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Chapter 1Surface Plasmon Resonance and Surface Plasmon Field-Enhanced Fluorescence Spectroscopy for Sensitive Detection of Tumor Markers Yusuke Arima, Yuji Teramura, Hiromi Takiguchi,

Series Editor

John M Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

Biosensors and Biodetection

Methods and Protocols Volume 503: Optical-Based Detectors

Edited by

Avraham Rasooly* and Keith E Herold †

*FDA Center for Devices and Radiological Health, Silver Spring, MD, USA

and

National Cancer Institute, Bethesda, MD, USA

† Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA

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

Avraham Rasooly Keith E Herold

FDA Center for Devices Fischell Department of Bioengineering

and Radiological Health University of Maryland

Silver Spring, MD College Park, MD

Library of Congress Control Number: 2008941063

© Humana Press, a part of Springer Science+Business Media, LLC 2009

All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science + Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.

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1 Biosensor Technologies

In recent years, many types of biosensors have been developed and used in a wide variety of analytical settings, including biomedical, environmental, research, and oth-ers 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, immunosystems, tissues, organelles, or whole cells to detect chemical com-

pounds usually by electrical, thermal, or optical signals” (1) Thus, almost all

biosen-sors are based on a two-component system: a biological recognition element (ligand) that facilitates specific binding to or biochemical reaction with a target, and a signal conversion unit (transducer) Although it is impossible to fully cover the fast-moving field of biosensing in one publication, this publication presents some of the many types of biosensors to give the reader a sense of the enormous potential for these devices

An early reference to the concept of a biosensor is from Dr Leland C Clark,

who worked on biosensors in the early 1960s (2) developing an “enzyme electrode”

for glucose concentration measurement with the enzyme glucose oxidase, a ment that is important in the diagnosis and treatment of disorders of carbohydrate metabolism in diabetes patients Still today, the most common biosensors used are for glucose analysis

measure-A large number of basic biosensors, all combining a biological recognition ment and a transducer, were subsequently developed Currently, the trend is toward more complex integrated multianalyte sensors capable of more comprehensive analy-ses Advances in electronics and microelectrical and mechanical systems (MEMS) have enabled the miniaturization of many biosensors and the newest generation biosensors include miniaturized multianalyte devices with high-throughput capabilities and more than 1,000 individually addressable sensor spots per square centimeter

ele-A useful categorization of biosensors is to divide them into two groups: direct ognition sensors, in which the biological interaction is directly measured, and indirect

rec-detection sensors, which rely on secondary elements for rec-detection Figure 1 shows a

schematic of the two groups of biosensors In each group, there are several types of transducers including optical, electrochemical, and mechanical For all of these tech-nologies, the recognition ligand plays a major role Although the most commonly used ligands are antibodies, other ligands are being developed including aptamers (protein-binding nucleic acids) and peptides

In the literature and in practice, there are numerous types of biosensors, and the choice of a suitable system for a particular application is complex, based on many fac-tors such as the nature of the application, the label molecule (if used), the sensitivity required, the number of channels (or area), cost, technical expertise, and the speed

of detection needed A primary purpose of this book is to provide more access to the

v

technical methods involved in using a variety of biosensors to facilitate such decision making.

Direct detection biosensors utilize direct measurement of the biological action Such detectors typically measure physical changes (e.g., changes in optical, mechanical, or electrical properties) induced by the biological interaction, and they

inter-do not require labeling (i.e., label free) for detection Direct biosensors can also be used in an indirect mode, typically to increase their sensitivity Direct detection sys-tems include optical-based systems (most common being surface plasmon resonance) and mechanical systems such as quartz crystal resonators

Indirect detection sensors rely on secondary elements (labels) for detection ples of such secondary elements are enzymes (e.g., alkaline phosphatase or glucose oxidase) and fluorescently tagged antibodies that enhance detection of a sandwich complex Unlike direct detectors, which directly measure changes induced by biologi-cal interactions and are “label free,” indirect detectors require a labeled molecule to bind to the target Most indirect sensors based on optical detection are designed to measure fluorescence The detection system can be based on a charge coupled device (CCD), photomultiplier tube (PMT), photodiode, or spectrometer Electrochemical transducers, which measure the oxidation or reduction of an electroactive compound

Exam-on the secExam-ondary ligand, are another commExam-on type of indirect detectiExam-on sensor eral types of electrochemical biosensors are in use including amperometric devices, which measure the electric current as a function of time while the electrode potential

Sev-is held constant

Ligands are recognition molecules that bind specifically with the target molecule

to be detected The most important characteristics for ligands are affinity and city Various types of ligands are used in biosensors Biosensors that use antibodies as recognition elements (immunosensors) are common because antibodies are highly specific, versatile, and bind strongly and stably to the antigen Several limitations of antibodies are long-term stability, and manufacturing costs, especially for multitarget biosensor applications where many ligands are needed

specifi-Fig 1 General schematic of biosensors: (a) direct detection biosensors where the recognition element is label free; (b) indirect detection biosensors using a “sandwich” assay where the analyte is detected by labeled molecule Direct detec- tion biosensors are simpler and faster but typically yield a higher limit of detection compared with indirect detection systems

Other types of ligands that show promise for high-throughput screening and chemical synthesis are aptamers and peptides Aptamers are protein-binding nucleic acids (DNA or RNA molecules) selected from random pools on the basis of their ability to bind other molecules with high affinity Peptides can be selected for affinity

to a target molecule by display methods (phage display and yeast display) However,

in general, the binding affinity of peptides is lower than the affinity of antibodies or aptamers

2 Biosensor Applications

Biosensors have several potential advantages over other methods of biodetection, especially increased assay speed and flexibility Rapid, essentially real-time analysis can provide immediate interactive information to users This speed of detection is an advantage in essentially all applications

Applications of biosensors include medical, environmental, public security, and food safety areas Medical applications include clinical, pharmaceutical and device manufacturing, and research Biosensor-based diagnostics might facilitate disease screening and improve the rates of earlier detection and attendant improved prog-nosis Such technology may be extremely useful for enhancing health care delivery in the community setting and to underserved populations Environmental applications include spill clean-up, monitoring, and regulatory instances Public safety applications include civil and military first responders as well as unattended monitoring Food safety applications include monitoring of food production, regulatory monitoring, and diagnosis of food poisoning Biosensors allow multitarget analyses, automation, and reduced costs of testing

The key strengths of biosensors are the following:

• Fast or real-time analysis: Fast or real-time detection provides almost immediate

interactive information about the sample tested, enabling users to take corrective measures before infection or contamination can spread

• Point-of-care detection: Biosensors can be used for point-of-care or on-site testing

where of-the-art molecular analysis is carried out without requiring a of-the-art laboratory

state-• Continuous flow analysis: Many biosensor technologies can be configured to allow

continuous flow analysis This is beneficial in food production, air quality, and water supply monitoring

• Miniaturization: Biosensors can be miniaturized so that they can be integrated

into powerful lab-on-a-chip tools that are very capable while minimizing cost of use

• Control and automation: Biosensors can be integrated with on-line process

mon-itoring schemes to provide real-time information about multiple parameters at each production step or at multiple time points during a process, enabling better control and automation of many industrial and critical monitoring facilities

Preface vii

3 Aims and Approach

The primary aim of this book is to describe the basic types and the basic elements of biosensors from methods point of view We tried to include manuscripts that represent the major technologies in the field and to include enough technical detail so that the informed reader can both understand the technology and also be able to build similar devices The target audience for this book includes engineering, chemical, and physical science researchers, who are developing biosensing technologies Other target groups are biologists and clinicians, who are the users and developers of applications for the technologies

In addition to supporting the research community, the book may also be useful

as a teaching tool for bioengineering, biomedical engineering, and biology faculty and students To better represent the field, most topics are covered by more than one chapter The purpose of this “redundancy” is to try to include several alternative approaches for the topics, so as to help the reader choose an appropriate design

4. Chapter Organization

This publication is divided into two volumes: Vol 503 is focused on Optical-Based Detectors and Vol 504 is focused on Electrochemical and Mechanical Detectors, Lateral Flow, and Ligands for Biosensors

4.1 Volume 503: Optical-Based Detectors

Optical detection is used in a broad array of biosensor technologies, including both

direct and indirect style sensors Volume 503 is organized in two parts Part I focuses

on direct optical detectors, while Part II concentrates on indirect optical detection

Probably, the most common approach for direct optical detection is based on nescent wave physics, where the interaction between the evanescent wave and the bound target generates a detection signal The most common technology in this

eva-group is surface plasmon resonance (SPR) and several chapters (see Chaps 1–5)

describe biosensors based on SPR Other important optical direct detection

meth-ods including resonant mirror (see Chap 6), optical ring resonator (see Chap 7), interferometric sensors (see Chaps 8 and 9) and grating coupler (see Chap 10) are

all included in Part I The second part of Vol 503 describes various indirect optical

detectors As discussed earlier, indirect detectors require a labeled molecule to bind

to the target generating a signal For optical sensors, the label molecule emits or modifies light Most indirect optical detectors are designed to measure fluorescence However, optical detectors can also measure optical density (densitometry), changes

in color (colorimetric), and chemoluminesence, depending on the type of label used

Optical signals can be measured in various ways (described in Part II) including

vari-ous CCD-based detectors, which are very versatile, inexpensive, and relatively simple

to construct and use (see Chaps 11–16 and 25) Other optical detectors discussed

in Part II are photodiodes (see Chaps 17–20), photomultipliers (see Chaps 21–23),

Preface ix

and spectrometers (see Chaps 24 and 25) Photomultipliers may offer higher

sensi-tivity, smaller footprint (the size of photodiode can be few millimeters) eters offer better interrogation of changes in light wavelengths

Spectrom-4.2 Volume 504: Electrochemical and Mechanical Detectors, Lateral Flow, and Ligands

Volume 504 describes various electrochemical and mechanical detectors, lateral flow devices, and ligands for biosensors As in Vol 503, we describe several direct measure-

ment sensors (in Part I), indirect methods (Parts II–III) Ligands are described in

Part IV and two related technologies are described in Part V.

In Part I, we describe several mechanical detectors that modify their mechanical

properties as a result of biological interactions Such mechanical direct biosensors typically sense resonance of the mechanical element, which changes when the target

molecule binds to the surface Piezoelectric biosensors (see Chaps 1–3) employ a

technology that is widely used in a variety of applications (e.g., vapor deposition of metals) and is thus readily available and relatively inexpensive Cantilever-based sys-

tems (see Chaps 4 and 5) can be miniaturized to micrometer dimensions with

attend-ant benefits for system and sample size

In Part II, we describe several electrochemical detectors (see Chaps 6–11)

Elec-trochemical biosensors were the first biosensors developed and are the most monly used biosensors today (e.g., glucose monitoring)

com-Part III covers lateral flow technologies (see Chaps 12–15) Although lateral flow

devices are not “classical” biosensors, with ligands and transducers, they are included

in this book because of their importance for biosensing Lateral flow assays are ple immunodetection (or DNA hybridization) devices, which utilize competitive or sandwich assays They are used mainly for medical diagnostics, including laboratory, home and point-of-care detection A common format is a “dipstick” in which the test sample diffuses through a porous matrix via capillary action followed by detection by

sim-a colorimetric resim-agent bound to sim-a secondsim-ary sim-antibody The primsim-ary sim-antibody is bound

to the matrix in a line, and the assay result is a color change at a particular location on the matrix Lateral flow assays can be dependable and inexpensive

Part IV focuses on recognition ligands, which are key elements in any biosensor

(see Chaps 16–22) The recognition ligands bind specifically with the target molecule

to be detected Various ligands described in Part IV include antibodies, aptamers,

and peptides Antibodies are the most commonly used ligands but advances in tion methods for aptamers (SELEX) and peptides (phage and yeast display) are cur-rently providing alternatives

selec-Part V includes two papers on protein (see Chap 23) and DNA preparation (see

Chap 24) These papers are relevant to the subject of biosensor technologies but did not fit elsewhere into the book organization outline

References

1 IUPAC Compendium of Chemical Terminology 2nd Edition (1997) (1992), 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

sur-gery Ann N Y Acad Sci 102:29–45.

Preface v Contributors xiii Contents of Volume 504 xvii

PART I: OPTICAL-BASED DETECTORS

1 Surface Plasmon Resonance and Surface Plasmon Field-Enhanced

Yusuke Arima, Yuji Teramura, Hiromi Takiguchi, Keiko Kawano,

Hidetoshi Kotera, and Hiroo Iwata

2 Surface Plasmon Resonance Biosensor for Biomolecular Interaction

Xiang Ding, Fangfang Liu, and Xinglong Yu

Jong Seol Yuk and Kwon-Soo Ha

Valery N Konopsky and Elena V Alieva

Marek Piliarik, Hana Vaisocherová, and Ji í Homola

Mohammed Zourob, Souna Elwary, Xudong Fan, Stephan Mohr,

and Nicholas J Goddard

7 Label-Free Detection with the Liquid Core Optical Ring

Resonator Sensing Platform 139

Ian M White, Hongying Zhu, Jonathan D Suter, Xudong Fan,

and Mohammed Zourob

8 Reflectometric Interference Spectroscopy 167

Guenther Proll, Goran Markovic, Lutz Steinle, and Guenter Gauglitz

9 Phase Sensitive Interferometry for Biosensing Applications 179

Digant P Davé

10 Label-Free Serodiagnosis on a Grating Coupler 189

Thomas Nagel, Eva Ehrentreich-Förster, and Frank F Bier

PART II: INDIRECT DETECTORS

11 CCD Camera Detection of HIV Infection 203

John R Day

12 Simple Luminescence Detector for Capillary Electrophoresis 221

Antonio Segura-Carretero, Jorge F Fernández-Sánchez,

and Alberto Fernández-Gutiérrez

13 Optical System Design for Biosensors Based on CCD Detection 239

Douglas A Christensen and James N Herron

xi

xii Contents

14 A Simple Portable Electroluminescence Illumination-Based CCD Detector 259

Yordan Kostov, Nikolay Sergeev, Sean Wilson, Keith E Herold,

and Avraham Rasooly

15 Fluoroimmunoassays Using the NRL Array Biosensor 273

Joel P Golden and Kim E Sapsford

16 Biosensors Technologies: Acousto-Optic Tunable Filter-Based Hyperspectral

and Polarization Imagers for Fluorescence and Spectroscopic Imaging 293

Neelam Gupta

17 Photodiode-Based Detection System for Biosensors 307

Yordan Kostov

18 Photodiode Array On-chip Biosensor for the Detection

of E coli O157:H7 Pathogenic Bacteria 325

Joon Myong Song and Ho Taik Kwon

19 DNA Analysis with a Photo-Diode Array Sensor 337

Hideki Kambara and Guohua Zhou

20 Miniaturized and Integrated Fluorescence Detectors

for Microfluidic Capillary Electrophoresis Devices 361

Toshihiro Kamei

21 Photomultiplier Tubes in Biosensors 375

Yafeng Guan

22 Integrating Waveguide Biosensor 389

Shuhong Li, Platte Amstutz III, Cha-Mei Tang, Jun Hang, Peixuan Zhu,

Yunqi Zhang, Daniel R Shelton, and Jeffrey S Karns

23 Detection of Fluorescence Generated in Microfluidic

Channel Using In-Fiber Grooves and In-Fiber Microchannel Sensors 403

Rudi Irawan and Swee Chuan Tjin

24 Multiplex Integrating Waveguide Sensor: Signalyte™-II 423

Shuhong Li, Yunqi Zhang, Platte Amstutz III, and Cha-Mei Tang

25 CCD Based Fiber-Optic Spectrometer Detection 435

Rakesh Kapoor

Index 447

ELENA V ALIEVA • Institute of Spectroscopy, Russian Academy of Sciences, Troitsk, Moscow Region, Russia

Japan

FRANK F BIER • Department of Molecular Bioanalytics & Bioelectronics, Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany

Electrical & Computer Engineering, University of Utah, Salt Lake City, UT, USA

JOHN R DAY • Gen-Probe Incorporated, San Diego, CA, USA

XIANG DING • Department of Precision Instruments and Mechanics, Tsinghua

University, Beijing, China

Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany

Missouri-Columbia, Columbia, MO, USA

Faculty of Sciences, University of Granada, Granada, Spain

JORGE F FERNÁNDEZ-SÁNCHEZ • Department of Analytical Chemistry,

Faculty of Sciences, University of Granada, Granada, Spain

University of Tuebingen, Tuebingen, Germany

(CEAS), The University of Manchester, Manchester, UK

JOEL P GOLDEN • Center for Bio/Molecular Science & Engineering,

US Naval Research Laboratory, Washington, DC, USA

Dalian Institute of Chemical Physics, Dalian, China

KWON-SOO HA • Department of Molecular and Cellular Biochemistry and

Nanobio Sensor Research Center, Kangwon National University College

of Medicine, Chuncheon, Kangwon-do, Korea

JUN HANG • Creatv MicroTech, Inc., Potomac, MD, USA

xiii

of Washington Alliance, Nanyang Technological University, Singapore

Department of Physics, University of Lampung, Bandar Lampung, Indonesia

HIROO IWATA • Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Technology (AIST), Ibaraki, Japan

Birmingham, AL, USA

of Agriculture-Agricultural Research Service, Beltsville, MD, USA

Institute of Kyoto, Kyoto, Japan

Moscow Region, Russia

Baltimore County (UMBC), Baltimore, MD, USA

Engineering, Kyoto University, Kyoto, Japan

HO TAIK KWON • Celltek Co., Ltd., Ansan-si, South Korea

Tsinghua University, Beijing, China

of Tuebingen, Tuebingen, Germany

The University of Manchester, Manchester, UK

Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany

Czech Republic, Prague, Czech Republic

of Tuebingen, Tuebingen, Germany

MD, USA, National Cancer Institute, Bethesda, MD, USA

KIM E SAPSFORD • Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DC, USA

Sciences, University of Granada, Granada, Spain

MD, USA

of Agriculture-Agricultural Research Service, Beltsville, MD, USA

JOON MYONG SONG • Research Institute of Pharmaceutical Sciences and College

of Pharmacy, Seoul National University, Seoul, South Korea

of Tuebingen, Tuebingen, Germany

of Missouri-Columbia, Columbia, MO, USA

Institute of Kyoto, Kyoto, Japan

CHA-MEI TANG • Creatv MicroTech, Inc., Potomac, MD, USA

Kyoto University, Kyoto, Japan

SWEE CHUAN TJIN • Photonics Research Centre, School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore

of the Czech Republic, Prague, Czech Republic

IAN M WHITE • Biological Engineering Department, University of

Missouri-Columbia, Columbia, MO, USA

Baltimore, MD, USA

Xinglong Yu • Department of Precision Instruments and Mechanics, Tsinghua University, Beijing, China

JONG SEOL YUK • Department of Molecular and Cellular Biochemistry and

Nanobio Sensor Research Center, Kangwon National University College

of Medicine, Chuncheon, Kangwon-do, Korea

YUNQI ZHANG • Creatv MicroTech, Inc., Potomac, MD, USA

Missouri-Columbia, Columbia, MO, USA

QC, Canada

Contents of Volume 504

Preface

Contributors

Contents of Volume 503

PART I: MECHANICAL DETECTORS

1 A Set of Piezoelectric Biosensors Using Cholinesterases

Carsten Teller, Jan Halámek, Alexander Makower, and Frieder W Scheller

2 Piezoelectric Biosensors for Aptamer–Protein Interaction

Sara Tombelli, Alessandra Bini, Maria Minunni, and Marco Mascini

3 Piezoelectric Quartz Crystal Resonators Applied for Immunosensing

and Affinity Interaction Studies

Petr Skládal

4 Biosensors Based on Cantilevers

Mar Álvarez, Laura G Carrascosa, Kiril Zinoviev, Jose A Plaza,

and Laura M Lechuga

5 Piezoelectric-Excited Millimeter-Sized Cantilever Biosensors

Raj Mutharasan

6 Preparation of Screen-Printed Electrochemical Immunosensors

for Estradiol, and Their Application in Biological Fluids

Roy M Pemberton and John P Hart

7 Electrochemical DNA Biosensors: Protocols for Intercalator-Based

Detection of Hybridization in Solution and at the Surface

Kagan Kerman, Mun’delanji Vestergaard, and Eiichi Tamiya

8 Electrochemical Biosensor Technology: Application to Pesticide Detection

Ilaria Palchetti, Serena Laschi, and Marco Mascini

9 Electrochemical Detection of DNA Hybridization Using Micro and Nanoparticles

María Teresa Castañeda, Salvador Alegret, and Arben Merkoçi

10 Electrochemical Immunosensing Using Micro and Nanoparticles

Alfredo de la Escosura-Muñiz, Adriano Ambrosi, Salvador Alegret,

and Arben Merkoçi

11 Methods for the Preparation of Electrochemical Composite

Biosensors Based on Gold Nanoparticles

A González-Cortés, P Yáñez-Sedeño, and J.M Pingarrón

PART III: LATERAL FLOW

12 Immunochromatographic Lateral Flow Strip Tests

Gaiping Zhang, Junqing Guo, and Xuannian Wang

xvii

13 Liposome-Enhanced Lateral-Flow Assays for the Sandwich-Hybridization Detection of RNA

Katie A Edwards and Antje J Baeumner

14 Rapid Prototyping of Lateral Flow Assays

Alexander Volkov, Michael Mauk, Paul Corstjens, and R Sam Niedbala

15 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide

Shuo Wang, Can Zhang, and Yan Zhang

PART IV: LIGANDS

16 Synthesis of a Virus Electrode for Measurement of Prostate

Specific Membrane Antigen

Juan E Diaz, Li-Mei C Yang, Jorge A Lamboy, Reginald M Penner, and Gregory A Weiss

17 In Vivo Bacteriophage Display for the Discovery of Novel

Peptide-Based Tumor-Targeting Agents

Jessica R Newton and Susan L Deutscher

18 Biopanning of Phage Displayed Peptide Libraries for the

Isolation of Cell-Specific Ligands

Michael J McGuire, Shunzi Li, and Kathlynn C Brown

19 Biosensor Detection Systems: Engineering Stable, High-Affinity

Bioreceptors by Yeast Surface Display

Sarah A Richman, David M Kranz, and Jennifer D Stone

20 Antibody Affinity Optimization Using Yeast Cell Surface Display

Robert W Siegel

21 Using RNA Aptamers and the Proximity Ligation Assay

for the Detection of Cell Surface Antigens

Supriya S Pai and Andrew D Ellington

22 In Vitro Selection of Protein-Binding DNA Aptamers

as Ligands for Biosensing Applications

Naveen K Navani, Wing Ki Mok, and Yingfu Li

23 Immobilization of Biomolecules onto Silica and Silica-Based Surfaces for Use in Planar Array Biosensors

Lisa C Shriver-Lake, Paul T Charles, and Chris R Taitt

24 Rapid DNA Amplification Using a Battery-Powered Thin-Film Resistive Thermocycler

Keith E Herold, Nikolay Sergeev, Andriy Matviyenko,

and Avraham Rasooly

Index

Chapter 1

Surface Plasmon Resonance and Surface Plasmon

Field-Enhanced Fluorescence Spectroscopy

for Sensitive Detection of Tumor Markers

Yusuke Arima, Yuji Teramura, Hiromi Takiguchi, Keiko Kawano,

Hidetoshi Kotera, and Hiroo Iwata

Summary

Surface plasmon resonance (SPR), which provides real-time, in situ analysis of dynamic surface events,

is a valuable tool for studying interactions between biomolecules In the clinical diagnosis of tumor markers in human blood, SPR is applied to detect the formation of a sandwich-type immune complex composed of a primary antibody immobilized on a sensor surface, the tumor marker, and a secondary antibody However, the SPR signal is quite low due to the minute amounts (ng–pg/mL) of most tumor markers in blood We have shown that the SPR signal can be amplified by applying an antibody against the secondary antibody or streptavidin-conjugated nanobeads that specifically accumulate on the secondary antibody Another method employed for highly sensitive detection is the surface plasmon field-enhanced fluorescence spectroscopy-based immunoassay, which utilizes the enhanced electric field intensity at a metal/water interface to excite a fluorophore Fluorescence intensity attributed to binding

of a fluorophore-labeled secondary antibody is increased due to the enhanced field in the SPR condition and can be monitored in real time.

Key words: Surface plasmon resonance, Immunosensing, Tumor marker, Signal amplification, Polyclonal antibody, Surface plasmon field-enhanced fluorescence spectroscopy.

Surface plasmon resonance (SPR)-based sensing has been used to

study interactions between biomolecules (1) The SPR method

is a sensitive technique to detect changes in the local refractive

index near the surface of a thin metal (typically gold) film (2)

1 Introduction

Avraham Rasooly and Keith E Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol 503

© Humana Press, a part of Springer Science + Business Media, LLC 2009

DOI: 10.1007/978-1-60327-567-5_1

3

Figure 1 shows an SPR apparatus of Kretchmann configuration

(a) and its schematic representation (b) A beam of p-polarized

light is used to illuminate the back side of a gold thin film on glass, and the front side of the film faces a solution of interest When the incident angle exceeds the critical angle, total internal reflection occurs An evanescent wave is generated on the surface facing the solution, which has a lower refractive index than glass

At a specific incident angle, the evanescent wave of the incoming light is able to couple with the free oscillating electrons (plas-mons) in the metal film, and the surface plasmon is resonantly excited This excitation causes energy from the incident light to

be lost to the metal film, resulting in a reduction in the intensity

of reflected light ( Fig 2a ) The resonance angle is a function of the refractive index at the interface of the metal film and solution Thus, a shift in resonance angle reflects events at the interface, such as protein adsorption on the surface and antigen–antibody interactions SPR offers rapid, label-free, and real-time monitoring

of binding events between biomolecules

10 cm(a)

(b)

He-Ne laser (632.8 nm) Iris

Gran-Thomson prism

Iris Glass plate

Lens Photodiode

Biaxial rotation

stage

Photodiode

Beam splitter Lens

Flow cell

Protein solution

Prism

Fig 1 a A surface plasmon resonance (SPR) apparatus and b its schematic representation.

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 5

Recently, SPR-based immunoassays for the clinical detection

of biomarkers in human blood have been investigated (3, 4)

The SPR-based immunoassay detects the specific interaction of

a biomarker with an antibody immobilized on the SPR sensor

( Fig 2b ) Primary antibodies against a particular biomarker are immobilized onto a gold surface modified with a self-assembled monolayer (SAM) of alkanethiols A blood sample is brought into contact with the sensor, and the biomarker in blood specifically binds to the antibodies immobilized on the sensor surface If the concentration of the biomarker in blood is high and its molecular weight is large, the SPR resonance angle shift can be detected easily without further modification However, the concentra-tions of most tumor markers are in the range ng/mL–pg/mL

( Table 1 ) Therefore, amplification of SPR signal intensity is needed to detect most tumor markers in clinical samples

Several methods for amplifying SPR signal intensity are shown schematically in Fig 3 In the method shown in

Fig 3a, the sensor surface is incubated with a solution containing

Secondary antibody

Reflected light Incident light

Gold thin film Self-assembled

Fig 2 a Relationship between incident angle q and intensity of reflected light before (solid line) and after (dashed line)

protein adsorption For real-time monitoring, the intensity of reflected light is monitored at a fixed angle throughout the

measurement (arrow) b Schematic representation of SPR-based sandwich-type immunoassay.

Table 1

Concentrations of some tumor markers

Cut-off level (ng/mL)

Cut-off level (pmol/L)

secondary antibodies, which bind to the tumor marker previously captured by the primary antibody on the SPR sensor surface As shown in Fig 3b, further amplification can be achieved by apply-

ing polyclonal antibodies against the secondary antibody (5)

Because the immobilization of nanobeads causes a large change

in the refractive index at the metal/solution interface, nanobeads are expected to be useful for inducing a substantial shift in the

SPR resonance angle ( Fig 3c ) In this method, biotin-labeled secondary antibodies are bound to tumor markers, which are trapped by primary antibodies immobilized on a sensor surface Streptavidin-conjugated nanobeads (50 nm in diameter) and biotin-labeled anti-streptavidin antibodies are alternately layered

on the surface via the specific biotin–streptavidin interaction (6)

All three of the previously described amplification methods lize an SPR angle shift caused by changes in the local refractive index near the sensor surface A fourth interesting method to amplify signal intensity utilizes the strong increase in surface light intensity at the metal/water interface around the SPR resonance angle, i.e., surface plasmon field-enhanced fluorescence spectros-

uti-copy (SPFS) (7) In this method, SPFS is employed to detect the

(a)

(c)

Fluorophore-conjugated secondary antibody

Primary antibody Tumor marker Detector for fluorescence

Secondary antibody

Primary antibody Tumor marker Antibody against secondary antibody

Biotin-labeled secondary antibody

Streptavidin-conjugated magnetic beads (50 nm)

Biotin-labeled

anti-streptavidin antibody

(b)Secondary antibody

Primary antibody Tumor marker

(d)

Fig 3 Signal amplification methods for detection of a minute amount of tumor marker a Binding of secondary antibodies

to tumor marker captured by immobilized primary antibodies b Binding of polyclonal antibodies to secondary antibody

c Accumulation of streptavidin-conjugated nanobeads and biotin-labeled antistreptavidin antibodies d Detection of

fluorophore-conjugated secondary antibodies by surface-plasmon field-enhanced fluorescence spectroscopy.

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 7

fluorescence due to binding of a fluorophore-labeled secondary

antibody ( Fig 3d ).Here, we introduce the methods of SPR-based and SPFS-based immunoassays for the detection of tumor markers

1 Hemicylindrical prism (custom-made, diameter: 25 mm, width: 10 mm; Sigma Koki Co., Ltd, Tokyo, Japan)

2 Index-matching fluid (n = 1.515; Cargille Laboratories,

7 Beam splitter (NPCH-10-6328; Sigma Koki)

8 Gran-Thomson prism (GTPC-08-20AN; Sigma Koki)

9 Photodiode detector (S3590-01; Hamamatsu Photonics K.K., Shizuoka, Japan)

10 Lens (SLB-30-300PM, f = 300 mm; Sigma Koki).

11 Iris (IH-30; Sigma Koki)

12 Optical rail (OBA-500SH; Sigma Koki)

13 Optical table (MB-PH; Sigma Koki)

12 mm

2 mm

PVC plate

Hemicylindrical prism Fig 4 Assembly of a flow cell.

14 Lens (SLB-30-60PM, f = 60 mm; Sigma Koki).

15 Biaxial rotation stage (SGSP-120YAW-W; Sigma Koki), which is operated through an intelligent driver (CSG-522R; Sigma Koki) by homemade software

The basal components of an SPFS apparatus are the same as for

an SPR apparatus ( Fig 1b ), except for the addition of a CCD camera on the sensor surface:

1 Laser diode (LD, VHK laser diode module l = 635 nm,

0.95 mW; Coherent, Santa Clara, CA)

2 Lens (SLB-20-25P, f = 25 mm; Sigma Koki, Tokyo, Japan)

for collimation of the laser light

3 Polarizing filter (TS0851-G; Sugitoh, Tokyo, Japan)

4 Neutral density filter (AND-20C-10, T = 10%; Sigma Koki).

5 Guide rail (OBS-200G; Sigma Koki) with appropriate holders

6 Rotational stage (MM-40θ; Chuo Seiki, Tokyo, Japan)

7 Lens (SLB-20-25P; Sigma Koki, Japan)

8 Photodiode detector (S2281-01; Hamamatsu Photonics, Hamamatsu, Japan)

9 Two-axis stage controller (QT-CM2; Chuo Seiki)

10 Objective lens (SLWD Plan20×; Nikon, Tokyo, Japan)

11 Interference filter (l = 670 nm, transmittance 75%, full-width

half length max (FWHM) = 7 nm; Optical Coatings Japan, Tokyo, Japan)

12 Extension barrel (TS0155-H; Sugitoh, Tokyo, Japan)

13 CCD camera with a charge multiplier (MC681-SPD; Texas

Instruments, Dallas, TX) (see Note 1)

14 Image capture board (MT-PCI2; Micro-Technica, Tokyo, Japan)

15 Homemade intensity scanning software (see Note 2)

16 A glass plate (S-LAL10, refractive index: 1.720; Sigma Koki) with a thin gold film (49 nm in thickness)

17 Triangular prism (25 × 25 × 25 mm, S-LAL10; Sigma Koki)

18 Index-matching fluid (n = 1.72, Cargille Laboratories).

1 Ethanol is deoxygenated with bubbling nitrogen gas for 20–30 min before use

2 (1-Mercaptoundecanoic-11-yl)tri(ethylene glycol) (TEG: HS–(CH2)11–(OCH2CH2)3–OH) and (1-mercaptounde-canoic11-yl)hexa(ethylene glycol)carboxylic acid (HEG:

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 9

HS–(CH2)11–(OCH2CH2)5–OCH2CH2OCH2COOH) (SensoPath Technologies, Inc., Bozeman, MT) are dissolved

in deoxygenated ethanol solution at 0.9 and 0.1 mM,

respec-tively (see Note 3)

3 α-Fetoprotein (AFP; Morinaga Institute of Biological Science, Inc., Yokohama, Japan) is dissolved in phos-phate buffer at 10 μg/mL and stored in single-use aliq-uots at −80°C

4 Whole blood is collected by drawing venous blood from healthy donors into Venoject® II blood collection tubes containing EDTA-2 Na (TERUMO Co., Tokyo, Japan) To

separate plasma, the tubes are centrifuged at 3,000 × g at

4°C for 30 min After centrifugation, supernatant is collected and stored at −80°C until use

5 Working solutions of AFP at prescribed concentrations are

prepared by dilution in human plasma (see Note 4)

6 A tablet of PBS-Tween® (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, 0.05% Tween® 20; Calbiochem, Inc., Darmstadt, Germany) is dissolved in 1 L pure water (one tablet per 1 L)

7 Phosphate buffer (pH 6.6) is prepared by dissolving 33 mM

Na2HPO4 and 33 mM KH2PO4 in pure water at a volume ratio of 2:1 This solution is degassed by a water aspirator and used for sample preparation

8 Anti-AFP antibody (mouse monoclonal, Clone number: ME-101, affinity = 6 × 109; Abcam Ltd, Cambridge, UK)

is dissolved in phosphate buffer at 3.2 mg/mL and stored in single-use aliquots at −80°C A primary antibody solution is prepared by diluting the solution to 10 μg/mL in phosphate

buffer (see Note 5)

9 Lyophilized powder of anti-AFP antibody with various salts (rabbit polyclonal; Monosan Ltd, Uden, Nether-land) is dissolved in pure water at 1.0 mg/mL and stored

at 4°C A secondary antibody solution is prepared by diluting the solution to 10 μg/mL in phosphate buffer

(see Note 5)

10 Anti-rabbit IgG antibody (goat polyclonal, 1.5 mg/mL; Zymed Laboratories, Inc., South San Francisco, CA) is dissolved at 10 μg/mL in phosphate buffer just before use

Various SPR instruments are commercially available from nies such as Biacore AB and Moritex Corp The optical construc-tion of an SPR instrument is simple, as shown schematically in

compa-Fig 1b We assembled an SPR instrument from optical parts as

described (8) The glass plate is coupled to a hemicylindrical prism

(custom-made, diameter: 25 mm, width: 10 mm; Sigma Koki

Co., Ltd, Tokyo, Japan) with an index-matching fluid (n = 1.515;

Cargille Laboratories, Ceder Grove, NJ), and the SPR flow cell

is set on the glass plate The flow cell is assembled with silicone rubber and a PVC plate and is connected to silicone tubes (inner

diameter: 0.5 mm, outer diameter: 1 mm) ( Fig 4 ) The ture of the flow cell is kept constant with a silicone rubber heater regulated by a digital controller (E5EK; Omron Corp., Kyoto, Japan) A peristaltic pump (MP-3 N; Tokyo Rikakikai Co., Ltd, Tokyo, Japan) delivers the liquid sample to the flow cell at the rate of 4 mL/min

tempera-A He–Ne laser beam (l = 633 nm, 05-LHP-151; Melles Griot,

Carlsbad, CA) is separated into two by a beam splitter

(NPCH-10-6328; Sigma Koki) One beam is linearly p-polarized using a

Gran-Thomson prism (GTPC-08-20AN; Sigma Koki), and the other is guided to a photodiode detector (S3590-01; Hamamatsu Photonics K.K., Shizuoka, Japan) to monitor the fluctuation of incident light intensity The polarized light is focused by a lens

(SLB-30-300PM, f = 300 mm; Sigma Koki) onto the backside of

a gold thin film evaporated on a glass plate The He–Ne laser, iris (IH-30; Sigma Koki), Gran-Thomson prism, beam splitter, and lens are placed on an optical rail (OBA-500SH; Sigma Koki) and fixed on an optical table by optical bases (MB-PH; Sigma Koki).The reflected light passes through a lens (SLB-30-60PM,

f = 60 mm; Sigma Koki), and its intensity is measured by a

photo-diode detector Reflectance is calculated from the intensities of the incident and reflected light (voltage), which are converted from the current of each photodiode The sample stage and the detector for reflected light are rotated at intervals of 0.03° using a biaxial rotation stage (SGSP-120YAW-W; Sigma Koki), which is operated through an intelligent driver (CSG-522R; Sigma Koki)

by homemade software, to obtain the relationship between

inci-dent angle and reflectance ( Fig 2a ) To precisely determine a resonance angle, a quadratic function was fitted to an incident angle–reflectance profile ranging from 0.4° lower to 0.1° higher than an apparent minimum in reflectance, and the minimum of the quadratic function was regarded as the resonance angle An SPR control and analysis program was developed on an integrated development environment using Object Pascal language (Boland Delphi 4 Pro Jpn ed.; Inprise Corp., Tokyo, Japan)

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 11

For real-time monitoring of binding processes, the change

in reflected light intensity is monitored at a fixed angle (in our system, 0.5° lower than the resonance angle) during a measure-ment (Fig 2a, arrows) Finally, the change in reflected light inten-

sity is converted to SPR angle shift by homemade software The amount of adsorbed protein is determined by the SPR angle shift using the following relationship:

The amount of adsorbed protein (ng/cm2) = 500 × increase

in resonance angle (degree),where the refractive index and density of protein are assumed

to be 1.45 and 1.0 g/cm3, respectively

1 Glass plates for SPR (material: BK7, refractive index: 1.515,

25 mm × 25 mm × 1 mm) are purchased from Arteglass ciates (Kyoto, Japan)

2 Piranha solution, a 7:3 mixture of concentrated sulfuric acid

and 30% hydrogen peroxide solution, is prepared (see Note 7)

3 BK7 glass plates are immersed in piranha solution for 5 min, rinsed twice with deionized water, rinsed with 2-propanol, and stored in 2-propanol until use

4 Glass plates are dried with a stream of nitrogen gas

5 Glass plates are placed in a thermal evaporation apparatus

(V-KS200; Osaka Vacuum, Osaka, Japan) (see Note 8)

6 A gold wire (purity: 99.99%, f = 0.5 mm) and a chromium

piece (purity: 99.99%) are placed in separate tungsten baskets

7 The pressure in the evaporation chamber is decreased to less than 3 × 10−4 Pa

8 A chromium layer of 1-nm thickness is deposited on the glass

plate at 0.02 nm/s (see Note 9)

9 A gold layer is deposited on the glass plate at 0.05 nm/s for

4 nm, 0.3 nm/s for 38 nm, and 0.05 nm/s for 7 nm (total

thickness: 49 nm) (see Note 10)

10 The gold-coated plates are immersed immediately in the TEG/

HEG mixture (see Subheading 1.2.3) for approximately 24 h

at room temperature to form a mixed SAM (see Note 11)

11 The glass plates modified with TEG/HEG-mixed SAM are washed thoroughly with pure water and 2-propanol before

use (see Note 12)

1 A TEG/HEG-mixed SAM-coated glass plate is dried with

a N2 gas stream and placed on a hemicylindrical prism with

index-matching fluid (n = 1.515; Cargille Laboratories,

Cedar Grove, NJ)

2 A flow cell chamber composed of a glass plate with prism,

a 0.5-mm-thick silicone rubber spacer, and an upper

plate is assembled and is placed in the SPR instrument

5 A mixture of 1-ethyl-3-(3-dimethylaminopropyl) ide hydrochloride (EDC; Dojindo Laboratories, Kumamoto,

carbodiim-Japan) and N-hydroxylsuccinimide (NHS; Nacalai Tesque,

Kyoto, Japan) (stored as a mixed powder) is dissolved in degassed phosphate buffer at 0.1 and 0.05 M, respectively, just before use

6 The mixture of 0.1 M EDC and 0.05 M NHS in phosphate buffer is flowed for 15 min to activate the COOH groups of the TEG/HEG-mixed SAM, and then the primary antibody solution (10 μg/mL) is immediately flowed for 25 min to achieve covalent immobilization

7 A BSA blocking solution is flowed for 15 min to block specific adsorption and to deactivate unreacted NHS ester

non-groups on the surface (see Note 15)

8 Human plasma containing AFP (50–500 ng/mL) or a cal sample is flowed into the SPF flow cell for 30 min

9 PBS-Tween® solution is flowed for 15 min after flowing

of plasma to remove nonspecifically adsorbed proteins

(see Note 15)

10 A secondary antibody solution (10 μg/mL, polyclonal) is applied for 30 min, followed by a solution of anti-rabbit IgG antibody (10 μg/mL, polyclonal) for 30 min for the SPR

signal enhancement (see Note 16)

11 As control experiments, the same procedures (steps 8–10)

are performed in the absence of AFP in human plasma

12 In this system, sample and buffer solutions (3 mL each) are circulated through the flow cell at 4.0 mL/min by a peri-staltic pump Between the different solution injections, the flow cell is washed with phosphate buffer for at least 5 min,

except for between the two injections in step 6 (see Notes

17 and 18)

The SPR immunoassay is based on the formation of a

sandwich-type immune complex with two kinds of antibody (Fig 1.2b),

which is also the basis of the widely used enzyme-linked sorbent assay (ELISA) Primary antibody is immobilized onto a SAM surface bearing carboxylic groups via covalent amide bond-ing by a NHS/EDC coupling method After blocking treatment

immuno-3.1.4 SPR Sensorgram

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 13

with BSA or inert materials to prevent nonspecific adsorption of serum proteins, AFP in human plasma and secondary antibody (rabbit IgG) are applied sequentially The SPR signal shift can be further enhanced by binding of anti-rabbit IgG antibody (poly-

clonal) (Fig 1.5; Subheading 1.3.1.3) The concentration of a

tumor marker in blood can be determined from the calibration curve, which is obtained from resonance angle shifts for solutions with various concentrations of tumor marker

Amplification of the SPR signal to detect low tions of AFP (25 pg/mL–1 ng/mL) can be accomplished using streptavidin-conjugated nanobeads and biotin-labeled antist-reptavidin antibodies, according to the method reported for

concentra-detection of brain natriuretic peptide (BNP) (6) Namely, after

flowing of biotin-labeled secondary antibody, nanobeads and biotin-labeled antistreptavidin antibody are flowed sequentially, followed by the flowing of 0.05% Tween

streptavidin-20 in PBS solution for the removal of nonspecific adsorbed teins and beads These procedures can be repeated to further

pro-amplify the SPR signal (see Note 19)

Secondary antibody Tween20

Time (min)

Secondary antibody

[AFP] =

50 ng / mL [AFP] = 500 ng / mL

Control (no AFP)

[AFP] = 50 ng / mL [AFP] = 500 ng / mL

0 100 200 300 400 500 600 700

183 173 163 153 1380 1390 1410 1420 1430 1440

Fig 5 SPR-based immunoassay for AFP in human plasma a SPR profiles of sequential reactions during AFP detection in

human plasma containing 50 or 500 ng/mL AFP (5) After immobilization of primary antibody by EDC/NHS and blocking

treatment with BSA, plasma containing AFP was perfused The sensor surface was washed with PBS-Tween ® Then, a solution of secondary antibody was flowed An SPR signal shift (155 mDA) was clearly observed when the solution of

secondary antibody was applied The inset shows an SPR signal shift for the plasma containing 50 ng/mL AFP b

Ampli-fication of SPR signal using goat polyclonal antibody against the secondary antibody (rabbit IgG) An SPR sensor surface was exposed to plasma supplemented with AFP ([AFP] = 50 or 500 ng/mL) or without AFP addition The sensor surface was washed with PBS-Tween ® , and then a solution of secondary antibody was flowed The time course of the SPR profile during application of the solution of anti-rabbit IgG antibody was recorded.

The basal components of an SPFS apparatus are the same as for

an SPR apparatus (Fig 1.1b), except for the addition of a CCD

camera on the sensor surface When the incident angle approaches the SPR angle, the surface electric field intensity at the water/metal interface strongly increases, as depicted by the dashed line

in Fig 1.6a (7) Peak intensities relative to the incoming

inten-sity can reach an enhancement factor of ~16 for gold and incident light l = 635 nm This enhanced field can be utilized to excite a

fluorophore to detect fluorophore-labeled secondary antibodies

in the SPFS-based immunoassay (Fig 1.6b) (9) The intensity

of fluorescence by SPFS is increased relative to that of the conventional total internal reflection due to the enhanced field

in the SPR condition

Figure 1.7a is a photo of our SPFS apparatus The optical construction of an SPFS instrument is simple as shown sche-

matically in Fig 1.7b A laser diode (LD, VHK laser diode

module l = 635 nm, 0.95 mW; Coherent, Santa Clara, CA), a lens (SLB-20-25P, f = 25 mm; Sigma Koki, Tokyo, Japan) for

collimation of the laser light, a polarizing filter (TS0851-G; toh, Tokyo, Japan), and a neutral density filter (AND-20C-10,

Sugi-T = 10%; Sigma Koki) are placed on the same guide rail

(OBS-200G; Sigma Koki) with appropriate holders The guide rail with these parts is placed on an arm of a rotational stage (MM-40θ; Chuo Seiki, Tokyo, Japan) Optical parts for detection of the reflected light, such as a lens (SLB-20-25P; Sigma Koki, Japan) and a photodiode detector (S2281-01; Hamamatsu Photonics, Hamamatsu, Japan), are placed on a guide rail and attached to

an arm of another rotational stage These rotational stages are operated by a two-axis stage controller (QT-CM2; Chuo Seiki) Reflectance is determined from the intensities of incident and

Incident light Reflected light

Gold thin film

θ

Self-assembled monolayer Primary antibody

Fig 6 Principle of surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and its application to

immu-noassays a Reflectance (solid line) and electric field enhancement (dashed line) relative to the incoming intensity as

a function of incident angle q at an Au/water interface b Schematic representation of SPFS-based sandwich-type

immunoassay.

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 15

reflected light (voltage), which are converted from currents of the photodiode detector by the same algorithm used for the SPR measurement apparatus

Fluorescent light from fluorophores on a sensor surface is collected by an objective lens (SLWD Plan20×; Nikon, Tokyo,

Japan), passed through an interference filter (l = 670 nm,

trans-mittance 75%, FWHM = 7 nm; Optical Coatings Japan, Tokyo, Japan) fixed in a extension barrel (TS0155-H; Sugitoh, Tokyo, Japan), and then guided to a CCD camera with a charge multi-

plier (MC681-SPD; Texas Instruments, Dallas, TX) (see Note 1)

A fluorescence image is acquired by an image capture board (MT-PCI2; Micro-Technica, Tokyo, Japan) and analyzed by

homemade intensity scanning software (see Note 2) The light intensity in a fixed area (100 × 100 pixels) at the center of the laser light is monitored for a certain integration time, and the average intensity is recorded

5 cm(a)

(b)

LD (635 nm, 0.95 mW) Flow cell

Photodiode

Interference filter (670 nm) CCD camera

SPFS

Glass plate (S-LAL10, Au:Cr = 49:1 nm)

Objective (x20)

Polarizing filter

Rotational stage θ

Fig 7 a A surface plasmon field-enhanced fluorescence spectroscopy apparatus and b its schematic representation.

The glass plate (S-LAL10, refractive index: 1.720; Sigma Koki) with a thin gold film (49 nm in thickness) is coupled to

a triangular prism (25 × 25 × 25 mm, S-LAL10; Sigma Koki)

with an index-matching fluid (n = 1.72, Cargille Laboratories)

The flow cell illustrated in Fig 1.4 is assembled on the glass

plate Instead of a hemicylindrical prism and a PVC plate, a triangular prism and a transparent PMMA plate are employed

(see Note 20)

1 Phosphate-buffered saline (PBS) consists of 10 mM phate buffer with 140 mM NaCl and 3 mM KCl in pure water (pH 7.4)

phos-2 Anti-AFP antibody used as a secondary antibody (1D5, mouse monoclonal; Japan Clinical Laboratories, Inc., Kyoto, Japan)

is dissolved at 2.5 mg/mL in PBS and stored at 4°C

3 Anti-AFP antibody used as a secondary antibody (6D2, mouse monoclonal; Japan Clinical Laboratories, Inc.) is dissolved at 2.5 mg/mL in PBS and stored at 4°C

4 The secondary antibody is conjugated with Alexa Fluor 647 dye according to the standard protocol of a labeling kit from Molecular Probes (Eugene, OR), and the concentration of conjugate is <1 mg/mL in PBS

5 Other working solutions are prepared by the methods

men-tioned in Subheading 1.2.3.

1 Glass plates (S-LAL10, refractive index: 1.720, 25 mm × 25 mm

× 1 mm) are purchased from Sigma Koki (Tokyo, Japan)

2 S-LAL10 glass plates are cleaned with oxygen plasma using

a plasma reactor (PA300AT; O-kuma Engineering, Fukuoka,

Japan) under 5 Pa for 1 min (see Note 21)

3 Glass plates with a gold thin layer are prepared, a mixed SAM is formed on the gold surface, and primary anti-bodies are immobilized on the mixed SAM by the methods

TEG/HEG-described in Subheading 1.3.1.3, steps 1–7.

4 Immobilization of primary antibody and blocking with BSA are monitored in the SPR mode

1 The incident angle is adjusted to the SPR condition by

moni-toring the intensity of reflected light (see Notes 22 and 23)

2 For one-step measurement of the AFP concentration in plasma,

20 μL of the 1 μg/mL Alexa Fluor 647-labeled secondary body solution is added to 4,980 μL of human plasma containing AFP and left for 20 min at room temperature to form an AFP-

anti-secondary antibody complex (see Note 24)

3 After incubation, human plasma is introduced into the flow cell at a rate of 1.3 mL/min for 5 min

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 17

4 The surface is rinsed with PBS for 5 min to remove plasma

from the flow cell (see Note 25)

5 Fluorescence intensity is monitored during the infusions of

plasma and PBS (Fig 1.8).

The SPFS immunoassay is based on the formation of a

sandwich-type immune complex with two different antibodies (Fig 1.7b)

Primary antibody is immobilized on the sensor surface by an NHS/EDC coupling method, and the surface is treated with a BSA solution Immobilization of the primary antibody and block-ing with BSA are monitored by SPR as described above A mix-ture of the AFP-containing plasma and the fluorophore-labeled secondary antibody is introduced into the flow cell The fluores-cent intensity excited by the enhanced electric field is increased

upon introduction of the plasma (Fig 1.8) After washing for

10 min with PBS, the specific complex bound to the surface remains The AFP concentration in plasma can be determined from the fluorescence intensity The SPFS-based immunoassay is

a sensitive, straightforward method for detecting tumor markers

50000 100000 150000 200000 250000

Fig 8 SPFS-based immunoassay for AFP (0, 1, or 10 ng/mL) Primary antibodies were immobilized on a sensor surface

as shown in Fig 5a After incubation of AFP and the secondary antibody for 20 min, the mixture was infused into the SPFS flow cell The sensor surface was washed with PBS-Tween ®

2 Although the intensity scanning software, which is used to determine the average pixel intensity of a fixed area in the fluorescence image, was programmed in our laboratory, the software can be substituted by other software.

3 Carboxylic groups of HEG are accessible without steric hindrance by TEG chains on the surface because the chain length of HEG is longer than that of TEG

4 Numerous competitive reagents are available from other commercial sources

5 We have used two kinds of antibodies that are recommended for ELISA Numerous competitive reagents are available from other commercial sources

6 Numerous competitive blocking reagents are available from other commercial sources

7 Piranha solution reacts violently with many organic materials and should be handled with extreme care

8 Electron beam evaporation may be employed in place of thermal evaporation

9 A chromium underlayer is needed to improve attachment of the gold layer to the glass plate A titanium underlayer may also be employed

10 A 49-nm-thick gold layer is optimal for this wavelength of incident light (633.5 nm) Optimal thickness varies with the wavelength of incident light

11 Although a SAM of alkanethiol on gold is formed within a few minutes, we usually choose to leave gold substrates in a solution of alkanethiol for approximately 24 h

12 Unless otherwise stated, all solutions should be prepared in water that has a resistance higher than 18.2 MΩ and a total organic content of less than five parts per billion This stand-ard is referred to as “water” in the text

13 SAM-coated glass plates should be used within a few days Otherwise, the glass plate is stored in a vacuum desiccator

14 A temperature change in the solution or flow cell also drifts the resonance angle A temperature increase of 1°C causes a decrease in resonance angle of 0.015° To improve the signal/noise ratio, temperatures should be kept constant

15 The apparent increase in SPR signal during the flowing of BSA and plasma solution is due to an increased refractive index caused by the bulk effect of high concentrations of components The increase in baseline signal after flowing plasma and washing with PBS-Tween® is caused not only by the binding of AFP to primary antibody but also by nonspe-cific adsorption of plasma components on the surface

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced 19

16 Polyclonal antibody binds polyvalently to AFP on the surface, which leads to a large enhancement of SPR signal

17 When the solution is exchanged, the introduction of air into the circulation tube should be avoided Otherwise, the baseline

is shifted substantially

18 Activated COOH groups are hydrolyzed and inactivated

in the presence of water Therefore, the sensor surface is washed with phosphate buffer for only 30 s after flowing EDC/NHS

19 Although the SPR signal can be amplified by repeating this procedure, the background noise level is also increased

20 When the collimated light is used to illuminates the back side

of a gold thin film on a glass plate, a triangular prism is used

to adjust the incident angle same over the illuminated area

21 Because S-LAL10 cannot resist strong acid, the plates must

be cleaned by plasma or UV-ozone treatment

22 Although there is a slight difference between the angle at the maximum enhancement of the electric field and the SPR angle, the angle of the incident light is set at the SPR condi-tion It is important to precisely set the incident angle at the SPR angle for the SPFS measurement because the intensity of fluorescence is highly sensitive to the electric field intensity

23 Although the electric field enhancement by the surface mons is strong, the excess intensity of the incident light causes photobleaching of the fluorescent dye For SPFS measurements in this system, the incident light is attenuated

plas-to 0.1 mW (~7.85 mW/cm2) with a neutral density filter (10% transmittance; Sigma Koki)

24 It is essential that an excess amount of the secondary body for AFP is present in the mixture

anti-25 As shown in Fig 1.8, the mixture of AFP and Alexa

Fluor-labeled secondary antibody is flowed for 5 min and adsorbed

to primary antibody on the surface After the washing step, the remaining fluorescence reflects the concentration of AFP, and quantitative analysis is possible

References

1 Homola, J (2003) Present and future of

sur-face plasmon resonance biosensors Anal

Bio-anal Chem 377, 528–539

2 Raether, H (1988) Surface Plasmons on Smooth

and Rough Surfaces and on Gratings Springer,

Berlin

3 Besselink, G A J., Kooyman, R P H., van

Os, P J H J., Engbers, G H M., and

Schas-foorta, R B M (2004) Signal amplification

on planar and gel-type sensor surfaces in face plasmon resonance-based detection of

sur-prostate-specific antigen Anal Biochem 333,

165–173

4 Chou, S F., Hsu, W L., Hwang, J M., and Chen, C Y (2004) Development of an immu- nosensor for human ferritin, a nonspecific

tumor marker, based on surface plasmon

res-onance Biosens Bioelectron 19, 999–1005

5 Teramura, Y and Iwata, H (2007) Label-free

immunosensing for α -fetoprotein in human

plasma using surface plasmon resonance Anal

Biochem 365, 201–207

6 Teramura, Y., Arima, Y., and Iwata, H (2006)

Surface plasmon resonance-based highly

sensi-tive immunosensing for brain natriuretic

pep-tide using nanobeads for signal amplification

Anal Biochem 357, 208–215

7 Liebermann, T and Knoll, W (2000)

Surface-plasmon field-enhanced

fluores-cence spectroscopy Colloid Surf A 171,

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Chapter 2

Surface Plasmon Resonance Biosensor for Biomolecular Interaction Analysis Based on Spatial Modulation Phase Detection

Xiang Ding, Fangfang Liu, and Xinglong Yu

Summary

Surface plasmon resonance (SPR) biosensor is a powerful tool for biomolecular interaction analysis in proteomics research and drug discovery But when it is used to analyze small molecules, the sensitivity still needs enhancement Phase detection is a potential solution, for phase changes more abruptly than intensity when SPR is excited An SPR system is developed based on spatial modulation phase detection (SMPD) In this system, collimated monochromatic light is used to excite SPR, and the phase of the reflected light is spatially modulated to generate an interference pattern By processing the interfer- ence pattern by certain algorithms, instantaneous phase distribution of the whole sensing area can be obtained Continuously detecting the phase change, the whole process of biomolecular interaction can

be recorded in the form of phase change, based on which we can make kinetic analysis and get kinetic parameters Antibody array chip is tested by this system Experimental results indicate that this technique

is capable of array detection and is more sensitive than intensity detection.

Key words: Surface plasmon resonance (SPR), Spatial modulation, Phase detection, Biomolecular interaction, Antibody array.

Present commercialized surface plasmon resonance (SPR) sensors are mostly based on intensity detection, which takes an advantage of simple system configuration But due to drifts of the light source, the photoreceiver, and the amplification circuit, the detection precision is degraded, commonly no better than 10−6

bio-refractive index unit (RIU)

1 Introduction

Avraham Rasooly and Keith E Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol 503

© Humana Press, a part of Springer Science + Business Media, LLC 2009

DOI: 10.1007/978-1-60327-567-5_2

21

In proteomics research and drug discovery, the voice for niques of small molecule detection is getting louder and louder Small molecules are usually defined as those whose molecular weights are below several hundred Daltons When interacting with other molecules, they generate very limited refractive index change, which is difficult to be detected So it is necessary to enhance the sensitivity of SPR biosensing techniques There are two solutions: one is to modify the analyzed molecules, for example, nanoparticle modification; the other is to excavate the potential

tech-of sensors As we know, modification to molecules may lead to

a lot of problems: poor repeatability, time wasting, influence on molecular activity, etc An approach to higher sensitivity without modification to molecules is preferable When SPR is excited, both the reflected light’s intensity and phase vary rapidly, but the phase changes much more abruptly than the intensity It means that detecting phase of the reflected light instead of its intensity

is a simple way to enhance the sensitivity of SPR biosensor retical analysis indicates that the sensitivity of phase detection is

Theo-higher than that of intensity detection by 1–2 magnitudes (1).

There are many phase detection techniques, including sometry, interferometry, and heterodyne, which have the simplest system configuration and the highest sensitivity among them Our group developed a SPR biosensing system and used heterodyne method to detect the interaction between ricin and its antibody

ellip-(2) However, it is difficult to realize array detection by heterodyne

Interferometry can detect the phase distribution over the whole sor area, and is appropriate for array detection We performed array detection based on spatial modulation phase detection (SMPD), and obtained phase distribution of the antibody-array chip and phase–time curves of all sensing units Experimental results indicate that this technique has a high sensitivity in array detection

sen-SPR technique can be used to analyze the interactions of a broad variety of biomolecules Among all biomolecular interaction model systems, antigen–antibody interaction model system is one

of the most common and simple one So we choose rabbit IgG as

an antigen and goat anti-rabbit-IgG IgG (as an antibody), a typical model system which can reflect common characteristics of antigen–antibody interaction, to illustrate the SPR biosensing technique

1 Piranha solution: 7:3 (volume ratio) mixture of concentrated sulfuric acid and 30% hydrogen peroxide

2 MUA solution: 11-mercapto-undecanoic acid (MUA, Sigma) dissolved in ethanol at 1 mM and stored at room temperature

2 Materials

2.1 Reagents and

Solutions

Surface Plasmon Resonance Biosensor for Biomolecular Interaction 23

3 Activation solution: 1:1 (volume ratio) mixture of N-hydroxy

succinimide (NHS, 0.1 M, Sigma) solution and fresh [3-dimethylaminopropyl]carbodiimide (EDC, 0.4 M, Sigma)

1-ethyl-3-solution (see Note 1)

4 Phosphate buffered saline (PBS) solution: 8 mM Na2HPO4, 1.5 mM KH2PO4, 27 mM KCl, and 1.37 M NaCl in high-purity H2O, pH 7.4

5 Regeneration solution: 0.2 M glycine, pH adjusted to 2.0 with hydrochloric acid

6 Rabbit IgG solution (Biodee, CN): dissolved in PBS at

2 The chip has a size of 12 mm × 12 mm × 1.2 mm, with both

surfaces polished (see Note 2)

3 A chromium (Cr) layer with a thickness of 2 nm is evaporated onto one surface of the chip, at a speed of 0.1 nm/s, in 5 ×

10−3 Pa vacuum (see Note 3)

4 A 40-nm gold (Au) layer is evaporated onto the chip surface with predeposition of Cr, at a speed of 0.1 nm/s, in 5 × 10−3

Pa vacuum (see Note 4)

5 Chips are stored in airtight container, protected from dirt and moisture

1 Before use, the gold surface of the chip should be cleaned thoroughly First, the chip is dipped in Piranha solution for

5 min, rinsed by pure water, and blown dry Then it is cleaned ultrasonically in ethanol for 5 min

2 Immerse the chip in MUA solution over 24 h to form a carboxyl terminated self-assembled monolayer on the gold surface Then rinse the chip by pure water and blow it dry

3 Carboxyl groups on the gold surface are activated by immersing the chip in the activation solution for 30 min before they can immobilize antibody molecules Rinse the chip by pure water and blow it dry

4 Rabbit IgG solution (1 mg/mL) is manually spotted on the activated carboxyl terminated surface by a pipette A 2 × 2

2.2 Sensing Chip

2.3 Preparation of

Antibody Array Chip

array of rabbit IgG spots is fabricated, with spot diameters of about 1 mm The antibody array chip is incubated at 35°C for

30 min and rinsed by PBS solution thoroughly

5 The chip is immersed in BSA solution (10 mg/mL) at 35°C for 30 min to block residual activated carboxyl groups, and rinsed by PBS thoroughly

6 The antibody array chip is glued to the flow cell

7 Fill the flow cell with PBS solution to keep the chip surface from becoming dry

The configuration of the SPR system is shown in Fig 2.1, which can be divided into three parts: the incident arm, the SPR sensing configuration, and the reflective arm

The incident arm consists of the following (numbers spond to those in Fig 2.1):

corre-1 A He–Ne laser (200-mm length and 50-mm diameter,

purchased from Qufu Normal University, CN), which

gener-ates frequency-stabilized laser with a beam diameter of about

2 mm, at wavelength of 632.8 nm

2 A pinhole of 10-μm diameter at the common focus of the lenses in the beam expander, made of aluminum

3 A beam expander that contains a microscope objective with

the focus length of 4.65 mm (Daheng, Inc., CN, GCO-2105) and a lens with the focus length of 30 mm (Daheng, Inc., CN,

Surface Plasmon Resonance Biosensor for Biomolecular Interaction 25

The SPR sensing configuration consists of the following:

6 A Kretschmann prism with an equiangular triangle section and three 20 mm × 12 mm side faces, made of ZF5 glass, whose refractive index is 1.740

7 A sensing chip (details in Subheading 2.2.2).

8 A flow cell (details in Subheading 2.3.3).

The reflective arm consists of the following:

9 A Wollaston prism that has a size of 12 mm × 12 mm × 12 mm

and a splitting angle of 0.3° (Qufu Normal University, CN,

LSP-03)

10 A polarizing prism that has a size of 10 mm × 10 mm × 27 mm

(Qufu Normal University, CN, LGP-01).

11 An imaging lens with focal length of 200 mm and diameter

Phase difference between the two light components can not

be measured directly But after passing through a polarizer, their phase difference is connected with the light intensity, defined as:

j

I I = + cos( ), 1 I2

where I is the intensity of interference light, I1 and I2 are mined by the intensities of the p-polarized light and the s-polar-

deter-ized light, and j is their phase difference Since I1 and I2 are

unknown, j cannot be obtained by just detecting I.

In SPR system based on SMPD, the Kretschmann

configu-ration (3) is employed The Kretschmann configuconfigu-ration is the

simplest method to excite SPR and has been widely used in mercialized and laboratory SPR systems, shown in Fig 2.3

com-In SMPD, the core optical element is a Wollaston prism, splitting the p-polarized component and s-polarized component of the reflected light by a small angle After passing through a polarizer,

the two light components interfere with each other and form

interference stripes on screen (see Fig 2.3), whose intensity is determined by:

I( , ) = + x y I1 I2cos[ ( , )x y fx ],

where f is spatial frequency of the stripes (see Note 5), x and y are

rectangular coordinates on the screen Collecting the ence images by a CCD camera and processing them with algo-rithms, phase distribution can be obtained Several algorithms can be used to calculate the phase difference, such as sinusoidal fitting, correlation, and Fourier transform process (FTP) FTP

interfer-Fig 2 Phase of p-polarized light against refractive index on SPR sensor surface at different gold film thicknesses The light

wavelength is 632.8 nm The prism’s dielectric constant is 3.08 and the gold’s dielectric constant is −10.92 + 1.49i.

Fig 3 The Kretschmann configuration and interference image in SPR system based on SMPD The reflected light passes through a Wollaston prism and a polarizing prism and forms interference stripes on screen.