical heavy chains (H) inside and two identical light chains (L) outside (Fig. 1.1). Each L chain contains a variable domain VL and a constant domain CL while each H chain has a variable domain, VH, and three constant domains CH1, CH2, and CH3. The amino terminal ends of the polypeptide chains, located at the top of Y-shaped Ab, are referred to as the variable (V) regions as they have significant variation in amino acid composition. In contrast, the remaining Ab structure is composed of relatively constant (C) regions. The V regions of H and L chains form the antigen-binding sites of IgG on both arms of Y-shaped Ab. Therefore, each IgG is bivalent as it contains two antigen-binding sites that are held together by covalent disulfide bonds between the H and L chains, and noncovalent interactions. The H chains at the hinge region are also held together by disulfide bonds. The total MW of an IgG is ∼150  kDa, which is composed of ∼440 amino acids. The heavy chain has an MW of ∼50  kDa and double the number of amino acids than the light chain having MW of ∼25  kDa. There are four subclasses of IgG in humans, i.e., IgG1, IgG2, IgG3, and IgG4.

Figure 1.1  The structure of an antibody.

The other classes of Ab have different heavy chains, i.e., μ-chains for IgM; α-chains for IgA; ε-chains for IgE; and δ-chains for IgD. They function in different types of immune responses and at particular stages of the immune response. The polypeptide protein sequences responsible for these differences are located primarily in the Fc fragment. However, there are only two main types of light chains, i.e., kappa (κ) and lambda (λ). These antibodies have a varying number of Y-like monomers. IgM is a pentamer containing 10 light chains, 10 heavy chains, and 10 antigen-binding sites. The IgM monomers are bound together by disulfide bonds and a joining (J) chain. It is the most efficient complement-fixing Ab that constitutes ∼10% of normal human serum Ab content and is majorly involved in the primary immune response to most antigens. IgA constituting ∼15% of the total serum Ab exists in both monomeric and dimeric forms. It is predominant in saliva and tears as a dimer, where it protects against local infections and prevents foreign substances from entering into the circulatory system. IgD and IgE are prevalent in serum at a very low concentration where IgD acts as a receptor for antigens on mature B lymphocytes while IgE prevents the invasion of parasites.

3. Need for Immunoassays

3.1. Clinical

A large number of diseases are diagnosed exclusively based on the determination of disease biomarkers' concentrations by IA. The monitoring and management of the disease and the effectiveness of the therapeutic regimen are also done via IA. Therefore, IAs play a critical role in the clinical decision-making. Most of the IA in developed countries are fully automated and performed using commercial clinical analyzers, such as those from Roche, Abbott, and Siemens. However, manual ELISA is still mainly used in developing nations due to the lack of resources and infrastructure. Most automated IAs have the sample-to-analysis time of about half an hour, a critically required feature for clinical diagnostics. In brief, commercial automated IAs are based on disease biomarkers, as specified in Table 1.1. Nevertheless, there is still a need to develop such automated IAs for less prevalent disease biomarkers. To date, a large number of manual ELISAs have been developed for numerous disease biomarkers. The leading companies in the manual ELISA are R&D Systems, BD Biosciences, Bio-Rad Laboratories, Life Technologies Corporation, Millipore Corporation, Thermo Fisher Scientific Inc., BioMerieux SA, EMD Biosciences Inc., Abbott Diagnostics, BioLegend, and Ortho-Clinical Diagnostics Inc. It is essential for all IA to be clinically validated before they can be used in healthcare settings. Therefore, the development of a clinically validated IA must follow stringent bioanalytical testing guidelines.

3.2. Industrial

IAs are being used routinely in biopharmaceuticals for the analysis of biopharmaceutical analytes [18]. The most widely used IAs are those for Chinese hamster ovary protein and monocyte chemotactic protein, which need to be monitored very frequently. Similarly, several biological compounds and biomarkers are determined in industries by IAs. These analytes include insulin, cytokines, prostate-specific antigen, troponin I, insulin-like growth factor 1, matrix metallopeptidase 9, C-reactive protein, N-terminal prohormone of brain natriuretic peptide, glycated hemoglobin, and other biomolecules/disease biomarkers. Further, the current trend in biopharmaceuticals is toward the manufacturing of biologics, i.e., biomolecules such as antibodies that are used as drugs. Therefore, there is a growing need for IAs in industry. Although most of the IAs used in industry are based on automated ELISA, there has been an evolving trend toward the continuously increasing use of real-time SPR-based IAs such as those using BiaCore instruments from GE Healthcare or similar instruments from other companies [43,54].

Table 1.1

Biomarkers for Various Clinical Areas That Are Determined by Automated Clinical Analyzer–Based Immunoassays

ACTH, adrenocorticotropic hormone; AFP, alpha-fetoprotein; ASLO, antistreptolysin O; AT III, antithrombin III; CA, carcinoma antigen; CCP, cyclic citrullinated peptide; CEA, carcinoembryonic antigen; CK, creatine kinase; CK-MB, creatine kinase MB isoenzyme; CMV, cytomegalovirus; CRP, C-reactive protein; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; EBV-EBNA, Epstein–Barr virus nuclear antigen; EBV-VCA, Epstein–Barr virus viral capsid antigen; ECP, eosinophil cationic protein; EPO, erythropoietin; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GAD, glutamic acid decarboxylase; HAV, hepatitis A virus; HBc, hepatitis B core; HBeAg, hepatitis B e antigen; HBs, hepatitis B surface; HBsAg, hepatitis B surface antigen; hCG, human chorionic gonadotropin; HCV, hepatitis C virus; HE4, human epididymis protein 4; hGH, human growth hormone; HIV, human immunodeficiency virus; HSV, herpes simplex virus; HTLV, human T-lymphotropic virus; IAA, indoleacetic acid; ICA, islet cell antibodies; IGF BP-3, insulin-like growth factor binding protein 3; IGF-1, insulin-like growth factor 1; IL, interleukin; LBP, lipopolysaccharide-binding protein; NSE, neuron specific enolase; NT-proBNP, N-terminal prohormone of brain natriuretic peptide; PAPP-A, pregnancy-associated plasma protein A; proGRP, Progastrin-releasing peptide; PSA, prostate-specific antigen; PTH, parathyroid hormone; SHBG, sex hormone–binding globulin; T3, 3,5′,3′-triiodo-L-thyronine; T4, 3,3′,5,5′-tetraiodo-L-thyronine; TBG, thyroxine-binding globulin; TG, thyroglobulin; TNF, tumor necrosis factor; TPA, tissue polypeptide antigen; TPO, thrombopoietin; TPS, tissue polypeptide–specific antigen; TSH, thyroid-stimulating hormone; TSI, thyroid-stimulating immunoglobulins.

3.3. Environment and Security

Several IAs need to be developed for the monitoring of the polluting substances such as harmful chemicals (pesticides, mercury, arsenic, etc.), toxins, or biowarfare agents. The outbreak of these substances could lead to epidemics. An example was the use of Anthrax spores as a biowarfare agent, which led to serious security concerns.

3.4. Food

The testing of the potential adulterants, toxins, or harmful substances in foods is done by IAs [1,47]. The most common are the IA for testing the food allergens [53], which are one of the main concerns in the Western society. There have been many outbreaks of Escherichia coli O157: H7 contamination of food products [14,44]. Similarly, there have been potential outbreaks of botulinum toxin [5,76] and other pathogens such as Salmonella [15,22] and Campylobacter [82] in food products that have led to numerous critical investigations.

3.5. Personalized Healthcare

The current trend deviates strongly toward the development of point-of-care IAs, such as those based on a disposable lab-on-a-chip and SP readout, which can be used at any place and time for the personalized monitoring of important health parameters [62,63,67]. Many prospective IAs have also been recently developed, which enable the monitoring of analytes in just a few minutes using an easy-to-use procedure. The bioanalytical performance of such IA is comparable or even better than the IAs performed in the central laboratories using advanced instruments.

4. Immunoassay Formats

ELISA has been the most widely used IA format, which has been used for the development of immunodiagnostic kits for numerous analytes due to its high sensitivity, high specificity, and high throughput. The ELISA procedure has been automated by employing robotic workstations equipped with washing and dispensing modules, and automated mechanical handling and analysis. There has been continuous innovation in antibody immobilization strategies [23,61,78–80], enzyme substrates [19], signal enhancement approaches [20,71,74], and novel IA concepts [38], which have considerably improved the bioanalytical performance of ELISA. The outstanding advance is the development of wash-free ELISA, i.e., AlphaLISA by Perkin Elmer [8] (Fig. 1.2A), which has considerably reduced the IA duration and complexity of the conventional ELISA. AlphaLISA employs streptavidin-coated alpha donor beads and anti-analyte-conjugated acceptor beads. The formation of the immune complex in the presence of analyte brings the acceptor and donor beads in close proximity leading to the generation of the chemiluminescent signal. It offers high sensitivity, broad dynamic range, and analytical performance superior to that of the conventional ELISA. Another prospective development is the Optimiser ELISA by Siloam Biosciences, USA, which employs a microfluidic ELISA protocol in a novel MTP [29] (Fig. 1.2B). The IA format has a sample-to-analysis time of only a few minutes due to the microfluidic IA procedure with reduced number of steps and lesser volume requirement of IA reagents. An interesting advance is the naked-eye ELISA using gold nanoparticles with attogram per mL range detection of analyte [9,16].

Figure 1.2  (A) AlphaLISA, a wash-free ELISA by Perkin Elmer [11] . (B) Microfluidic ELISA-based Optimiser microplate by Siloam Biosciences [15] for rapid ELISA. (A) Reproduced with permission from Nature publishing group. (B) Reproduced with permission from the Royal Society of Chemistry.

Most ELISA procedures employ a multistep IA procedure such as sandwich ELISA and competitive ELISA. However, many prospective one-step kinetics-based rapid IA procedures have been demonstrated [58,64]. The most prominent developments are the SP-based readout devices, which enables the readout of colorimetric or chemiluminescent ELISA with the same or increased sensitivity as microplate readers [65,69]. These novel readout devices have further extended the outreach of ELISA to remote and decentralized locations as they do not require any power supply and can store, transmit, and analyze data at any place and time. Of interest is the SP-based EasyELISA platform technology, which enables the one-step kinetics-based rapid IA to be performed within 30  min [72].

Microfluidics and lab-on-a-chip technologies have led to several automated rapid IA formats that employ minimal volumes of reagents [77]. LFIA is the most widely used format for the remote semiquantitative measurement of analytes in just a few minutes. Although it has been used predominantly for pregnancy testing of human chorionic gonadotropin, many prospective IAs have been developed for other potential biomarkers. The most recent developments are the LFIA formats for the quantitative analyte measurement and the SP-based integrated test reader by Cellmic, USA [42] (Fig. 1.3A). Another prospective IA format is the paper-based IA [20,26], an ideal immunodiagnostic solution for the developing nations because of its low-cost and mass production. However, these formats could not match the high analytical sensitivity and performance of established clinically accredited IA that are being used in the central laboratories [63]. The most prominent microfluidic IA developed for clinical diagnostics have been developed by Abaxis Inc. (Fig. 1.3B) and Gyros AB (Fig. 1.3C). They employ centrifugal microfluidics-based LabDisk platforms and analyzers, which can automatically determine many analytes via fully automated microfluidic operations. Abaxis provides a large variety of Clinical Laboratory Improvement Amendments–waived multianalyte tests. Similarly, the IAs developed by Gyros AB are being widely used by the pharmaceutical industry as they have a broad dynamic range and short sample-to-answer time using only minimal reagent volumes.

Figure 1.3  (A) Holomic rapid diagnostic reader (HRDR) for the readout of lateral flow immunoassays (IAs). Left: HRDR-200, Right: HRDR-300. Centrifugal microfluidics-based LabDisk platform and analyzer for fully automated IAs by (B) Abaxis Inc., USA, and (C) Gyros AB, Sweden. (A) Reproduced with permission from Cellmic, USA. (B, C) Reproduced with permission from Abaxis Inc. and Gyros AB.

SPR-based real-time IA using BiaCore instruments from GE Healthcare have become a global biopharmaceutical and clinical research standard [27,43,54] (Fig. 1.4). They require the gold-coated SPR chips, which could also be prefunctionalized with specific biomolecules or chemical entities as required to facilitate the binding of capture antibodies. The fully automated microfluidic IA procedure enables the determination of analytes in just a few minutes. Several prospective surface functionalization and antibody immobilization chemistries [59,68], and strategies for signal-enhanced IA have been developed during the last two decades, which have significantly improved the performance of SPR-based IA. Several prospective SPR-based instruments are commercially available by IBIS Technologies, Bio-Rad Laboratories, NanoSPR Devices, Reichert Technologies, SensiQ, etc. As the eyes of analysts, in diverse bioanalytical settings, they provide a thorough understanding of the complex biomolecular interactions and enable rapid screening of antibodies and components for an IA. They also enable effective and rapid troubleshooting of an IA, which can otherwise take a lot of time and costs.

Figure 1.4  Surface plasmon resonance–based BiaCore systems by GE Healthcare for fully automated label-free and real-time rapid immunoassays. Reproduced with permission from GE Healthcare.

Several biosensing concepts, based on microcantilevers [21,57,60], quartz crystal microbalance [70], interferometry [39], electrochemistry [55], and acoustic waves [33] have also been used for the development of a wide range of IAs. Despite a good bioanalytical performance shown by such biosensors, they are still extensive research efforts required to develop commercially and clinically viable biosensor-based IAs. In particular, mass spectroscopy–based assays have been demonstrated for a large number of analytes in healthcare and industrial settings [25,36]. Considering their high analytical precision, high sensitivity and specificity, and short analysis time, they are the established standard for clinical diagnostics. In fact, they are being used for the method comparison and determination of precision of new IAs.

There is considerable interest in the multiplex detection of biomarkers [32] and the clinical scoring–based diagnosis of a particular disease [30,45,56], which can be used in predictive diagnostics. If the biomarkers responsible for a particular disease are known and weightage can be assigned to them based on their relevance in the diagnosis of a disease, the clinical score can be calculated based on the multiplex detection of biomarkers. It will provide an easy-to-use clinical indicator for the monitoring and management of a disease, and analyze the effectiveness of therapeutic treatment and/or personalized/medical intervention. Various algorithms for the clinical scoring have been demonstrated and used clinically for the diagnosis and differentiation of various diseases. Several IA formats have been demonstrated for multiplexed detection of biomarkers, such as the multiplex bead-based Luminex IA by Thermo Fisher Scientific [4,11,17,49] and multispot electrochemiluminescence-based IA by Meso Scale Discovery [11,13].

SPs have become ubiquitous and personalized and have advanced biosensors, processing speed, better connectivity, and other desired features, which make them the ideal device for point-of-care IAs [62,63,66,67]. In brief, a large number of IAs with readout by SP have been developed. Several prospective SP-based devices are based on the detection of colorimetric [65,69,73], absorbance [37,75], chemiluminescent [6,81], fluorescent [7,12], electrochemical [28,50–52], SPR [34,46], and other optical signals [10,48]. They show superior bioanalytical performances in comparison with central laboratory-based IAs.

The near future would witness continuous innovation in IA formats, diagnostic platforms, biosensing concepts, IA chemistries, and complementary technologies, which would provide an impetus to the development of next-generation IA technologies [63].

5. Conclusions and Future Trends

A wide range of IA formats, platforms, and devices have been developed during the last two decades for various bioanalytical applications. The most widely used IA format being used in clinical diagnostics is the automated ELISA using commercial clinical analyzers. However, most of the clinical IAs in the developing nations with limited resources and infrastructure are still performed using manual ELISA. The last decade has witnessed an evolving trend toward the use of fully automated SPR-based IA in biopharmaceuticals and research establishments. The extensive research efforts have led to the emergence of many prospective IA formats, based on microcantilevers, QCM, micro/nanomaterials, lab-on-a-chip formats, and microfluidics. The mass spectroscopy–based clinically validated assays have been developed for a large number of analytes and serve as the established assays for determining the precision of newly developed IA. Moreover, several new biosensing concepts have emerged during the last two decades, which have been demonstrated for a wide range of IAs.

The field of point-of-care testing has also evolved drastically and is undergoing a constant change as there is an extensive need for immunodiagnostics for decentralized and remote settings. Similarly, there is a requirement for low-cost point-of-care diagnostics in the developing nations. The most prospective IA formats for these are the paper, lab-on-a-chip, and SPIA. Despite intensive efforts, the paper-based IAs have not been successful for the clinical diagnostics due to the lack of precision and sensitivity. Some lab-on-a-chip-based IAs have already become commercially successful and are being used for clinical diagnostics. As the latest trend, the SP-based devices are being seen as the ideal device for personalized healthcare monitoring and management. Many of such SP-based mobile healthcare devices are already commercial and are being used by millions of users worldwide. However, the development of a clinically accredited SPIA would still require extensive efforts before they can be used by analysts and the general population. It would require more rigorous clinical validation and testing to validate that the SPIAs are as precise as the established clinically accredited IAs being used in the central laboratory. Similarly, there are concerns about the enormous data such as data storage, data security, data transmission, and data ownership, which need to be counteracted effectively.

There is a need for multiplex IA formats so that all the biomarkers involved in the diagnosis of a disease can be quantitatively detected at the same time. Further, the clinical score for a disease can be determined by various clinical scoring algorithms. This would enable the rapid screening of a disease in a population and would facilitate the continuous monitoring and management of the disease via effective medical and personal intervention.

The continuous improvements in IA formats, bioanalytical platforms, novel biosensing concepts, portable detectors, and complementary technologies would lead to the development of several prospective IA and devices in the near future, which would change the landscape of immunodiagnostics. Such advances would pave the way to next generation of IA that would have rapid analysis time, less cost, high-throughput, and superior bioanalytical performance. This would lead to much better healthcare as diseases could be detected and treated at a very early stage, which could significantly cut down the healthcare costs.

References

[1] Alocilja E.C, Radke S.M. Market analysis of biosensors for food safety. Biosens. Bioelectron. 2003;18:841–846.

[2] Anonymous. Antibody Production and Purification Technical Handbook. Thermo Fisher Scientific Inc.; 2010 (version 2).

[3] Anonymous. Measuring the Information Society Report. 2016. http://www.itu.int/en/ITU-D/Statistics/Pages/publications/mis2016.aspx.

[4] Arellano-Garcia M.E, Hu S, Wang J, et al. Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva. Oral Dis. 2008;14:705–712.

[5] Arnon S.S, Schechter R, Inglesby T.V, et al. Botulinum toxin as a biological weapon: medical and public health management. J. Am. Med. Assoc. 2001;285:1059–1070.

[6] Arts R, Den Hartog I, Zijlema S.E, et al. Detection of antibodies in blood plasma using bioluminescent sensor proteins and a smartphone. Anal. Chem. 2016;88:4525–4532.

[7] Barbosa A.I, Gehlot P, Sidapra K, et al. Portable smartphone quantitation of prostate specific antigen (PSA) in a fluoropolymer microfluidic device. Biosens. Bioelectron. 2015;70:5–14.

[8] Beaudet L, Rodriguez-Suarez R, Venne M.-H, et al. AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery. Nat. Methods. 2008;5 an8an9.

[9] Cecchin D, De La Rica R, Bain R, et al. Plasmonic ELISA for the detection of gp120 at ultralow concentrations with the naked eye. Nanoscale. 2014;6:9559–9562.

[10] Cho S, Park T.S, Nahapetian T.G, et al. Smartphone-based, sensitive microPAD detection of urinary tract infection and gonorrhea. Biosens. Bioelectron. 2015;74:601–611.

[11] Chowdhury F, Williams A, Johnson P. Validation and comparison of two multiplex technologies, Luminex® and Mesoscale discovery, for human cytokine profiling. J. Immunol. Methods. 2009;340:55–64.

[12] Coskun A.F, Nagi R, Sadeghi K, et al. Albumin testing in urine using a smart-phone. Lab Chip. 2013;13:4231–4238.

[13] Dabitao D, Margolick J.B, Lopez J, et al. Multiplex measurement of proinflammatory cytokines in human serum: comparison of the meso scale discovery electrochemiluminescence assay and the cytometric bead array. J. Immunol. Methods. 2011;372:71–77.

[14] Davis B, Brogan R. A widespread community outbreak of E. coli 0157 infection in Scotland. Public Health. 1995;109:381–388.

[15] De Buyser M.L, Dufour B, Maire M, et al. Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. Int. J. Food Microbiol. 2001;67:1–17.

[16] De La Rica R, Stevens M.M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat. Nanotechnol. 2012;7:821–824.

[17] Dunbar S.A, Hoffmeyer M.R. Microsphere-based multiplex immunoassays: development and applications using Luminex® xMAP® technology. In: The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques. vol. 157. 2013.

[18] Findlay J.W, Smith W.C, Lee J.W, et al. Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. J. Pharm. Biomed. Anal. 2000;21:1249–1273. .

[19] Frey A, Meckelein B, Externest D, et al. A stable and highly sensitive 3,3',5,5'-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays. J. Immunol. Methods. 2000;233:47–56.

[20] Ge X, Asiri A.M, Du D, et al. Nanomaterial-enhanced paper-based biosensors. Trends Anal. Chem. 2014;58:31–39.

[21] Goeders K.M, Colton J.S, Bottomley L.A. Microcantilevers: sensing chemical interactions via mechanical motion. Chem. Rev. 2008;108:522–542.

[22] Hennessy T.W, Hedberg C.W, Slutsker L, et al. A national outbreak of Salmonella enteritidis infections from ice cream. The Investigation Team. N. Engl. J. Med. 1996;334:1281–1286.

[23] Hermanson G.T. Bioconjugate Techniques. Academic press; 2013.

[24] Herr A.E, Hatch A.V, Throckmorton D.J, et al. Microfluidic immunoassays as rapid saliva-based clinical diagnostics. Proc. Natl. Acad. Sci. U.S.A. 2007;104:5268–5273.

[25] Hoofnagle A.N, Wener M.H. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J. Immunol. Methods. 2009;347:3–11.

[26] Hu J, Wang S, Wang L, et al. Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 2014;54:585–597.

[27] Jason-Moller L, Murphy M, Bruno J. Overview of Biacore systems and their applications. Curr. Protoc. Protein Sci. 2006;45 19.13:19.13.1–19.13.14.

[28] Jiang H, Sun A, Govindaraj V, et al. An audio jack based electrochemical impedance spectroscopy sensor for point-of-care diagnostics. IEEE Sens. J. 2016;17:589–597.

[29] Kai J, Puntambekar A, Santiago N, et al. A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA). Lab Chip. 2012;12:4257–4262.

[30] Kalantar-Zadeh K, Block G, Mcallister C.J, et al. Appetite and inflammation, nutrition, anemia, and clinical outcome in hemodialysis patients. Am. J. Clin. Nutr. 2004;80:299–307.

[31] Karle M, Vashist S.K, Zengerle R, et al. Microfluidic solutions enabling continuous processing and monitoring of biological samples: a review. Anal. Chim. Acta. 2016;929:1–22.

[32] Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discov. 2006;5:310–320.

[33] Lange K, Rapp B.E, Rapp M. Surface acoustic wave biosensors: a review. Anal. Bioanal. Chem. 2008;391:1509–1519.

[34] Lee K.L, You M.L, Tsai C.H, et al. Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone. Biosens. Bioelectron. 2016;75:88–95.

[35] Lin C.-C, Wang J.-H, Wu H.-W, et al. Microfluidic immunoassays. J. Lab. Autom. 2010;15:253–274.

[36] Liu R, Wu P, Yang L, et al. Inductively coupled plasma mass spectrometry-based immunoassay: a review. Mass Spectrom. Rev. 2014;33:373–393.

[37] Long K.D, Yu H, Cunningham B.T. Smartphone instrument for portable enzyme-linked immunosorbent assays. Biomed. Opt. Express. 2014;5:3792–3806.

[38] Luppa P.B, Sokoll L.J, Chan D.W. Immunosensors-principles and applications to clinical chemistry. Clin. Chim. Acta. 2001;314:1–26.

[39] Makarona E, Petrou P, Kakabakos S, et al. Point-of-need bioanalytics based on planar optical interferometry. Biotechnol. Adv. 2016;34:209–233.

[40] Martinez A.W, Phillips S.T, Whitesides G.M, et al. Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 2009;82:3–10. .

[41] Mohammed M.I, Desmulliez M.P. Lab-on-a-chip based immunosensor principles and technologies for the detection of cardiac biomarkers: a review. Lab Chip. 2011;11:569–595.

[42] Mudanyali O, Dimitrov S, Sikora U, et al. Integrated rapid-diagnostic-test reader platform on a cellphone. Lab Chip. 2012;12:2678–2686.

[43] Myszka D.G, Rich R.L. Implementing surface plasmon resonance biosensors in drug discovery. Pharm. Sci. Technol. Today. 2000;3:310–317.

[44] Ostroff S.M, Griffin P.M, Tauxe R.V, et al. A statewide outbreak of Escherichia coli 0157: H7 infections in Washington State. Am. J. Epidemiol. 1990;132:239–247.

[45] Papachristou G.I, Papachristou D.J, Avula H, et al. Obesity increases the severity of acute pancreatitis: performance of APACHE-O score and correlation with the inflammatory response. Pancreatology. 2006;6:279–285.

[46] Preechaburana P, Gonzalez M.C, Suska A, et al. Surface plasmon resonance chemical sensing on cell phones. Angew. Chem. Int. Ed. Engl. 2012;51:11585–11588.

[47] Samarajeewa U, Wei C.I, Huang T.S, et al. Application of immunoassay in the food industry. Crit. Rev. Food Sci. Nutr. 1991;29:403–434.

[48] San Park T, Li W, Mccracken K.E, et al. Smartphone quantifies Salmonella from paper microfluidics. Lab Chip. 2013;13:4832–4840.

[49] Siawaya J.F.D, Roberts T, Babb C, et al. An evaluation of commercial fluorescent bead-based luminex cytokine assays. PLoS One. 2008;3:e2535.

[50] Sun A, Venkatesh A, Hall D.A. A multi-technique reconfigurable electrochemical biosensor: enabling personal health monitoring in mobile devices. IEEE Trans. Biomed. Circuits Syst. 2016;10:945–954.

[51] Sun A, Wambach T, Venkatesh A, et al. A low-cost smartphone-based electrochemical biosensor for point-of-care diagnostics. In: Biomedical Circuits and Systems Conference (BioCAS), 2014 IEEE. 2014:312–315.

[52] Sun A.C, Yao C, Venkatesh A.G, et al. An efficient power harvesting mobile phone-based electrochemical biosensor for point-of-care health monitoring. Sens. Actuators B Chem. 2016;235:126–135.

[53] Taylor S.L, Nordlee J.A, Niemann L.M, et al. Allergen immunoassays-considerations for use of naturally incurred standards. Anal. Bioanal. Chem. 2009;395:83–92.

[54] Thillaivinayagalingam P, Gommeaux J, Mcloughlin M, et al. Biopharmaceutical production: applications of surface plasmon resonance biosensors. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010;878:149–153.

[55] Turner A.P. Biosensors: sense and sensibility. Chem. Soc. Rev. 2013;42:3184–3196.

[56] Van Zee K.J, Kohno T, Fischer E, et al. Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A. 1992;89:4845–4849.

[57] Vashist S.K. A review of microcantilevers for sensing applications. AzoNano J. Nanotech. 2007;3:1–18.

[58] Vashist S.K, Czilwik G, Van Oordt T, et al. One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min. Anal. Biochem. 2014;456:32–37.

[59] Vashist S.K, Dixit C.K, Maccraith B.D, et al. Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay. Analyst. 2011;136:4431–4436. .

[60] Vashist S.K, Holthöfer H. Microcantilevers for sensing applications. Meas. Control. 2010;43:84–88.

[61] Vashist S.K, Lam E, Hrapovic S, et al. Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chem. Rev. 2014;114:11083–11130.

[62] Vashist S.K, Luong J.H.T. Trends in in vitro diagnostics and mobile healthcare. Biotechnol. Adv. 2016;34:137–138.

[63] Vashist S.K, Luppa P.B, Yeo L.Y, et al. Emerging technologies for next-generation point-of-care testing. Trends Biotechnol. 2015;33:692–705.

[64] Vashist S.K, Marion Schneider E, Luong J.H.T. Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of Human fetuin A. Biosens. Bioelectron. 2015;67:73–78.

[65] Vashist S.K, Marion Schneider E, Zengerle R, et al. Graphene-based rapid and highly-sensitive immunoassay for