Journal of Medical and Biological Engineering, 26(4): 149-153 149

Heterodyne Interferometric Surface Plasmon

Resonance Biosensor
Shen-Fen Joe Li-Zen Hsieh* Liann-Be Chang1 Chih-Chung Hsieh2

Chien-Ming Wu2
Department of Electrical Engineering, Chung-Cheng Institute of Technology, National Defense University, Tao-Yuan, Taiwan, 335 ROC
1
Department of Electronic Engineering, Chang Gung University, Tao-Yuan, Taiwan, 333, ROC
2
Department of Biomedical Engineering & Environmental Sciences, National Tsing-Hua University, Hsin-Chu, Taiwan, 300 ROC

Received 30 Jun 2006; Accepted 3 Nov 2006

Abstract
A high-sensitivity surface plasmon resonance (SPR) biosensor is constructed by using a common-path heterodyne
interferometric system. The beam combiner is employed to combine the modulated He-Ne TE-wave and TM-wave into
a heterodyne light source with a frequency difference of 60 kHz. Using the common-path heterodyne interferometric
system to obtain the phase shift between the two waves has the advantage of high-sensitivity real-time phase detection
for monitoring molecular interactions. By detecting the phase shift, the proposed SPR biosensor is employed to monitor
the interactions between the sheep IgG covalently immobilized on the sensor chip surface and the anti-sheep IgG
contained in the running buffer.

Keywords: Surface Plasmon Resonance, Biosensor, Heterodyne interferometry

occur, thus resulting in changes in the refractive index and

Introduction
Surface plasmon resonance (SPR) has the advantages of
being highly sensitive, requires no fluorescent-labeled analytes
or receptors, and is capable of monitoring biomolecular
interactions in real time. This technique has been employed in
commercially available optical biosensors [1-2]. SPR is the
collective oscillation of electrons that occurs at the interface
between a dielectric and a good conductor. To make it resonant,
the parallel polarization wave vector of the incident light
matches the wave vector of the surface plasmon [3-4]. Factors
affecting the resonance include the incident angle and
wavelength of the light source, as well as the permittivity of
the dielectric material near the metal surface. During
resonance, electrons from the metal absorb most of the energy,
thus rapidly decreasing the intensity of the reflected light.
According to this principle, with other parameters of the
reflected light held constant, changes in the intensity of the
reflected light will indicate the changes in the refractive index
of the metal-adjoining dielectric material.
In SPR biosensing, the ligand is immobilized on a metal
surface to form a biosensing film. The sample containing the
target analyte is passed over the surface of the biosensing film,
where interactions between the target analyte and ligand may
* Corresponding author: Li-Zen Hsieh
Tel: +866-3-3800301 ext.265; Fax: +886-3-3801407
E-mail: lzhsieh@ccit.edu.tw
thickness of the dielectric biosensing layer. Therefore,
examining the changes in the intensity of the reflected light
can reveal the molecule interactions [5-7]. Methods for
detecting the intensity of reflected light have been applied to
SPR biosensor [8-9]. The angle of incidence and wavelength
of the reflected light at its lowest intensity are taken as the
resonance angle and resonance wavelength, respectively.
During resonance, there is also a significant phase change of
the reflected TM-wave [10]. The phase detection is more
sensitive than those of angle or wavelength measurement [11].
There have been a few studies on the phase measurement
[12-16], and among these, the common-path interferometers
method has been found to be less affected by external
disturbance [17]. The TE-wave and TM-wave of different
frequencies are employed to obtain the reference and measured
beating frequency signals. Most of the noise is eliminated by
means of a high-quality filter, thus enhancing the purity of the
signal [18].
In this study, the SPR biosensor is developed using a
common-path heterodyne interferometer that is employed to
detect the interactions between the sheep IgG and its antibody
at concentrations ranging from 0.055 to 110µg/ml. Two
acousto-optical modulators are utilized to modulate the
TE-wave and TM-wave, thus forming a heterodyne light
source with a frequency difference of 60 kHz. Observing the
phase difference between the reference and measured signals
obtained by the dual-phase lock-in amplifier can reveal the
150 J. Med. Biol. Eng., Vol. 26. No. 4 2006
phase shift between the TE-wave and TM-wave. To examine
the performance of the proposed SPR biosensor, comparison is
also made with the commercial optical sensor Biacore X.
Principle
Figure 1 displays the five-layer Kretschmann-Raether
configuration of a SPR sensor chip. The five layers from top to
bottom are prism, gold film, linker, hydrogel and liquid, all of
which serve different functions. The prism provides total
internal reflection for inducing SPR. The linker layer, which is
attached to the gold film, provides a hydrophilic environment
for the immobilization of molecules passing through the
hydrogel layer. The antigen and antibody from the solution
interact and are bound covalently when passing through the
hydrogel layer. When the incident light reaches the interface
between the prism and the metal, the component of the wave
vector in the x direction is
kx = ko np Sinθ (1)
where k0=2 π λ , np is the refractive index of the prism,
and θ is the angle of incidence. The wave vector of surface
plasmon resonance is
ω ε mε d
K SP = ε m +ε d (2)
c
where ε m is the permittivity of gold and ε d is the
permittivity of the dielectric sample.
When both the TE-wave and TM-wave hit the interface of
the dielectric material, the reflection coefficient of the
TE-wave, rs, and the reflection coefficient of the TM-wave, rp,
can be expressed respectively as
iδ s
rs = rs e (3)
rp = rp eδ p (4)
i
where δs and δp denote the phase shift of the TE-wave and
TM-wave, respectively. Since the TE-wave cannot induce SPR,
there is little variation in the phase shift near the resonance
angle; hence, δs can be regarded as constant. In fact, phase shift
of the TM-wave δp shows great variation near the resonance
angle. The phase difference, Φ, between the TM-wave and
TE-wave can be expressed as
Φ=δp-δs. (5)
Since there exists a linear relationship between the phase
difference, Φ, and phase shift of TM-wave, δp, molecular
interactions on the sensor surface can be detected by measuring
the phase difference.
Experiment
3.1 Buffers and reagents
Sensor chip CM5, amine-coupling kits, HBS Buffer
(0.01M HEPES pH=7.4, 0.15M NaCl, 3mM EDTA, 0.005%
Surfactant P20), sodium acetate (pH=4.0,10mM) and
glycine-HCl (10mM pH= 2.0) were all purchased from
Pharmacia Biosensor AB (Uppsala, Sweden). Sheep IgG and
θ
Prism
Gold film
Linker
Hydrogel
Liquid
Figure 1. The five-layer Kretschmann-Raether configuration
M1 AOM1 PBS1
Laser
AOM2
PBS2
M2
Flow cell
Rotary stage
BS
Pol2
Pol1 PhD2
PhD1 Lock-in amplifier
Figure 2. Schematic diagram of the common-path heterodyne
interferometer
donkey anti-sheep IgG (whole molecule) were obtained from
SIGMA (St. Louis, MO, USA).
3.2 Experimental Setup
Figure 2 is the schematic diagram of the common-path
heterodyne interferometer used for detecting molecular
interactions. The light source is a linear polarized stabilized
He-Ne laser of 632 nm wavelength. The incident light is split
equally into two beams, TE-wave and TM-wave, by the
polarization beam splitter (PBS1). The TE-wave and TM-wave
are then modulated by two acoustooptic modulators AOM1 and
AOM2 (both are InterAction Corp, 400 series), with
frequencies of 40 MHz and 40.06 MHz, respectively. After
modulation, the two beams are combined into the heterodyne
light source with a frequency difference of 60 KHz by the beam
combiner (PBS2). Passing through a beam splitter (BS), the
heterodyne light source is again divided into two beams. One
passes through a polarizer (Pol1) and enters the photo-detector
(PhD1) as a reference signal, Ir, for the lock-in amplifier
(Standford Research System, model CR850). The other is
transmitted through two prisms, both of which are fixed on the
rotary stage (Newport, URM100PP). One prism has its side in
close contact with the sensor chip and flow cell, while the other
provides total internal reflection of the incident light beam. In
this way, the incident light beam transmitted onto the rotary
stage will be parallel to that reflected from the rotary stage.
 Heterodyne Interferometric SPR Biosensor 151

Finally, this beam passes through another polarizer (Pol2) and

enters the photo-detector (PhD2) to become a measurement
signal, Im, for the lock-in amplifier in detecting phase shift. The
reference signal, Ir, and measurement signal, Im, can be
expressed respectively as

I r ∝ Eso Epo Cos( ∆ ωt) (6)

I m ∝ ｒ Ep0Ep0 Cos( ∆ ωt+ Φ )

Ｐ
(7)

Eso and Epo denote the amplitudes of the TE-wave and

TM-wave, respectively. ∆ ω is the difference in beating
frequency between the TE-wave and TM-wave. Φ , the phase
difference between TM-wave and TE-wave, is detected by the Figure 3. Phase shift of sheep IgG immobilized on the sensor chip
senor chip and that after being reflected by the prism. surface using the amine-coupling method
Therefore, the difference between the measurement signal and
the reference signal obtained by the dual-phase lock-in
amplifier can provide real-time detection of the interaction
between biological molecules on the sensor chip surface.

Results and Discussion

4.1 Sheep IgG immobilization
To immobilize IgG involves binding it covalently to the
CM5 sensor chip using the amine-coupling method. All
reagents are injected by a peristaltic pump. First, the running
buffer, HEPES, is injected into the sensor chip, providing a
standard for future comparison. The sensor chip is then
activated by a mixture of EDC and NHS at a 1:1 ratio. The
sheep IgG is mixed with sodium acetate (pH = 4.0) at a 1:100
ratio, and then injected into the flow cell for covalent
immobilization on the CM5 sensor chip. The running buffer,
HEPES, is again injected into the flow cell to remove free
sheep IgG not bounded on the sensor chip. To prevent
non-specific binding, ethanolamine is employed to deactivate
the remaining active esters of the unbound sheep IgG.
Non-specific or unwanted binding can reduce overall assay
performance and sensitivity at extremely low detection
concentrations.
Figure 3 shows the phase shift and time taken for
immobilization of sheep IgG on the sensor chip surface. As
can be seen, there is an increase of 85 degree in phase shift as
a result of sheep IgG immobilization, indicating that the sheep
IgG has been successfully immobilized on the sensor chip
surface.
4-2 Biosensing Protocol
To analyze the antigen-antibody interaction, the running
buffer HEPES is first injected into the flow cell at a rate of 150
ml/min, followed by the injection of the anti-sheep IgG of
different concentrations stored in the sample loop after 4
minutes. Finally, glycine-HCl (pH = 2.0) is injected to
disassociate the anti-sheep IgG immobilized on the sensor chip
to measure the phase shift at various concentrations of
anti-sheep IgG. Regeneration of the sensor surface with
non-specifically bound anti-body removed is important for
maintaining binding reproducibility.
Figure 4 The sensorgram obtained using the common-path
heterodyne interferometer
4.3 Measurement with different concentration
Figure 4 displays the binding of anti-sheep IgG at various
concentrations to immobilized sheep IgG obtained using the
phase-measurement-based SPR biosensor. As can be seen,
there are increases in phase shift of 4, 14, 33, 62 and 107
degrees at anti-sheep IgG concentrations of 0.055, 0.11, 1.1, 11
and 110 µg/ml. At concentrations below 1.1 µg/ml, the
increase is slow and gradual, while at concentrations of 110
µg/ml, the increase is sharp and marked.
Figure 5 shows the relationship between phase shift and
concentration obtained using the developed SPR biosensor. As
can be seen, there exists a linear relationship between the
phase difference and various concentrations of anti-sheep IgG.
Their correlation can be expressed as Y = 5.595 logX + 38.225
with R2 =0.9686, where X is the concentration of anti-sheep
IgG, and Y is the phase difference.
T h e sa m e e xp e r i me n t wa s c o n d uc t e d usi n g a
commercially available optical biosensor, Biacore X (Biacore
International SA, Uppsala, Sweden), and the results are shown
in Figure 6. As can be seen, no change in the resonance unit
can be detected at anti-sheep IgG concentrations of 55ng/ml
and 110ng/ml. However, at anti-sheep IgG concentrations of
1.1, 11 and 110 µg/ml, there are significant changes in the
resonance unit that can be attributed to molecular interactions.
152 J. Med. Biol. Eng., Vol. 26. No. 4 2006
Figure 5 Plot depicting the relationship between phase difference
and concentration of the sensorgram data from Fig.4
Figure 6 The sensorgram obtained using Biacore X
Figure 7 Plot depicting the relationship between change in resonance
unit and concentration of the sensorgram data from Fig.6
Figure 7 is the plot of concentration of anti-sheep IgG against
changes in resonance units, showing a linear relationship
between the two. Their correlation can be expressed as Y =
1.5699 logX + 1.475 with R2 =0.9949, where X is the
concentration of anti-sheep IgG and Y is the change in the
resonance unit.
Comparing the results shown in Figure 5 and Figure 7
reveals that the phase-measurement-based SPR biosensor is
superior in its capacity to detect phase difference at lower
concentrations of anti-sheep IgG, while the results obtained
from Biacore X better fit the linear relationship between
changes in resonance unit and concentrations of IgG.
Conclusion
This study develops a sensitive SPR biosensor with the
common-path heterodyne interferometric system to detect the
molecular interactions between sheep IgG and its antibody.
The proposed biosensor utilizes a pair of acoustooptic
modulators to modulate the TE-wave and TM-wave, thus
forming a heterodyne light source with a frequency difference
of 60 kHz. The phase difference between the TE-wave and
TM-wave can be obtained by using the dual-phase lock-in
amplifier to obtain the difference in beating frequency between
the reference signal and the measured signal. The obtained
phase difference of 4, 14, 33, 62 and 107 degrees between the
sheep IgG immobilized on the sensor chip and the anti-sheep
IgG with a corresponding concentration ranges from 0.055 to
110 µg/ml. The proposed SPR biosensor constructed with the
common-path heterodyne interferometric system does not
require fluorescent-labeled analytes or receptors. Not only can
it reduce disturbance from the surrounding environment, it can
also provide high-sensitivity real-time phase detection for
monitoring molecular interactions. Compared with the
commercial optical biosensor, Biacore X, this proposed SPR
biosensor has better performance in detecting the anti-sheep
IgG at concentrations as low as 55ng/ml.
Acknowledgement
This work was supported by the National Science Council
of ROC under Grant No. NSC-94-2215-E-007-015.
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