Methods in molecular biology vol 1571 biosensors and biodetection methods and protocols, volume 1

Commonevanescent wave biosensors are surface plasmon resonance SPR or resonant mirror sensors.Other direct optical detectors include interferometric sensors or grating coupler.. For exam

Biosensors

and Biodetection

Avraham Rasooly

Ben Prickril Editors

Methods and Protocols

Volume 1: Optical-Based Detectors

Second Edition

Molecular Biology 1571

ME T H O D S I N MO L E C U L A R BI O L O G Y

Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

Methods and Protocols Volume 1:

Optical-Based Detectors

Second Edition

Edited by Avraham Rasooly National Cancer Institute National Institutes of Health Rockville, MD, USA Ben Prickril National Cancer Institute National Institutes of Health Rockville, MD, USA

Avraham Rasooly

National Cancer Institute

National Institutes of Health

Rockville, MD, USA

Ben Prickril National Cancer Institute National Institutes of Health Rockville, MD, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

ISBN 978-1-4939-6846-6 ISBN 978-1-4939-6848-0 (eBook)

DOI 10.1007/978-1-4939-6848-0

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and Ilan Rasooly.

Biosensor Technologies

A biosensor is defined by the International Union of Pure and Applied Chemistry (IUPAC)

as “a device that uses specific biochemical reactions mediated by isolated enzymes, nosystems, tissues, organelles or whole cells to detect chemical compounds usually byelectrical, thermal or optical signals” [1]; all biosensors are based on a two-componentsystem:

immu-1 Biological recognition element (ligand) that facilitates specific binding or biochemicalreaction with the target analyte

2 Signal conversion unit (transducer)

Since the publication of the first edition of this book in 2009, “classical” biosensormodalities such as electrochemical or surface plasmon resonance (SPR) continue to bedeveloped New biosensing technologies and modalities have also been developed, includ-ing the use of nanomaterials for biosensors, fiber-optic-based biosensors, genetic code basedsensors, field effect transistors, and the use of mobile communication device-based biosen-sors Although it is impossible to describe the fast-moving field of biosensing in a singlepublication, this book presents descriptions of methods and uses for some of the basic types

of biosensors while also providing the reader a sense of the enormous importance andpotential for these devices In order to present a more comprehensive overview, the bookalso describes other biodetection technologies

Dr Leland C Clark, who worked on biosensors in the early 1960s, provided an earlyreference to the concept of a biosensor by developing an “enzyme electrode” for glucoseconcentration measurement using the enzyme glucose oxidase (GOD) [2] Glucose moni-toring is essential for diabetes patients, and even today the most common clinical biosensortechnology for glucose analysis is the electrochemical detection method envisioned by Clarkmore than 50 years ago Today, glucose monitoring is performed using rapid point of carebiosensors made possible through advances in electronics that have enabled sensor minia-turization The newest generation of biosensors includes phone-based optical detectors withhigh-throughput capabilities

The Use of Biosensors

Biosensors have several potential advantages over other methods of biodetection, includingincreased assay speed and flexibility Rapid, real-time analysis can provide immediate inter-active information to health-care providers that can be incorporated into the planning ofpatient care In addition, biosensors allow multi-target analyses, automation, and reducedtesting costs Biosensor-based diagnostics may also facilitate screening for cancer and otherdiseases by improving early detection and therefore improving prognosis Such technologymay be extremely useful for enhancing health-care delivery to underserved populations and

in community settings

vii

The main advantages of biosensors include:

Rapid or real-time analysis: Direct biosensors such as those employing surface plasmonresonance (SPR) enable rapid or real-time label-free detection and provide almostimmediate interactive sample information This enables facilities to take correctivemeasures before a product is further processed or released for consumption

Point of care detection capabilities: Biosensors can be used for point of care testing Thisenables state-of-the-art molecular analysis without requiring a laboratory

Continuous flow analysis: Many biosensors are designed to allow analysis of bulk liquids Insuch biosensors the target analyte is injected onto the sensor using a continuous flowsystem immobilized in a flow cell or column, thereby enhancing the efficiency of analytebinding to the sensor and enabling continuous monitoring

Miniaturization: Increasingly, biosensors are being miniaturized for incorporation intoequipment for a wide variety of applications including clinical care, food and dairyanalyses, agricultural and environmental monitoring, and in vivo detection of a variety

of diseases and conditions

Control and automation: Biosensors can be integrated into online process monitoringschemes to provide real-time information about multiple parameters at each productionstep or at multiple time points during a process, enabling better control and automation

of biochemical facilities

Biosensor Classification

In general biosensors can be divided into two groups: direct recognition sensors in whichthe biological interaction is directly measured and indirect detection sensors which rely onsecondary elements (often catalytic) such as enzymes or fluorescent tags for measurements.Figure 1 illustrates the two types of biosensors In each group there are several types of

optical, electrochemical, or mechanical transducers Although the most commonly usedligands are antibodies, other ligands are being developed including aptamers (protein-binding nucleic acids) and peptides.

There are numerous types of direct and indirect recognition biosensors, and choice of asuitable detector is complex and based on many factors These include the nature of theapplication, type of labeled molecule (if used), sensitivity required, number of channels (orarea) measured, cost, technical expertise, and speed of detection In this book we describemany of these detectors, their application to biosensing, and their fabrication

The transducer element of biosensors converts the biochemical interactions of theligand into a measurable electronic signal The most important types of transducer usedtoday are optical, electrochemical, and mechanical

Direct Label-Free Detection Biosensors

Direct recognition sensors, in which the biological interaction is directly measured in realtime, typically use non-catalytic ligands such as cell receptors or antibodies Such detectorstypically measure directly physical changes (e.g., changes in optical, mechanical, or electricalproperties) induced by the biological interaction and do not require additional labeledmolecules (i.e., are label-free) for detection The most common direct detection biosensorsare optical biosensors including biosensors which employ evanescent waves generated when

a beam of light is incident on a surface at an angle yielding total reflection Commonevanescent wave biosensors are surface plasmon resonance (SPR) or resonant mirror sensors.Other direct optical detectors include interferometric sensors or grating coupler Nonopticaldirect detection sensors are quartz resonator transducers that measure change in resonantfrequency of an oscillating piezoelectric crystal as a function of mass (e.g., analyte binding)

on the crystal surface, microcantilevers used in microelectromechanical systems (MEMS)measuring bending induced by the biomolecular interactions, or field effect transistor (FET)biosensors, a transistor gated by biological molecules When biological molecules bind tothe FET gate, they can change the gate charge distribution resulting in a change inconductance of the FET

Indirect Label-Based Detection Biosensors

Indirect detection sensors rely on secondary elements for detection and utilize labeling orcatalytic elements such as enzymes Examples of such secondary elements are the enzymealkaline phosphatase and fluorescently tagged antibodies that enhance detection of a sand-wich complex Unlike direct sensors, which directly measure changes induced by biologicalinteraction and are “label-free,” indirect sensors require a labeled molecule bound to thetarget Most optical indirect sensors are designed to measure fluorescence; however, suchsensors can also measure densitometric and colorimetric changes as well as chemilumines-cence, depending on the type of label used

Electrochemical transducers measure the oxidation or reduction of an electroactivecompound on the secondary ligand and are one common type of indirect detection sensor.Several types of electrochemical biosensors have been developed including amperometricdevices, which detect ions in a solution based on electric current or changes in electriccurrent when an analyte is oxidized or reduced Another common indirect detectionbiosensor employs optical fluorescence, detecting fluorescence of the secondary ligand viaCCD, PMT, photodiode, and spectrofluorometric analysis In addition, visual measurementsuch as change of color or appearance of bands (e.g., lateral flow detection) can be used forindirect detection

Indirect detection can be combined with direct detection to increase sensitivity or tovalidate results; for example, the use of secondary antibody in combination with an SPRimmunosensor Using a sandwich assay, the analyte captured by the primary antibody isimmobilized on the SPR sensor and generates a signal which can be amplified by the binding

of a secondary antibody to the captured analyte

Ligands for Biosensors

Ligands are molecules that bind specifically with the target molecule to be detected Themost important properties of ligands are affinity and specificity Of the various types ofligands used in biosensors, immunosensors—particularly antibodies—are the most commonbiosensor recognition element Antibodies (Abs) are highly specific and versatile and bindstrongly and stably to specific antigens However, Ab ligands have limited long-term stabilityand are difficult to produce in large quantities for multi-target biosensor applications wheremany ligands are needed

Other types of ligands such as aptamers and peptides are more suited to throughput screening and chemical synthesis Aptamers are protein-binding nucleic acids(DNA or RNA molecules) selected from random pools based on their ability to bind othermolecules with high affinity Peptides are another potentially important class of ligandsuitable for high-throughput screening due to their ease of selection However, the affinity

high-of peptides is high-often lower than that high-of antibodies or aptamers, and peptides vary widely instructural stability and thermal sensitivity

New Trends in Biosensing

While the fundamental principles and the basic configuration of biosensors have notchanged in the last decade, this book expands the application of these principles usingnew technologies such as nanotechnology, integrated optics (IO) bioelectronics, portableimaging, new fluidics and fabrication methodologies, and new cellular and molecularapproaches

Integration of nanotechnology: There has been great progress in nanotechnology and material in recent years New nanoparticles have been developed having unique electricconductivity, optical, and surface properties For example, in several chapters newoptical biosensors are described that integrate nanomaterials in SPR biosensor config-urations such as localized surface plasmon resonance (LSPR), 3D SPR plasmonicnanogap arrays, or gold nanoparticle SPR plasmonic peak shift In addition to SPRbiosensors, nanomaterials are also applied to fluorescence detection utilizing fluores-cence quantum dot or silica nanoparticles to increase uniform distribution of enzymeand color intensity in colorimetric biosensors or to improve lateral flow detection Inaddition to optical sensors, gold nanoparticles (AuNPs) have been integrated intoelectrochemical biosensors to improve electrochemical performance, and magneticnanoparticles (mNPs) have been used to improve sample preparation Nanoparticle-modified gate electrodes have been used in the fabrication of organic electrochemicaltransistors

nano-Bioelectronics: Several chapters described the integration of biological elements in electronictechnology including the use of semiconductors in several configurations of field effecttransistors and light-addressable potentiometric sensors

Application of imaging technologies: The proliferation of high-resolution imagingtechnologies has enabled better 2D image analysis and increases in the number ofanalytical channels available for various modalities of optical detection These includetwo-dimensional surface plasmon resonance imaging (2D-SPRi) utilizing CCD cameras

or 2D photodiode arrays The use of smartphones for both fluorescence and ric detectors is described in several manuscripts

colorimet-Integrated optics (IO): Devices with photonic integrated circuits are presented which grate several optical and often electronic components Examples include an integratedoptical (IO) nano-immunosensor based on a bimodal waveguide (BiMW) interferomet-ric transducer integrated into a complete lab-on-a-chip (LOC) platform

inte-New fluidics and fabrication methodologies: Fluidics and fluid delivery are important ponents of many biosensors In addition to traditional polymer fabricated microfluidicssystems, inkjet-printed paper fluidics are described that may play an important role inLOCs and medical diagnostics Such technologies enable low-cost mass production ofLOCs In addition, several chapters describe the use of screen printing for devicefabrication

com-Cellular and molecular approaches: Molecular approaches are described for aptamer-basedbiosensors (aptasensors), synthetic cell-based sensors, loop-mediated DNA amplifica-tion, and circular strand displacement for point mutation analysis

While “classic” transducer modalities such as SPR, electrochemical, or piezoelectricremain the predominant biosensor platforms, new technologies such as nanotechnology,integrated optics, or advanced fluidics are providing new capabilities and improvedsensitivity

Aims and Approaches

This book attempts to describe the basic types, designs, and applications of biosensors andother biodetectors from an experimental point of view We have assembled manuscriptsrepresenting the major technologies in the field and have included enough technicalinformation so that the reader can both understand the technology and carry out theexperiments described

The target audience for this book includes engineering, chemistry, biomedical, andphysics researchers who are developing biosensing technologies Other target groups arebiologists and clinicians who ultimately benefit from development and application of thetechnologies

In addition to research scientists, the book may also be useful as a teaching tool forbioengineering, biomedical engineering, and biology faculty and students To better repre-sent the field, most topics are described in more than one chapter The purpose of thisredundancy is to bring several experimental approaches to each topic, to enable the reader tochoose an appropriate design, to combine elements from different designs in order to betterstandardize methodologies, and to provide readers more detailed protocols

Organization

The publication is divided into two volumes Volume I (Springer Vol 1571) focuses onoptical-based detectors, while Vol II (Springer Vol 1572) focuses on electrochemical,bioelectronic, piezoelectric, cellular, and molecular biosensors

Volume I (Springer Vol 1571)

Optical-based detection encompasses a broad array of technologies including direct andindirect methods as discussed above Part I of Vol I describes various optical-based directdetectors, while Part II focuses on indirect optical detection Three types of direct opticaldetection biosensors are described: evanescent wave (SPR and resonant waveguide grating),interferometers, and Raman spectroscopy sensors

The second part of Vol I describes various indirect optical detectors as discussed above.Indirect directors require a labeled molecule to be bound to the signal-generating target.For optical sensors such molecules emit or modify light signals Most indirect opticaldetectors are designed to measure fluorescence; however, such detectors can also measuredensitometric and colorimetric changes as well as chemiluminescence, and detectiondepends on the type of label used Such optical signals can be measured in various ways asdescribed in Part II These include various CCD-based detectors which are very versatile,inexpensive, and relatively simple to construct and use Other optical detectors discussed inPart II are photodiode-based detection systems and mobile phone detectors Lateral flowsystems that rely on visual detection are included in this section Although lateral flowdevices are not “classical” biosensors with ligands and transducers, they are included inthis book because of their importance for biosensing Lateral flow assays use simple immu-nodetection (or DNA hybridization) devices, such as competitive or sandwich assays, andare used mainly for medical diagnostics such as laboratory and home testing or any otherpoint of care (POC) detection A common format is a “dipstick” in which the test sampleflows on an absorbant matrix via capillary action; detection is accomplished by mixing acolorimetric reagent with the sample and binding to a secondary antibody to produce lines

or zones at specific locations on the absorbing matrix

Volume II (Springer Vol 1572)

Volume II describes various electrochemical, bioelectronic, piezoelectric, and cellular- andmolecular-based biosensors

In Part I of Vol II, we describe several types of electrochemical and bioelectronicdetectors Electrochemical biosensors were the first biosensors developed and are the mostcommonly used biosensors in clinical settings (e.g., glucose monitoring) Also included areseveral electronic/semiconductor sensors based on the field effect Unlike electrochemicalsensors, which are indirect detectors and require labeling, electronic/semiconductor bio-sensors are label-free

In Part II we describe “mechanical detectors” which modify their mechanical properties

as a result of biological interactions Such mechanical direct biosensors include piezoelectricbiosensors which change their acoustical resonance and cantilevers which modify theirmovement

Part III describes a variety of biological sensors including aptamer-based sensors andcellular and phage display technologies

Part IV describes several microfluidics technologies for cell isolation In addition, anumber of related technologies including Raman spectroscopy and high-resolution micro-ultrasound are described

The two volumes provide comprehensive and detailed technical protocols on currentbiosensor and biodetection technologies and examples of their applications and capabilities

1 International Union of Pure and Applied Chemistry (1992) IUPAC compendium of chemical terminology, 2nd edn (1997) International Union of Pure and Applied Chemistry, Research Triangle Park, NC

2 Clark LC Jr, Lyons C (1962) Electrode systems for continuous monitoring in cardiovascular surgery Ann N Y Acad Sci 102:29–45

Preface viiContributors xix

1 Localized Surface Plasmon Resonance (LSPR)-Coupled Fiber-Optic

Nanoprobe for the Detection of Protein Biomarkers 1Jianjun Wei, Zheng Zeng, and Yongbin Lin

2 Ultra-Sensitive Surface Plasmon Resonance Detection by Colocalized

3D Plasmonic Nanogap Arrays 15Wonju Lee, Taehwang Son, Changhun Lee, Yongjin Oh,

and Donghyun Kim

3 Two-Dimensional Surface Plasmon Resonance Imaging System

for Cellular Analysis 31Tanveer Ahmad Mir and Hiroaki Shinohara

4 Immunosensing with Near-Infrared Plasmonic Optical Fiber Gratings 47Christophe Caucheteur, Clotilde Ribaut, Viera Malachovska,

and Ruddy Wattiez

5 Biosensing Based on Magneto-Optical Surface Plasmon Resonance 73Sorin David, Cristina Polonschii, Mihaela Gheorghiu,

Dumitru Bratu, and Eugen Gheorghiu

6 Nanoplasmonic Biosensor Using Localized Surface Plasmon

Resonance Spectroscopy for Biochemical Detection 89Diming Zhang, Qian Zhang, Yanli Lu, Yao Yao,

Shuang Li, and Qingjun Liu

7 Plasmonics-Based Detection of Virus Using Sialic Acid Functionalized

Gold Nanoparticles 109Changwon Lee, Peng Wang, Marsha A Gaston, Alison A Weiss,

and Peng Zhang

8 MicroRNA Biosensing with Two-Dimensional Surface

Plasmon Resonance Imaging 117

Ho Pui Ho, Fong Chuen Loo, Shu Yuen Wu, Dayong Gu,

Ken-Tye Yong, and Siu Kai Kong

9 Gold Nanorod Array Biochip for Label-Free, Multiplexed

Biological Detection 129Zhong Mei, Yanyan Wang, and Liang Tang

10 Resonant Waveguide Grating Imager for Single Cell Monitoring

of the Invasion of 3D Speheroid Cancer Cells Through Matrigel 143Nicole K Febles, Siddarth Chandrasekaran, and Ye Fang

xv

11 Label-Free Biosensors Based on Bimodal Waveguide

(BiMW) Interferometers 161Sonia Herranz, Adria´n Ferna´ndez Gavela, and Laura M Lechuga

12 DNA-Directed Antibody Immobilization for Robust Protein Microarrays:

Application to Single Particle Detection ‘DNA-Directed Antibody

Immobilization 187Nese Lortlar €Unl€u, Fulya Ekiz Kanik, Elif Seymour,

John H Connor, and M Selim €Unl€u

13 Reflectometric Interference Spectroscopy 207Guenther Proll, Goran Markovic, Peter Fechner, Florian Proell,

and Guenter Gauglitz

14 Hypermulticolor Detector for Quantum-Antibody Based

Concurrent Detection of Intracellular Markers for HIV Diagnosis 221Annie Agnes Suganya Samson and Joon Myong Song

15 Low-Cost Charged-Coupled Device (CCD) Based Detectors for Shiga

Toxins Activity Analysis 233Reuven Rasooly, Ben Prickril, Hugh A Bruck, and Avraham Rasooly

16 Smartphone-Enabled Detection Strategies for Portable

PCR–Based Diagnostics 251Aashish Priye and Victor M Ugaz

17 Streak Imaging Flow Cytometer for Rare Cell Analysis 267Joshua Balsam, Hugh Alan Bruck, Miguel Ossandon, Ben Prickril,

and Avraham Rasooly

18 Rapid Detection of Microbial Contamination Using

a Microfluidic Device 287Mustafa Al-Adhami, Dagmawi Tilahun, Govind Rao,

Chandrasekhar Gurramkonda, and Yordan Kostov

19 Resonance Energy Transfer-Based Nucleic Acid Hybridization

Assays on Paper-Based Platforms Using Emissive

Nanoparticles as Donors 301Samer Doughan, M Omair Noor, Yi Han, and Ulrich J Krull

20 Enhanced Performance of Colorimetric Biosensing on Paper

Microfluidic Platforms Through Chemical Modification

and Incorporation of Nanoparticles 327Ellen Fla´via Moreira Gabriel, Paulo T Garcia, Elizabeth Evans,

Thiago M.G Cardoso, Carlos D Garcia,

and Wendell K.T Coltro

21 A Smartphone-Based Colorimetric Reader for Human C-Reactive

Protein Immunoassay 343A.G Venkatesh, Thomas van Oordt, E Marion Schneider,

Roland Zengerle, Felix von Stetten, John H.T Luong,

and Sandeep Kumar Vashist

22 A Novel Colorimetric PCR-Based Biosensor for Detection

and Quantification of Hepatitis B Virus 357

Li Yang, Mei Li, Feng Du, Gangyi Chen, Afshan Yasmeen,

and Zhuo Tang

23 CCD Camera Detection of HIV Infection 371John R Day

24 “Dipstick” Colorimetric Detection of Metal Ions Based

on Immobilization of DNAzyme and Gold Nanoparticles

onto a Lateral Flow Device 389Debapriya Mazumdar, Tian Lan, and Yi Lu

25 Liposome-Enhanced Lateral-Flow Assays for Clinical Analyses 407Katie A Edwards, Ricki Korff, and Antje J Baeumner

26 Development of Dual Quantitative Lateral Flow Immunoassay

for the Detection of Mycotoxins 435Yuan-Kai Wang, Ya-Xian Yan, and Jian-He Sun

Index 449

University, Ithaca, NY, USA; Institute for Analytical Chemistry, Chemo- and Biosensors,University of Regensburg, Regensburg, Germany

USA

HUGHA BRUCK  University of Maryland College Park (UMCP), College Park, MD, USA

GO, Brazil

Corporation, Corning Incorporated, Corning, NY, USA; Department of BiomedicalEngineering, Cornell University, Ithaca, NY, USA

Academy of Science, Chengdu, China

GO, Brazil; Instituto Nacional de Cieˆncia e Tecnologia de Bioanalı´tica (INCTBio),Campinas, SP, Brazil

MA, USA

SORINDAVID  International Centre of Biodynamics, Bucharest, Romania

JOHNR DAY  Illumina, Inc., San Diego, CA, USA

University of Toronto Mississauga, Mississauga, ON, Canada

FENGDU  Natural Products Research Center, Chengdu Institution of Biology, ChineseAcademy of Science, Chengdu, China

University, Ithaca, NY, USA

YEFANG  Biochemical Technologies, Corning Research and Development Corporation,Corning Incorporated, Corning, NY, USA

Corporation, Corning Incorporated, Corning, NY, USA; NanoScience Technology Center,Department of Mechanical, Materials and Aerospace Engineering, University of CentralFlorida, Orlando, FL, USA

Goiaˆnia, GO, Brazil

Brazil

xix

MARSHAA GASTON  Department of Molecular Genetics, Biochemistry and Microbiology,University of Cincinnati, Cincinnati, OH, USA

University of Tuebingen, Tuebingen, Germany

Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, The BarcelonaInstitute of Science and Technology, Bellaterra, Barcelona, Spain

of Bucharest, Bucharest, Romania

Photonics, The Chinese University of Hong Kong, N.T Hong Kong SAR, China

MD, USA

YIHAN  Chemical Sensors Group, Department of Chemical and Physical Sciences,

University of Toronto Mississauga, Mississauga, ON, Canada

Institute of Nanoscience and Nanotechnology (ICN2), CSIC, The Barcelona Institute

of Science and Technology, Bellaterra, Barcelona, Spain

HOPUIHO  Department of Electronic Engineering, Center for Advanced Research inPhotonics, The Chinese University of Hong Kong, N.T Hong Kong SAR, China

FULYAEKIZKANIK  Electrical and Computer Engineering Department, Boston University,Boston, MA, USA

Seoul, Republic of Korea

SIUKAIKONG  Department of Electronic Engineering, Center for Advanced Research inPhotonics, The Chinese University of Hong Kong, N.T Hong Kong SAR, China

RICKIKORFF  Department of Biological and Environmental Engineering, Cornell

University, Ithaca, NY, USA

and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada

TIANLAN  Glucosentient Inc., Champaign, IL, USA

Institute of Nanoscience and Nanotechnology (ICN2), CSIC, The Barcelona

Institute of Science and Technology, Bellaterra, Barcelona, Spain; CIBER-BBN,

Campus UAB, Ed-ICN2, Bellaterra, Barcelona, Spain

Engineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

MEILI  Natural Products Research Center, Chengdu Institution of Biology, ChineseAcademy of Science, Chengdu, China

YONGBINLIN  Center for Applied Optics, University of Alabama at Huntsville,

Huntsville, AL, USA

Engineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

FONGCHUENLOO  Department of Electronic Engineering, Center for Advanced Research

in Photonics, The Chinese University of Hong Kong, N.T Hong

Kong SAR, China

YANLILU  Biosensor National Special Laboratory, Key Laboratory for Biomedical

Engineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

YILU  Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana,

IL, USA

JOHNH.T LUONG  Innovative Chromatography Group, Irish Separation Science Cluster(ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility(ABCRF), University College Cork, Cork, Ireland

Ulm, Germany

ZHONGMEI  Department of Biomedical Engineering, University of Texas at San Antonio,San Antonio, TX, USA

of Toyama, Toyama, Japan; Graduate School of Innovative Life Sciences for Education,University of Toyama, Toyama, Japan; Institutes for Analytical Chemistry, Chemo- andBiosensor, University of Regensburg, Regensburg, Germany; Institute of BioPhysio SensorTechnology (IBST), Pusan National University, Busan, South Korea

M OMAIRNOOR  Chemical Sensors Group, Department of Chemical and Physical Sciences,University of Toronto Mississauga, Mississauga, ON, Canada

Republic of Korea

USA

University, College Station, TX, USA

Tuebingen, Germany; Biametrics GmbH, Tuebingen, Germany

Tuebingen, Germany; Biametrics GmbH, Tuebingen, Germany

U.S Department of Agriculture, Albany, CA, USA

MD, USA

CLOTILDERIBAUT  B-SENS, Mons, Belgium

Ankara, Turkey

of Toyama, Toyama, Japan; Graduate School of Innovative Life Sciences for Education,University of Toyama, Toyama, Japan

Republic of Korea

JOONMYONGSONG  College of Pharmacy, Seoul National University, Seoul, South Korea

Applications, Department of Microsystems Engineering—IMTEK, University of Freiburg,Freiburg, Germany

University, College Station, TX, USA; Department of Biomedical Engineering, TexasA&M University, College Station, TX, USA

USA; Faculty of Medicine, Bahcesehir University, Istanbul, Turkey

M SELIMU€NL €U  Biomedical Engineering Department, Boston University, Boston, MA,USA; Electrical and Computer Engineering Department, Boston University, Boston,

MA, USA; Microbiology Department, Boston University School of Medicine, BostonUniversity, Boston, MA, USA

Applications, Department of Microsystems Engineering—IMTEK, University of Freiburg,Freiburg, Germany; Immunodiagnostics Systems, Liege, Belgium

A.G VENKATESH  Department of Electrical and Computer Engineering, Jacobs School ofEngineering, University of California San Diego, San Diego, CA, USA

PENGWANG  Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA

San Antonio, TX, USA

YUAN-KAIWANG  Key Laboratory of Veterinary Biotechnology, School of Agriculture andBiology, Shanghai Jiao Tong University, Shanghai, China

Nanoengineering (JSNN), University of North Carolina at Greensboro, Greensboro,

NC, USA

University of Cincinnati, Cincinnati, OH, USA

SHUYUENWU  Department of Electronic Engineering, Center for Advanced Research inPhotonics, The Chinese University of Hong Kong, N.T Hong Kong SAR, China

YA-XIANYAN  Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology,Shanghai Jiao Tong University, Shanghai, China

LIYANG  Natural Products Research Center, Chengdu Institution of Biology, ChineseAcademy of Science, Chengdu, China

YAOYAO  Biosensor National Special Laboratory, Key Laboratory for Biomedical

Engineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

Chinese Academy of Science, Chengdu, China

KEN-TYEYONG  Department of Electronic Engineering, Center for Advanced Research inPhotonics, The Chinese University of Hong Kong, N.T Hong Kong SAR, China

ZHENGZENG  Department of Nanoscience, Joint School of Nanoscience and

Nanoengineering (JSNN), University of North Carolina at Greensboro, Greensboro,

NC, USA

Applications, Department of Microsystems Engineering—IMTEK, University of Freiburg,Freiburg, Germany

QIANZHANG  Biosensor National Special Laboratory, Key Laboratory for BiomedicalEngineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

PENGZHANG  Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA

Engineering of Education Ministry, Department of Biomedical Engineering, ZhejiangUniversity, Hangzhou, China

Localized Surface Plasmon Resonance (LSPR)-Coupled

Fiber-Optic Nanoprobe for the Detection of Protein

Key words Fiber optics, Protein biomarker biosensors, Nanofabrication, Au nanodisk array, Localized surface plasmon resonance (LSPR), Signal transduction

1 Introduction

Recent advances of biomarker detection have been made in opticalfluorescence [1], light scattering [2], surface enhanced Ramanspectroscopy (SERS) [3], electrochemical [4], functional quartzcrystal microbalance [5], microcantilevers [6], and surface plasmonresonance (SPR) imaging [7] sensors Harnessing the advances inbiological ligand interactions, the fiber-optic (FO) technologyincorporating localized surface plasmon resonance (LSPR) nanop-robe sensing may provide an alternative tool for effective biomarkerdiagnosis via a compact, label-free format that does not require a

Avraham Rasooly and Ben Prickril (eds.), Biosensors and Biodetection: Methods and Protocols Volume 1:

Optical-Based Detectors, Methods in Molecular Biology, vol 1571, DOI 10.1007/978-1-4939-6848-0_1,

© Springer Science+Business Media LLC 2017

1

dedicated laboratory facility or highly trained personnel more, there is a need to develop advanced LSPR-FO biosensorsthat may avoid the use of bulky optics and high-precision mechanicsfor angular or wavelength interrogation of metal films in contactwith analytes, and provide high-performance, e.g., enhanced stabil-ity and high RI sensitivity, and overcome unwanted doping or weakadhesion.

Further-Surface plasmon resonance (SPR) is the resonance oscillation ofconduction electrons at the interface between a negative and apositive permittivity material excited by an electromagnetic radia-tion, e.g., light The surface plasmon polaritons (SPPs) launchedupon the radiation can be propagating along the metal-dielectricinterface and decay evanescently at the normal direction for a flatsurface Surface plasmons [8] (SPs) are very sensitive to the nearsurface refractive index (RI) changes and well suited to the detec-tion of surface-binding events The basic methodology of SPRsensing is based on the Kretschmann configuration (Fig.1) where

a prism is used for the light-SP coupling at the surface of a thinmetal film The probe light undergoes total internal reflection onthe inner surface of the prism At a defined SPR angle, an evanes-cent light field travels through the thin gold film and excites SPs atthe metal-dielectric interface, reducing the intensity of the reflectedlight at the resonance wavelength or changing the phase of theincident light The intensities of scattered and transmitted lightfields are used to determine the thickness and/or dielectric con-stant of the coating [9] The control variables for SPR sensorapplications, i.e., the wavelength of incident light, the thickness ofthe metal film, the physical and optical properties of the prism, andthe RI of the medium near the metallic interface have been wellstudied [10] However, the advantages of averaging over a largesurface area and the challenges of miniaturizing the optics limit theintegration of SPR-based sensing

Fig 1 A conventional surface plasmon resonance (SPR) configuration and setupfor biological sensing

LSPR is caused by resonant surface plasmons localized in scale systems when light interacts with particles much smaller thanthe incident wavelength (Fig.2a) Similar to the SPR, the LSPR issensitive to changes in the local dielectric environment near thenanoparticle surfaces Usually, the sense changes are measured inthe local environment through an LSPR wavelength shift of thescattering light via reflection or transmission (Fig.2b) NanoscaleLSPR makes possible for the development of a portable device forpoint-of-care (POC) detection regarding its requirements, such asrobust to handle, small volume sample, and little to no samplepretreatment, label-free and rapid response time, and compact size.Incorporation of the LSPR to fiber optics (FO) offers a fewadvantages in terms of avoiding the use of bulky optics and high-precision mechanics for angular or wavelength interrogation of

nano-Fig 2 Schematic diagrams illustrating (a) a localized surface plasmon [18], (b) aconfiguration of representative experimental setup and procedure for LSPRsensing [19]

metal films in contact with analytes, including immobilization of Au

or Ag nanoparticles (NPs) to optical fiber probes for LSPR tion [11,12] It may allow realizing an optical communication andanalytical tool for a wide spectrum of applications However, morecontrollable and stable LSPR nanoscale systems for fiber opticintegration are desirable to develop robust, reliable, portableLSPR biosensors

detec-In this work, the progress of developing a miniaturized

LSPR-FO probe and demonstration of sensitive, label-free detection of aprotein cancer biomarker, free prostate-specific antigen (f-PSA) arereported, which involves three major steps: (1) a low-cost lift-offprocess adapted to fabricate gold nanodisk arrays at the end of tip-facet, providing a very stable, robust, and clean LSPR fiber tipprobe, (2) the probe was functionalized via a facile self-assembledmonolayer (SAM) of alkanethiolates on the gold nanodisk array toattach a capture ligand, anti-PSA antibody, as a selective immuno-assay for the detection of the f-PSA, (3) the FO-based sensingtechnology was used as a powerful analytical tool by integratingthe LSPR nanoprobe to communicative fiber optics The sensingprinciple and configuration of the LSPR nanoprobe is shown inFig.3 The white light guided in the optical fiber using as incidentlight to the gold nanodisk arrays at the end of the fiber tip surfacefor excitation of the LSPR The reflectance of the light scatteringfrom the nanodisk arrays was recorded before and after thebiological binding The correlation of the changes of reflectancespectra to the binding of the analytes to the nanodisk arrays,corresponding to the analyte concentrations in samples, was estab-lished for detection The reported label-free LSPR fiber biosensormay allow an alternative approach for direct discrimination of the

140 120 100 80 60 40 20 0

Au Nanodisk

Optical Fiber Tip

LSPR Reflectance

White Light

Core

Cladding

Fig 3 A diagram illustrating the principle of LSPR coupling on fiber optic probe for biosensing The nanodiskarrays are fabricated on optical fiber tip end The LSPR reflectance is recorded with a white incident lightguided in the fiber

cancer biomarkers, and potentially developing a miniaturized,point-of-care (POC) device for early disease diagnosis.

This work suggests that: (1) as an emerging technology, theLSPR-FO sensor has ultra-high sensitivity in molecular adsorptiondetection; (2) the LSPR Au nano-array fabricated at the end facet ofthe fiber tip for sensing is very robust and reusable; (3) the primaryresonant wavelength can be tuned to a desired range (e.g., NIR) bytailoring the nano-array structure to enhance the sensitivity; (4)tailored surface functionalization harnessing the advances inbiological ligand interactions (e.g., immunoassays) enables signalamplification and a label-free, selective detection; and (5) the targetmolecules are immobilized by dipping the fiber-optic probe insample/reagent solution, contrary to pouring of sample solution

in conventional methods (ELISA, Electrochemiluminscence,Radioimmunoassay-RIA), resulting in drastic reduction of theamount of sample/reagents needed and decreases the washingtime of probes

2 Materials and Equipment

1 Single-mode optical fiber for 633 nm wavelength was chased from Newport Corporation

pur-2 Electron beam resist (ZEP-520A), thinner (ZEP-A), developer(ZED-N50), resist remover (ZEDMAC) were purchased fromZEON Corporation, Japan, and used without furtherpurification

8-Mercapto-1-Octanol (HSC8OH, 98%), minopropyl)-N0-ethylcarbodiimide hydrochloride (EDC),N-Hydroxysuccinimide (NHS), and glycine were purchasedfrom Sigma-Aldrich (Milwaukee, WI) and used withoutfurther purification

N-(3-Dimethyla-4 Mouse anti-human PSA monoclonal antibody (capture mAb),human free-PSA, and ELISA kits for CA125 were obtainedfrom Anogen-YES Biotech Laboratories Ltd (Mississauga,Canada)

5 The 5 ng/mL free-PSA standard solution was used for ration of free-PSA solutions with lower concentrationsobtained using sample dilutant provided in the ELISA kit.The 5 ng/mL free-PSA standard solution was prepared in aprotein matrix solution according to the World Health Orga-nization (WHO) standard [13] by the vendor

prepa-6 Chrome etchant was obtained from Microchem GmbH,Germany

7 The fiber clamp for hoisting fiber for vibration was obtainedfrom Newport Corporation, CA, USA.

8 Ultra clean convection oven (Ultra-clean 100) was obtainedfrom Thermo Fisher Scientific, OH, USA

9 Nano pattern generation system (NPGS) was obtained from JCNabity Lithography Systems, MT, USA

10 Field emission scanning electron microscope (FESEM) was aLEO 1550 model

11 Three cathode vacuum sputter system (Denton Discovery-18Sputter System) was obtained from Denton Vacuum LLC, NJ,USA

12 Vacuum thermal evaporate system was donated to University

of Alabama in Huntsville by US Army

13 Reactive Ion Etching (RIE) system (Plasma-Therm 790) wasobtained from Plasma-Therm, FL, USA

14 Optical fiber coupled Tungsten Halogen light source (LS-1)was obtained from Ocean Optics, FL, USA

15 Mini-spectrometer (USB2000-VIS-NIR) was obtained fromOcean Optics, FL, USA

16 2 2 Single-mode fiber optic couplers for 633 nm wavelengthwere obtained from Newport Corporation, CA, USA

Au nanostructures on fiber end face, as schematically illustrated

in Fig.4 There are four main technological steps: (1) tion of positive electron beam resist (ZEP520A, Zeon Chemi-cals, Japan) on the fiber end face with uniform thickness(Fig 4a–c), (2) nano-patterning on the E-beam resist usingEBL method (Fig.4d), (3) vacuum deposition of functionalmetallic materials over the e-beam resist using cleanroom

deposi-Fig 4 A schematic flow-chart displays the fabrication procedure for nanodisk arrays at the optical fiber endfacet (a) optical fiber tip, (b) 2 nm Cr deposition, (c) resist deposition, (d) EBL process, (e) gold film deposition,(f) lift-off process, (g) Cr etch to get the nanodisk arrays

thermal evaporation (Fig 4e), and (4) nano-pattern transferusing standard liftoff method (Fig.4f, g).

2 An optical fiber for single-mode wavelength of 633 nm isemployed in this work, which has a core diameter of 4μm, acladding diameter of 125 μm, and a polymer buffer coatingdiameter of 250μm (Fig 4a) Compared to standard opticalfiber operated at single-mode wavelength of 1310 nm, theadvantage of using this small core fiber is that it providesmore spectral stability in the wavelength ranges of600–750 nm, in which locate the resonance peak of our fabri-cated fiber tip LSPR sensors Small core size also means higherfabrication yield and less nanoparticles involved in the sensing,thus requires smaller amount of target samples

3 The preparation of optical fiber tip includes stripping off thepolymer buffer layer 4 cm from the end and cleaving the endface with a fiber cleaver, followed by cleaning with acetone andisopropyl alcohol (IPA) rinse for 5 min

4 Two nanometers of Cr film is firstly deposited on the fiber end faceusing the vacuum sputtering method [16] to provide a conductivelayer for the e-beam resist in the EBL process (Fig.4b)

5 A simple and unique wet resist coating method called “dip andvibration” technique has been developed in a nanofabrica-tion lab to deposit e-beam resist on the optical fiber tip(Fig 4c), and its procedure is schematically represented inFig.5 The optical fiber is dipped into the diluted e-beam resist(ZEP520A diluted with ZEP thinner at a ratio of 1:3) for 10 s(Fig.5a) Then, it is removed from the resist and hoisted into avertically upward position using a Newport fiber clamp, with

~15 mm of fiber tip outside the clamp at the top (Fig.5b, c)

Fig 5 Illustrations of the procedure called “dip and vibration” technique, (a) dip in the diluted e-beam resist,(b) after dip, (c) vibration, and (d) after bake

The fiber tip is then vibrated manually by pulling the fiber tip toone side and then releasing to get rid of excessive resist bymeans of cantilever beam free vibration The vibration fre-quency and strength is controlled by the length of fiber tipoutside the fiber clamp and the initial displacement of the fibertip The thickness of resist on the optical fiber tip is dependent

on the ZEP dilution ratio and vibration strength The iterativemethod is used to optimize the vibration process The initialfiber tip displacement for the vibration is 2–5 mm, and the fibertip length is 15–25 mm The ZEP dilution ratio used is from20% to 40%, diluted in ZEP thinner The resulted e-beam resistlayer thickness on the fiber tip is 100–200 nm, measured bySEM observation The fiber tip is held vertically upward andbaked in a 120C oven for 60 min (Fig.5d)

6 The EBL process based on the Nano Pattern Generation tem (NPGS) and a field emission scanning electron microscope(FESEM) is used to create nanodot array pattern on the e-beamresist on the fiber end face (Fig.4d) A Newport fiber clamp isused to hold the fiber vertically on the translation stage in theSEM chamber The EBL voltage is 30 kV and the exposuredose is 70 μC/cm2

Sys- The fiber tip is developed by dipping inresist developer (ZEP N50) for 1 min The fiber tip is thenrinsed in DI water and baked in the 120C oven for 10 min fordehydration

7 An oxygen plasma descum procedure in a reactive ion etcher(RIE, 25 watts power for 3 min) is used to remove the thinresidual layers of photoresist following the photoresist devel-opment step (see Note 1)

8 The deposition of 40 nm Au overlay over the patterned area iscarried out by using the standard thermal evaporation coatingtechnique (Fig.4e) The 2 nm Cr layer previously deposited as

a conductive layer for the EBL process is now served as anadhesive layer for Au overlay To lift off the e-beam resist, thefiber tip is dipped in the ZEP remover for 10 min, followed by a1-min ultrasonic bath to assist the lift-off process (Fig.4f) Thefiber tip is rinsed in DI water and checked under an opticalmicroscope to make sure that the resist layer on the fiber endface has been removed (see Note 2)

9 The fiber tip is dipped into the Cr remover solution for 30 s toremove the Cr layer that is not covered by the Au nanoparticles(Fig.4g) The fiber tip is rinsed again in DI water and baked inthe 120C oven for 10 min for dehydration Figure6showsthe scanning electron micrographs of a gold nanodisks array on

an optical fiber tip end facet

10 Finally, the Au nanodisk array on fiber end face is annealed at

530C for 5 min and ready for next usage (see Note 3)

3.2 Optical

Measurement

1 In this optical setup (Fig.7), a 2 1 single-mode optical fibercoupler for 633 nm wavelength is used, which has three lightconnection ports In Fig 7, port (a) and port (b) are twoconnections on one side of optical fiber coupler, and port (c)

is connection on the opposite side Port (a) is connected to thetungsten halogen white light source; port (b) is connected to amini-spectrometer; and port (c) is connected to the LSPR-FOprobe using a fusion splicer The white light (450–950 nm) islaunched from port (a), propagated to port (c), and reflectedfrom the end facet with the Au nanodisks arrays The reflectionlight propagated to port (b), where the spectrum of thereflection light is measured by the mini-spectrometer, which

is connected to a computer for data acquisition and processing.Figure 8 shows the photograph of the Optical setup forthe fiber tip LSPR sensor based on reflection spectrummeasurement

2 During fiber optic LSPR detection, the light is launched andguided to the LSPR fiber tip facet The light wave propagatesalong the core in the center of the optical fiber tip where the

Fig 6 Images of (a) overview of the optical fiber tip end, (b) SEM images of gold nanodisk arrays on the opticalfiber facet after the lift-off process

Fig 7 A schematic diagram illustrating optical setup for the fiber tip LSPR sensor based on reflection spectrummeasurement

nanodisk array is located The guided white light interacts withthe Au nanodisk array and excites the localized surface plasmongiving raise to enhanced scattering around the resonance wave-length The LSPR wavelength strongly depends on interfacerefractive properties [16] The light reflected from the LSPRinterface is guided in the fiber and propagated to the mini-spectrometer, and the spectrum of the reflected light isrecorded as a function of wavelength The detection of RIchange at the interface is accomplished by observing the pri-mary resonance peak shift in the spectrum.

3 Before starting the experiment, the sensor tip must be cleaned

of any impurities or other contaminants The sensor tip isrinsed with ethanol to clean the tip A baseline wavelength isachieved if the sensor tip returns to this wavelength three timesafter being washed in deionized water If the tip spectra do notreturn to this line, acetone and isopropyl alcohol (IPA) dip for

5 min may be used to further clean the tip of lingeringimpurities

4 In order to get the LSPR spectrum response due to Au tructures on the fiber end facet, a reference spectrum and a darkspectrum need to be recorded The reference spectrum isacquired without fiber LSPR probe on port (c), and the fiberend face of port (c) is perpendicularly cleaved The dark spec-trum is obtained by turning off both the tungsten halogen lightsource and room light The measured reflection spectra (Mλ) ofthe fiber tip sensor probe is obtained by the equation:

nanos-Mλ¼ (Sλ Dλ)/(Rλ Dλ) 100 % ,whereSλis the sample intensity,Dλis the dark intensity, andRλ

is the reference intensity at wavelengthλ (the intensity defines

as photon counts)

Fig 8 Photograph of the optical components (optical fiber, fiber coupler, spectrometer, light source) and setup for the fiber tip LSPR sensor Computerconnection to the mini spectrometer via USB cable is not shown

mini-3.3 LSPR-FO

Nanoprobe Sensitivity

Characterization

1 The nanoprobe sensitivity is used to determine if the sensor tip

in experimentation would be sensitive enough to determinesmall wavelength shifts accurately

2 The solvents used in determining the bulk RI sensitivity of theLSPR tip are acetone, methanol, ethanol, isopropyl alcohol,and water of different RIs

3 The LSPR-FO nanoprobe is dipped in the various solvents, andthe spectra of the reflected light were recorded, respectively, asshown in Fig.9a(see Note 4)

4 To determine the peak wavelength in the LSPR reflectionspectrum, a Matlab program was created to fit the data to aneighth-order polynomial function over a range of 80 nm [17],centered at the wavelength of maximum reflection in the rawdata

5 The calculated LSPR wavelength red shifted as the RI of thesolvent increased A linear relationship between the LSPR peakwavelength and the bulk RI of the medium is obtained (Fig.9b

black dots), with the sensitivity of 226 nm/RIU (RIU, tive index unit) used in this study

refrac-6 The light intensity-based RI sensitivity is investigated as well(Fig 9b red diamonds) The return light intensities at theLSPR peak wavelength are plotted against the bulk RI of themedia The LSPR peak intensity is seen to be linearly propor-tional to the RI, and the gradient of the line is 123 per RIU.The return light intensity of a single-wavelength laser can beused as a probing light for the refractive index change due to

Fig 9 Sensitivity measurements of the LSPR-FO nanoprobe (a) Measured reflectance spectra for the LSPR-FOprobe in various solvents of different refractive index (b) Correlation of the LSPR peak wavelengths (left axis)and LSPR peak intensity (right axis) with the refractive index (RI) of the solvents, the linear fit of the changes

vs the RI gives the sensing sensitivity In the inset equation, x and y represent the values of x and y axis R2isthe coefficient of determination in the linear fitting

biochemistry binding events, and thus eliminate the need for abroadband light source and optical spectrometer for an LSPRbiosensing.

at the gold nanodisck surfaces

2 The self-assembled monolayer (SAM) tethered with carboxylicacid groups then is activated by incubation in a pH 7.0, 10 mMphosphate buffer solution containing 0.5 mM of EDC/NHS,respectively, for 1 h

3 The activated SAM is rinsed with the distilled water and diately moved to a freshly prepared 10 mM PBS containing

imme-10μg/mL of the detector mAb for 1 h incubation

4 The fiber probe is finally rinsed with the PBS and followed bydipping in a 0.2 M glycine PBS solution for 10 min to deacti-vate the remaining active sites at the SAM

3.5 Detection of

f-PSA Biomarker

1 During the experiments, PBS (containing 0.05% tween-20) isused as a running buffer to help minimize the nonspecificadsorption of f-PSA at the fiber tips

2 In order to test binding of PSA to the anti-PSA mAb modifiedsurfaces, five tenfold-dilutions of a 5 ng/mL concentration off-PSA are prepared in PBS solution for the desired concentra-tions (i.e., 5 ng/mL, 0.5 ng/mL, 0.05 ng/mL, 5 pg/mL,0.5 pg/mL, 0.05 pg/mL) The sensor tip is placed in thef-PSA PBS for 10 min in the sequence at the lowest concentra-tion of 50 fg/mL to measure the reflectance spectrum

3 After each measurement, the fiber tip is rinsed thoroughly with

DI water and dried in air All spectra are obtained after the fibertip was cleaned and dried in air Figure10ashows the represen-tative reflectance spectra of f-PSA detection sensing variousf-PSA concentrations from 0 to 0.5 ng/mL

4 Figure10bshows the dependence of the wavelength peak shift

on the f-PSA concentration The peak shift for each point isobtained by averaging three measurements A wavelength shift

of twice the largest standard deviation (1.2 nm) is used todetermine LOD of the fiber LSPR sensors, which corresponds

to 100 fg/mL of f-PSA in PBS solution

5 Control experiments have been designed and carried out toevaluate the specificity/selectivity of the immunoassay usingthe LSPR-FO devices Specifically, the binding between thedetector mAb and bovine serum albumin (BSA), similar to

human serum albumin, at concentrations of 5 mg/mL hasbeen evaluated Additionally, to evaluate the nonspecific bind-ing to the SAM, the LSPR-FO devices modified only with themix SAM of HSC10COOH and HSC8OH SAM withoutantibody attached are tested by following the f-PSA detectionprocess, as shown in Fig.10b(blue diamond markers).

1 The optional step after the oxygen descum is to dip the fiber tip

in the chrome remover solution for 60 s to remove the thin Crlayer on the nanodisk area This step will eliminate the Cradhesion layer under the Au nanodisk, avoiding the undesiredside effects of Cr layer under Au nanodisk in the LSPR sensingprocess, such as spectrum broadening and dephasing How-ever, this step will weaken the adhesion strength between the

Au nanodisks and the fiber end glass substrate, and will reducethe reusability of the fiber LSPR probe

2 If the lift-off process is not complete, ZEP remover and sonic bath may be used to further lift off the e-beam resist

ultra-3 The fiber probe is inserted horizontally into the oven through

an access hole on the sidewall of a high-temperature oven Inorder to protect the polymer buffer on the fiber, only10–15 mm of fiber probe needs to be inside the oven

4 Note that there is a noisy region in all spectra from ~760 to

800 nm, which is caused by the transmission minimum for theoptical fiber coupler (single mode at 633 nm) in the samewavelength range

Fig 10 Detection of protein biomarker (a) The representative reflectance spectra of the anti-PSA mAbmodified LSPR-FO nanoprobe in various concentrations of f-PSA (from 0 up to 0.5 ng/mL) Insert plot showsspectra normalized to peak intensity to show the LSPR reflectance peak shifts (b) Correlation of f-PSAconcentration to the reflectance light of resonance peak wavelength shift

1 Mukundan H et al (2009) Optimizing a

wave-guide-based sandwich immunoassay for tumor

biomarkers: evaluating fluorescent labels and

functional surfaces Bioconjug Chem 20

(2):222–230

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immunoassay for cancer biomarker detection

using gold nanoparticle probes coupled with

dynamic light scattering J Am Chem Soc 130

(9):2780–2782

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Metal-decorated silica nanowires: an active

sur-face-enhanced raman substrate for cancer

bio-marker detection J Phys Chem C 112

(6):1729–1734

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immunosen-sor for cancer biomarker proteins using gold

nanoparticle film electrodes and

multienzyme-particle amplification ACS Nano 3

(3):585–594

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binding protein with enhanced sensitivity

using a functionalized quartz crystal

microbal-ance sensor Anal Chem 78(14):4880–4884

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Label-free sugar detection using

phenylboro-nic acid-functionalized piezoresistive

micro-cantilevers Anal Chem 80(13):4860–4865

7 Lee HJ, Nedelkov D, Corn RM (2006) Surface

plasmon resonance imaging measurements of

antibody arrays for the multiplexed detection

of low molecular weight protein biomarkers.

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smooth and rough surfaces and on gratings.

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interpreta-11 Mitsui K, Handa Y, Kajikawa K (2004) Optical fiber affinity biosensor based on localized sur- face plasmon resonance Appl Phys Lett 85 (18):4231–4233

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19 Zhao J et al (2006) Localized surface plasmon resonance biosensors Nanomedicine 1 (2):219–228

Ultra-Sensitive Surface Plasmon Resonance Detection

by Colocalized 3D Plasmonic Nanogap Arrays

Wonju Lee, Taehwang Son, Changhun Lee, Yongjin Oh,

and Donghyun Kim

Abstract

Ultra-sensitive detection based on surface plasmon resonance (SPR) was investigated using 3D nanogap arrays for colocalization of target molecular distribution and localized plasmon wave in the near-field Colocalization was performed by oblique deposition of a dielectric mask layer to create nanogap at the side

of circular and triangular nanoaperture, where fields localized by surface plasmon localization coincide with the spatial distribution of target molecular interactions The feasibility of ultra-sensitivity was experimen- tally verified by measuring DNA hybridization Triangular nanopattern provided an optimum to achieve highly amplified angular shifts and led to enhanced detection sensitivity on the order of 1 fg/mm2in terms

of molecular binding capacity We confirmed improvement of SPR sensitivity by three orders of magnitude, compared with conventional SPR sensors, using 3D plasmonic nanogap arrays.

Key words Surface plasmon resonance, Localized surface plasmon resonance, Surface plasmon nance detection, Nanogap arrays, Nanoapertures, Colocalization, DNA hybridization

reso-1 Introduction

In recent years, diverse optical techniques have attracted dous interests for ultra-sensitive real-time detection of variousphenomena involving biomolecular interactions Most of thesetechniques have been based on fluorescence Fluorescence-basedsensing, however, suffers from fluorescence interference or chemo-toxicity issues In contrast, surface plasmon resonance (SPR) bio-sensing has been widely investigated as a representative label-freetechnique, by which specific molecular interactions can be moni-tored in real time and kinetic characteristics related to molecularbinding are conveniently measured on a quantitative basis

tremen-Surface plasmon (SP) is a collective logitudinal oscillation ofelectrons existing at the metal/dielectric interface The oscillation

of electron can be coupled with a TM-polarized incident light

Avraham Rasooly and Ben Prickril (eds.), Biosensors and Biodetection: Methods and Protocols Volume 1:

Optical-Based Detectors, Methods in Molecular Biology, vol 1571, DOI 10.1007/978-1-4939-6848-0_2,

© Springer Science+Business Media LLC 2017

15

By tuning angle of incidence or incident wavelength, the tum matching condition between incident light and SP can besatisfied At resonance, due to energy transfer from incident light

momen-to a longitudinal surface wave, a narrow dip in the reflection acteristics is observed with respect to wavelength or angle of inci-dence A small change of dielectric medium refractive indexinduced from a biochemical interaction affects momentum match-ing condition, followed by the shift of resonance dip in the reflec-tion characteristics [1]

char-Despite broad uses of SPR sensors, conventional SPR sensorshave relatively poor detection limit on the order of 1 pg/mm2inbinding capacity [2] As an optical sensing technique other thanSPR, metal nanoparticle-based surface-enhanced Raman scatteringand microresonator-based whispering gallery modes have emergedfor ultra-sensitive label-free detection [3,4]

In this chapter, we describe self-aligned colocalization usingthree-dimensional plasmonic nanogap arrays for ultra-sensitive SPRbiosensors [5] Note that colocalization indicates the spatial overlapbetween the area of localized electromagnetic field and molecularinteraction Colocalization is preferred in terms of sensitivityenhancement per molecule, because much smaller number of mole-cules are involved at the interaction In previous studies, use ofplasmonic nanostructures was investigated to localize SP and eva-nescent near-field to enhance detection sensitivity [6,7] Randomand periodic nanostructure was also employed for specific andnonspecific detection based on enhanced surface plasmon reso-nance [8,9] It was also shown that when biomolecules are spatiallyaligned for colocalization with the localized field that is defined by2D linear nanopattern arrays, SPR signals can be effectively ampli-fied, which leads to efficient improvement of detection sensitivity[10–12] Moreover, theoretical investigation of nonspecific, non-colocalized, and colocalized detection models was performedbased on silver nanoislands, which confirmed possibility of furtheramplification of optical signature [13] Meanwhile, SPR sensorcharacteristics using grapheme-related materials were also studied[14,15]

Here, we describe use of 3D plasmonic nanogap arrays, strated in a schematic diagram of Fig.1, for colocalized detection ofbio-interactions of target molecules to achieve even more dramaticenhancement of sensitivity in SPR biosensors In particular, circularand triangular nanoholes were developed lithographically and 3Dnanogaps were then formed for colocalization by the shadowing ofobliquely evaporated dielectric mask layer on the metallic nanos-tructures This enables target molecules to directly access theunderlying metal film Oblique evaporation was previously used

illu-to fabricate molecular electronic devices based on nanoscale gapstructures [16] Since localized near-field is formed at the ridge ofthe nanostructure, localized fields and nanogap where target

molecules can bind are self-aligned for colocalization 3D nic nanostructures induce colocalization area to be much smallerthan what 2D linear grating patterns may produce Therefore,detection of much weaker interactions and/or those involve asmaller number of molecules would be feasible, which allowsextreme sensitivity enhancement in SPR sensing.

2 Electron beam (VEGA II LSH; TESCAN, Brno, Czech)

3 Scanning electron microscope (Elphy Quantum; Raith, mund, Germany)

Dort-4 A polymethylmethacrylate (PMMA) photoresist (AR-N7520.18; ALLRESIST, Strausberg, Germany)

5 Spin-coater (ACE-200; DONG AH Trade Corp, Seoul, Korea)

6 A dielectric mask layer using ITO or SiO2

7 Developer (AR 300-47; ALLRESIST, Strausberg, Germany)

8 Remover (AR 300-70; ALLRESIST, Strausberg, Germany)

Fig 1 (a) Schematic illustration of 3D triangular nanogap aperture arrays thatmay be produced by oblique evaporation for colocalized biomolecularinteraction (b) Lateral profile across the solid line (A–B) in (a)

2.2 Optical Setup A schematic illustration of the optical setup for colocalized SPR

detection is presented in Fig.2(see Note 1)

1 He-Ne laser (36 mW,λ ¼ 632.8 nm, Nominal beam diameter:

650μm; Melles-Griot, Carlsbad, CA)

2 Glan-Thompson Linear Polarizer (Thorlabs, Inc., Newton, NJ)

3 Chopper and controller (SR540; Stanford Research Systems,Sunnyvale, CA)

4 Two concentric motorized rotation stages (URS75PP andESP330; Newport, Irvine, CA) (see Note 2)

5 Low-noise lock-in amplifier (Model SR830; Stanford ResearchSystems, Sunnyvale, CA)

6 A p-i-n photodiode (818-UV; Newport, Irvine, CA)

7 Lab ViewLabVIEW (National Instruments, Austin, TX)

8 Index-matching gel (n ¼ 1.725; Cargille Laboratories, CedarGrove, NJ)

Fig 2 (a) Schematics of optical setup for a SPR sensor based on colocalizationusing plasmonic nanogap arrays and (b) photograph of the experimental setup

2.3 DNA Preparation 1 HPLC purified 24-mer sequence length capture probe and

target oligonucleotides (IDT, Coralville, IA)

2 The sequence of single-stranded probe DNA (p-DNA) was 50TTT TTT CGG TAT GCA TGC CAT GGC-3 modified withthiol at 50

-3 The sequence of single-stranded target DNA (t-DNA) was 50GCC ATG GCA TGC ATA CCG AAA AAA-30

-4 Plasma cleaner (Harrick Scientific Products, Pleasantville, NY)

5 Micropump (KD Scientific, Holliston, MA)

6 Acetone (1009-4404; DAEJUNG CHEMICAL & METALSCO., Siheung, Korea)

7 Phosphate Buffered Saline (PBS) buffer (pH ¼ 7.4, BP3991;Fisher Scientific, Pittsburgh, PA)

3 Metallic nanograting arrays were transferred by a lift-off processafter gold evaporation (see Fig.3a)

4 2D nanogap arrays were created on nanograting arrays byoblique evaporation of a dielectric mask layer using ITO orSiO2(see Fig.3b)

Fig 3 (a) SEM image of fabricated linear nanograting arrays and (b) magnified image of 2D nanogap arrays

To create 3D nanogap arrays, the overall fabrication process ispresented in Fig.4.

1 A 40-nm gold film was evaporated on an SF10 glass with a 2-nmchromium adhesion layer

2 Circular and triangular nanostructures with a 2-μm array periodand an aperture size of 600 nm were defined by electron beamlithography

3 20-nm-thick nanohole arrays were formed after a lift-off processthat involves e-beam lithography, evaporation of gold, andremoval of polymer resist

4 5-nm-thick ITO layer was obliquely deposited with the tion angle varied at 30, 45, and 60 to adjust the gap areadifferently (see Note 3)

diameter: 650 μm, Melles-Griot, Carlsbad, CA) is TM-polarized

by linear polarizer and temporally modulated by optical chopper(SR540; Stanford Research Systems, Sunnyvale, CA), which altersthe state of light on and off at a specific frequency The choppingfrequency is used as the reference input of a lock-in amplifiercomposed with usually set to 0.6 kHz lock-in amplifier and feed-back system Among the signals captured at a photodiode, noisesignal at frequency other than the chopping can be removed Twomotorized rotation stages (URS75PP; Newport, Irvine, CA) areemployed; one is for rotating a prism on which a plasmonic nanos-tructured sample is located, and the other is for rotating the pho-todiode to keep laser alignment to photodiode The angle of thestage on which the nanostructure sample located can be corrected

by using the calibration constant and the other stage with theFig 4 Fabrication procedure to implement 3D plasmonic nanogap arrays