Selected Papers

Chemical Sensors and Surface Plasmon
Resonance Biosensors
Yuzuru Iwasaki†, Tatsuya Tobita, Tsutomu Horiuchi,
and Michiko Seyama
Abstract
We have developed optical sensors for detecting biological samples using surface plasmon resonance(SPR), which is a basic platform for many kinds of chemical sensors. In this paper, we introduce the principle of chemical sensors, especially SPR sensors for biological substances. SPR can detect the specificbinding of biological molecules but was previously unable to detect enzymatic reactions, which are amajor class of biological reactions. To overcome this limitation, we developed electrochemical SPR sensor techniques and made an enzyme-based sensor array.

1. Introduction
Chemical substances affect our lives both directly
and indirectly. Since all living things take in chemicals and air and then metabolize them, our vital functions are strongly dependent on the chemicals aroundus. For example, heavy metals can poison the foodchain, acid rain damages trees, endocrine-disrupting
chemicals are suspected of having long-term effects
on human and animal health, and some people haveallergic reactions to particular chemicals. Therefore,
the detection and identification of chemicals is very
important.
At the cellular level, all information is transmitted
in the form of molecular signals, and genetic information is stored in the form of DNA sequences.
These days, the information transmitted overtelecommunication networks is acquired by physicaldevices such as keyboards, computer mice, microphones, and cameras. However, in the coming era ofubiquitous communication, this physical information
will be supplemented by information about chemical
substances (Fig. 1). For example, continuous monitoring of our personal health by measuring biomarkers will assist in the early diagnosis and treatment of† NTT Microsystem Integration Laboratories
Atsugi-shi, 243-0198 Japan
E-mail: iwasaki@aecl.ntt.co.jp

Vol. 4 No. 1 Jan. 2006

lifestyle diseases. These personal health conditions
vary from person to person and vary over time, so
chemicals should be measured easily with realtime
performance. To achieve such communication weneed to develop devices to obtain information about
these chemical substances, such as the class of chemical and its concentration. When we can identify andmeasure the concentration of chemical substances
anytime and anywhere in the real world using a small(ubiquitous) sensor, we will be able to take innovative
steps toward solving environmental and medical
problems and improving our daily lives.
Our research group has studied chemical sensors
designed to have a small size and high performance.
To meet these two requirements, we have studied
microfabrication technology to allow us to measuresmall sample volumes, surface science and biochemistry to improve detection sensitivity, and opticalchemical sensing technologies to enable the integration of several different types of sensors, such as anelectrochemical micro-sensor, enzyme-based sensor,
and optical sensor. In this report, we introduce ourrecent development of an optical sensor for biochemical analysis that we constructed using surface plasmon resonance (SPR) as the first step towards ubiquitous chemical sensors. SPR sensors have alreadybeen built as analytical instruments and used as a tool
for searching for candidates of pharmaceuticals. The
main features of an SPR sensor are its simple struc21

Selected Papers

Chemical sensors
Biological, environmental
chemicals

Physical sensors
Visual, auditory, tactile

Ubiquitous sensors
Analytical techniques
Spectroscopy
Mass spectroscopy
Chromatography

Chemical analysis

Current communication appliancesFig. 1. Ubiquitous sensors.

ture and its potential to be made small enough to be
integrated into a headset or cellular phone by meansof optical microfabrication technologies in the future.
Several classes of small sensors, such as immunosensors, DNA sensors, and enzyme-based sensors, havealready been developed and arrays of sensors of one
class have also been developed. The SPR sensor can
detect the specific binding of biological molecules
such as antigen-antibody and DNAs, but it has notbeen able to detect enzymatic reactions. However, aubiquitous chemical sensor must be able to integratedifferent kinds of detection schemes in a single platform. We show that electrochemical SPR sensor technology can be used to overcome this limitation, andwe apply it to an array of enzyme sensors.
2. Chemical sensors
2.1 Overview
When detecting molecules we want to identify a
specific molecule and quantify its amount. A sample
obtained from the environment or a sample of body
fluid is a mixture of many molecules, so a critical stepin conventional chemical analysis is to separate thedifferent molecules. Molecular separation techniquessuch as chromatography and mass spectroscopy havebeen developed. They allow the precise measurementof natural samples, and they are already contributing
to the medical, environmental, and fundamental
chemical analysis needs of a number of industries.
However, these separation techniques require large
instruments. A chemical sensor, on the other hand,
can detect a specific molecule without separation.
22

Chemical sensors consist of a molecular recognition unit and a transducer unit. Instead of separating
all the molecules, the molecular recognition unit
identifies only a specific group of molecules bymeans of a chemical reaction. The physical change
resulting from the reaction is converted into a physical signal by a transducer (Fig. 2). The physical signals used by chemical sensors include current, voltage, light intensity, and refractive index. Chemicalsensors can be made small and used at the location
where the specimen is sampled. NTT Microsystem
Integration Laboratories has recently developed
small chemical sensors for an environmental gas
monitoring system [1], for an odor recognition sensor[2], and for a realtime monitoring system for cardiac
patients [3]. The molecule identification performanceof chemical sensors depends on the molecular recognition unit, whereas the sensitivity depends mainly on
the transducer unit. The former can be improved byusing biological molecules and the latter can be
improved by using a technique for measuring theoptical refractive index.
2.2 Measurement of biomolecular interactions
Biological reactions are primarily controlled by the
specific binding reaction of two molecules. For
example (Fig. 3), an antibody finds a specific molecule with a characteristic structure (antigen) andbinds firmly to it. This binding triggers a chain of
immunoreactions. A sequence of single-strand DNA
binds with a complementary sequence of DNA toform double-strand DNA. These natural biologicalmolecule-specific reactions, which are called biomolNTT Technical Review

Selected Papers

Current
VoltageTransducer

Light intensity
Mass
Refractive index

Molecular recognition

Conversion into physical signal

Fig. 2. Elements of chemical sensor.

Mismatched DNA

Matched(complementary)DNA

Immobilizedprobe DNAA) Hybridization of DNA or RNA
AntigenInterfering
molecule

Immobilized
antibodyB) Antigen-antibody reaction

Fig. 3. Increase in refractive index caused by binding reaction.

ecular interactions, can be used to detect a specificmolecule without a separation step. A sensor basedon this idea is called a biosensor [4]. When one molecule of the pair is immobilized on a substrate and asample solution containing the other molecule of thepair is brought into contact with it, the immobilized
molecule captures the complementary molecule by amolecule-specific recognition reaction such as an
antigen-antibody reaction, DNA hybridization, or an
enzymatic reaction. Molecules that are not recogVol. 4 No. 1 Jan. 2006

nized by these reactions are not held on the substrate.
As a result, the target molecule is concentrated on thesubstrate and then detected by a transducer unit. For
example, a detection scheme using a fluorescent molecule and a photodetector as a transducer unit is widely used in biological analysis. A fluorescent molecule
is chemically bonded to all the molecules in a sample
before they come into contact with the substrate.
When the sample reacts with the substrate, fluorescence is observed on the substrate if the sample con23

Selected Papers

2.3 Refractive index measurement using SPR
The refractive index of a solution correlates with its
concentration. A commonly used method of determining the refractive index is to measure the criticalangle. The measurement sensitivity can be improvedby using surface plasmon resonance (SPR) measurement. SPR is a strong coupling phenomenon between
light and the free electrons in a metal surface [5], andit also refers to the measurement method. The mostcommonly used metal for sensor applications is gold.
The principle of SPR is shown in Fig. 4. A gold sensing film is formed on a prism having a high refractive
index. The gold film is 40 to 50 nm thick and is semitransparent. The refractive index of the gold surface(sensing layer) is measured. The gold surface is illuminated from the prism side with monochromatic
light and the reflection intensity is measured as afunction of the angle of incidence. Because therefractive index of the prism is higher than that of thesensing layer, the incident light undergoes totalreflection. However, the reflected p-polarized light(the electric field of the light is parallel to the plane
including the incident and the reflected light path) is
diminished at a certain incidence angle when thingold film is present at the prism surface. As shown in
Fig. 5, depending on the refractive index of the sensing layer, the curve of reflectivity versus incidence
angle moves to a larger angle as the refractive index
of the sensing layer increases. This curve is called an
SPR curve and the incidence angle of the minimum
reflectivity is called the SPR angle. When the prism
material is BK7, a refractive index of up to 1.5 can bemeasured. Because many biological molecules insolution phase have refractive indexes between 1.0
and 1.4, SPR can detect a wide range of biologicalmolecules. The dependence of the SPR angle shift on
the refractive index change is determined by a Fresnel multilayer reflectivity calculation. As shown in
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Sensing layer
Gold film

High-refractiveindex prism
Monochromatic
light source

Detector

Angle ofincidence

Fig. 4. Measurement of refractive index by SPR.

1

P-reflectivity

tains the target molecule. The molecular concentration can be calculated from the fluorescence intensity. This method does not require a separation step but
does need the pre-reaction or pretreatment of the molecules in the sample with fluorescent molecules.
Because the binding of a pair of molecules causes
an increase in molecular density, the recognition
reaction can be detected by measuring the change inrefractive index of the substrate surface. In a liquidsample, a bound molecular complex has a higher
refractive index than an unbound molecular set. Thisis a simple procedure, but it requires high-resolution
measurement of the refractive index.

Ethanoln = 1.36

0.5
Air
n = 1.0

Polypeptide filmn ≈ 1.4

Water
n = 1.304060Angle of incidence

80Fig. 5. SPR curves for different molecules (n = refractive
index).

Fig. 6(a), the SPR curve shows a large reflectivity
change in a narrow incidence angle range, and the
SPR angle can be determined with high resolution.
Moreover, the refractive index of the sensing layer isdirectly related to the SPR angle, as shown in Fig.
6(b).
A practical optical setup for SPR experiments isshown in Fig. 7. An inexpensive LED (light emitting
diode) light source is commonly used. After being
collimated with a lens system, the light emitted fromthe LED enters the prism and is focused on its centerwith a certain range of incidence angles. The reflected light is imaged by an array photodetector such as a
CCD (charge-coupled device) camera. In this opticalsetup, the incidence angle corresponds to the positionof the array detector, and the reflectivity is determined from the measured brightness of the image.
The SPR angle can be calculated from the image. As
shown in Fig. 7, SPR can be carried out using a simNTT Technical Review

Selected Papers

1.40

P-reflectivity

Refractive index ofsensing layer

1

0
40

60

80

1.35

1.30
65.0

70.0

(a) Angle of incidence

75.0(b) SPR angle

Fig. 6. Refractive index and SPR angle.

Sensing layer

Prism
Surface plasmon
Gold film
P-polarizer

De

te

cto

r

Light source

Fig. 7. Optical setup for SPR.

ple optical setup. Moreover, the measuring system issmall enough to fit in the palm of your hand. In the
future, it will be possible to integrate all of the opticalelements using optical fabrication technologies.
2.4 Refractive index mapping using SPR
For biological analysis, it is desirable to measure
many samples in a single operation. A chemical sensor can detect not only a specific molecule, but also
multiple different molecules if it is configured as anarray of chemical sensors for different targets. Furthermore, an array of a single type of chemical sensor
can be used to measure the spatial distribution of aVol. 4 No. 1 Jan. 2006

biological reaction such as cell metabolism. The optical setup shown in Fig. 8 enables SPR to be used forthis purpose. In this setup, an area of gold film is illuminated with collimated light with a fixed incidence
angle and the reflected light forms the image of thereflecting surface on an imaging device. The incidence angle is set slightly smaller than the SPR angle,
and a small refractive index change or variation isobserved as a change in reflection intensity. For
example, in Fig. 9, the SPR curve shows the reflection minimum incidence angle at 63.15° when therefractive index of the sample layer was 1.32 (redcurve). The biological binding reaction increased the25Selected Papers

Sensing layer

Gold film

Lens
Collimator

Prism

Polarizer

Angle of incidence
Optical fiber

ImagingdeviceLens

Monochromatic light source

Fig. 8. Optical configuration for imaging SPR.

3. SPR enzyme-based sensor

1

P-reflectivity

n = 1.322
Rp = 0.44

0.5

SPR = 63.15°

n = 1.320
Rp = 0.23
0
62

63

64

Angle of incidence (degrees)Fig. 9. Refractive index mapping.

refractive index of the sample to 1.322, and the SPR
curve shifted to the right (green curve). The observedreflectivity increased from 0.23 to 0.44 when thereflected light was observed at an incidence angle of
63.0°. Therefore, small variations in the refractive
index can be mapped or imaged. Although many biological molecules are transparent at visible to nearinfrared wavelengths, a slight change in a biologicalreaction can be detected and visualized using thismethod based on refractive index difference.

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Molecule-specific binding reactions can be detected by SPR. This is because the binding constants ofantigens and antibodies and of the complementary
sequences of DNA are very high, so the lifetimes of
the complexes are sufficiently long for them to be
detected by SPR as a refractive index increase. Enzymatic reactions are another major class of molecular
recognition reactions in biological systems. An
enzyme recognizes its specific substrate moleculeand forms an enzyme-substrate intermediate, whichthen catalyzes the chemical reaction of the substrate
and releases the molecule. The lifetime of the intermediate is too short to provide a sufficient differencein the refractive index, so previous efforts to detect
enzymatic reactions by SPR were ineffective. Nevertheless, since SPR is already used as an immunosensor and DNA sensor, if it were possible to detect
enzymatic reactions by SPR, then all the major biological reactions could be detected by SPR.
We have already developed a novel electrochemical
reaction analysis method using SPR. By using gold
film as a working electrode for an electrochemical
reaction, we were able to perform a realtime in-situ
analysis of the surface status of the gold electrode [6].
Because electrochemical reactions involve achange in the electron states of a molecule and arearrangement of the double-layer structure on thesurface of the gold film, the rate of the electrochemical reaction can be determined by SPR-based refracNTT Technical Review

Selected Papers

Gold film
Enzyme film
Glucose(substrate)

LED

Os-polymer + HRP film
H2O

HRP (ox.)Os2+

Glucoseoxidase

Prism
(BK7)
H2O2

HRP (red.)

Polarizer

Os3+

C

C

D

Gluconolactone
Sensing layer

Current
Glucose

GOx

H2O2

HRP

Os2+/3+
Refractive
index

Fig. 10. Structure of SPR enzyme-based sensor.

tive index measurement. We used this method to construct an enzyme-based sensor. As an example, weconstructed a glucose sensor. The determination of
glucose concentration is important in the treatment ofdiabetes and the analysis of food. We constructed
multiple enzyme layers and an electron mediatorlayer in the sensing layer of the SPR sensor, as shownin Fig. 10. The optical setup was the same as thatshown in Fig. 7. As the electron mediator, we used an
osmium ion complex that shuttles electrons between
the gold and horseradish peroxidase (HRP), which isan enzyme that catalyzes the reduction of hydrogen
peroxide. On the HRP layer, we formed a layer of
glucose oxidase (GOx), which recognizes the glucose
molecule, catalyzes the oxidation of glucose, andproduces hydrogen peroxide. On the gold electrode,the osmium ion complex underwent a redox reaction,and the refractive index of the film measured by SPR
exhibited a large change. SPR enables the redox status of the electron mediator film to be monitored inreal time [7]. When a sample containing glucose wasintroduced on the sensor surface, GOx producedhydrogen peroxide at a rate proportional to the glucose concentration. The hydrogen peroxide reactedwith the HRP in the sensing layer, and the HRP reacted with the osmium ion complex. As a result, theredox state of the osmium complex was quantitatively changed at a rate proportional to the glucose concentration. Using this scheme, we were able to determine the glucose concentration from the rate ofVol. 4 No. 1 Jan. 2006

change of the redox state of the osmium complex
layer measured by SPR [8]. The sensitivity of thissensor to glucose was 104 times higher than when theconcentration was determined from the refractive
index increase of the glucose concentration itself.
This result shows that an enzymatic reaction can bemeasured by SPR, which is the first step towards thetotal integration of biological sensors.
4. SPR imaging of biochemical reactions
Using the optical setup shown in Fig. 7 and the SPR
enzyme sensor described in the previous section, we
can visualize a biological reaction or construct an
enzyme sensor array. The glucose sensor structure
shown in Fig. 10 was constructed on a gold thin film.
The HRP and osmium ion complex layer wereformed on the surface. Then a GOx layer was spincoated onto the surface. The resulting multi-layer
film was observed. The refractive index changes ofthis sensor film are shown in Fig. 11 [9]. The osmium
ion complex layer was visualized as a ring-shaped
region. The reflected light intensity corresponds to
the refractive index. By controlling the redox state of
the film with the electrode potential and differentiating two images taken at different redox states, wewere able to visualize the redox state of the osmium
ion complex layer (Fig. 11(a)). The taller regions correspond to high redox reaction activity while thelower regions represent little or no redox reaction.
27

Selected Papers(a) Os2+ ↔ Os3+(b) Glucose → Gluconolactone

Fig. 11. Visualization of enzyme reaction.

Initially, we fully reduced the film electrochemicallyand then applied a glucose solution to the sensor surface. The glucose gradually reacted with the enzymes
on the sensor and the osmium ion complex was oxidized. The difference between the refractive indicesof the initial state (before enzymatic reaction) andfinal state (after the reaction) is shown in Fig. 11(b).
The zero level is shown as dark yellow (fringe area)in this picture. When other sugars that are not recognized by GOx were used, no spatial variation wasobserved in this experiment, so the structure shown inthis picture visualizes the enzymatic reaction. This
result also shows that we could measure an enzymatic reaction in a region as small as a single pixel of the
CCD camera we used and that a sensor array can beformed using this technique.
5. Summary
This paper explained the principle of chemical sensors and described an SPR sensor for detecting biological molecules. By combining it with electrochemical methods, we were able to use SPR to detectboth the binding reactions of biological moleculesand enzymatic catalytic reactions. Furthermore, we
demonstrated the visualization of an enzymatic reaction, which is a fundamental technique for an integrated biological sensor array. SPR is now commonly used in the field of bioinformatics research. A
portable system and an SPR imaging system havealready been commercialized by the NTT group [10].
SPR sensors can be made small and inexpensive andfurther improvements in sensitivity and usability will
make this sensor a platform for ubiquitous chemicalsensors.

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Acknowledgments
We thank Professor Koji Suzuki of Keio University, Kazuyoshi Kurihara of NICT, and Osamu Niwa of
AIST for their collaboration in developing the SPR
imaging system.
References[1]

Y. Ueno, T. Horiuchi, M. Tomita, O. Niwa, H-S. Zhou, T. Yamada,and I. Honma, “Separate detection of BTX mixture gas by a microfluidic device using a function of nanosized pores of mesoporous silica
adsorbent,” Anal. Chem. Vol. 74, 5257-5262, 2002.
[2] M. Seyama, Y. Iwasaki, S. Ogawa, I. Sugimoto, A. Tate, and O. Niwa,“Discriminative detection of volatile sulfur compound mixtures witha plasma-polymerized film-based sensor array installed in a humidity-control system,” Anal. Chem. Vol. 77, 4228-4234, 2005.
[3] K. Hayashi, Y. Iwasaki, R. Kurita, K. Sunagawa, O. Niwa, and A.
Tate, “The highly sensitive detection of catecholamines using a
microfluidic device integrated with an enzyme-modified pre-reactor
for interferent elimination and an interdigitated array electrode,” J. of
Electroanalytical Chem. Vol. 579, 215-222, 2005.
[4] L. J. Blum and P. R. Coulet, “Biosensor Principles and Applications(Biotechnology and Bioprocessing Series),” Marcel Dekker Inc. 1991.
[5] V. M. Agranovich and D. L. Mills, “Surface Polaritons,” North-Holland Publishing Company, 1982.
[6] Y. Iwasaki, T. Horiuchi, M. Morita, and O. Niwa, “Time differential
surface plasmon resonance measurements applied for electrochemical
analysis,” Electroanalysis, Vol. 9, No. 16, 1239-1241, 1997.
[7] S. Koide, Y. Iwasaki, T. Horiuchi, O. Niwa, E. Tamiya, and K.
Yokoyama, “A novel biosensor using electrochemical surface plasmon resonance measurements,” Chem. Commun, No. 9, 741-742,
2000.
[8] Y. Iwasaki, T. Horiuchi, and O. Niwa, “Detection of electrochemical
enzymatic reactions by surface plasmon resonance measurement,”
Anal. Chem. Vol. 73, 7373, 2001.
[9] Y. Iwasaki, T. Tobita, K. Kurihara, T. Horiuchi, K. Suzuki, and O.
Niwa, “Imaging of electrochemical enzyme sensor on gold electrodeusing surface plasmon resonance,” Biosensors and Bioelectronics,
Vol. 17, 783-788, 2002.
[10] http://www.ntt-at.com/products_e/handy-spr/

NTT Technical Review

Selected Papers

Yuzuru Iwasaki

Michiko Seyama

Research Scientist, NTT Microsystem Integration Laboratories.
He received the B.E. and M.E. degrees in polymer science and bio-electrochemistry from
Tokyo Institute of Technology, Tokyo and theDr.Eng. degree in synthetic chemistry from the
University of Tokyo, Tokyo in 1986, 1989, and1992, respectively. He joined NTT Basic
Research Laboratories in 1992 and moved to
NTT Environmental and Energy Technology
Laboratories, NTT Lifestyle and Environmental
Technology Laboratories, and finally to NTT
Microsystem Integration Laboratories in 2002.
He has been involved in R&D of electrochemistry including the analysis of electron transfer
reactions of proteins, development of ion transfer
battery materials, and optical biosensors.

Network Hardware Integration Laboratory,
NTT Microsystem Integration Laboratories.
She received the B.S., M.S., and Ph.D. degrees
in applied chemistry from Waseda University,
Tokyo in 1995, 1997, and 2004, respectively.
She joined the NTT Integrated Information andEnergy Systems Laboratories, Tokyo in 1997.
She moved to the Lifestyle and Environmental
Laboratories in 1999 and to the Microsystem
Integration Laboratories in 2002. She has
researched optical chemical sensing and odor
sensing systems. She is a member of the Electrochemical Society of Japan, the Japan Society for
Environmental Chemistry (JEC), and the Societyof Polymer Science (Japan). She received a
research promotion award and paper award from
JEC in 1999 and 2001, respectively.

Tatsuya Tobita

Tsutomu Horiuchi

Engineer, Materials Analysis Center, NTT
Advanced Technology Corporation.
He received the B.E. and M.E. degrees from
Shinshu University, Nagano in 1993 and 1995,
respectively. Since joining NTT Advanced Technology Corporation in 1995, he has been
involved in the development of an optical fiber
coupler machine and SPR measurement equipment.

Senior Research Scientist, Network Hardware
Integration Laboratory, NTT Microsystem Integration Laboratories.
He received the B.S. and M.S. degrees inphysics from Waseda University, Tokyo and the
Ph.D. degree in chemistry from Keio University,
Kanagawa in 1983, 1985, and 1994, respectively.
He joined NTT Ibaraki Electrical Communication Laboratories in 1985. He has been engaged
in research of electrochemistry and biosensors
and chemical sensors. He was a visiting scientist
at New Mexico State University, Las Cruces,
NM, USA, in 1997. He is a member of the Japan
Society of Applied Physics.

Vol. 4 No. 1 Jan. 2006

29