Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease
The influence of the sensitive element fabrication technology and noise performance of surface plasmon resonance (SPR) immunosensor on sensitivity and stability of operation inherent to the “Plasmon” series instrument has been investigated to improve reliability of diagnosis and treatment of the Eps...
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Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України
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irk-123456789-1215312017-06-15T03:05:21Z Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease Khrystosenko, R.V. The influence of the sensitive element fabrication technology and noise performance of surface plasmon resonance (SPR) immunosensor on sensitivity and stability of operation inherent to the “Plasmon” series instrument has been investigated to improve reliability of diagnosis and treatment of the Epstein–Barr herpes virus disease. To minimize the effect of the temperature change induced noise, compensation (introduction of the reference channel) and stabilization (active thermal control) methods were applied, allowing to substantially enhance resolution of the used SPR sensor system. 2016 Article Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease / R.V. Khrystosenko // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2016. — Т. 19, № 1. — С. 84-89. — Бібліогр.: 19 назв. — англ. 1560-8034 DOI: 10.15407/spqeo19.01.084 PACS 73.20.Mf, 87.55.fk http://dspace.nbuv.gov.ua/handle/123456789/121531 en Semiconductor Physics Quantum Electronics & Optoelectronics Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України |
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The influence of the sensitive element fabrication technology and noise performance of surface plasmon resonance (SPR) immunosensor on sensitivity and stability of operation inherent to the “Plasmon” series instrument has been investigated to improve reliability of diagnosis and treatment of the Epstein–Barr herpes virus disease. To minimize the effect of the temperature change induced noise, compensation (introduction of the reference channel) and stabilization (active thermal control) methods were applied, allowing to substantially enhance resolution of the used SPR sensor system. |
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Khrystosenko, R.V. |
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Khrystosenko, R.V. Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease Semiconductor Physics Quantum Electronics & Optoelectronics |
| author_facet |
Khrystosenko, R.V. |
| author_sort |
Khrystosenko, R.V. |
| title |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease |
| title_short |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease |
| title_full |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease |
| title_fullStr |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease |
| title_full_unstemmed |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease |
| title_sort |
optimization of surface plasmon resonance based biosensor for clinical diagnosis of the epstein–barr herpes virus disease |
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Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України |
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2016 |
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| citation_txt |
Optimization of surface plasmon resonance based biosensor for clinical diagnosis of the Epstein–Barr herpes virus disease / R.V. Khrystosenko // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2016. — Т. 19, № 1. — С. 84-89. — Бібліогр.: 19 назв. — англ. |
| series |
Semiconductor Physics Quantum Electronics & Optoelectronics |
| work_keys_str_mv |
AT khrystosenkorv optimizationofsurfaceplasmonresonancebasedbiosensorforclinicaldiagnosisoftheepsteinbarrherpesvirusdisease |
| first_indexed |
2025-07-08T20:03:12Z |
| last_indexed |
2025-07-08T20:03:12Z |
| _version_ |
1837110390614392832 |
| fulltext |
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2016. V. 19, N 1. P. 84-89.
doi: 10.15407/spqeo19.01.084
© 2016, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
84
PACS 73.20.Mf, 87.55.fk
Optimization of surface plasmon resonance based biosensor
for clinical diagnosis of the Epstein–Barr herpes virus disease
R.V. Khrystosenko
V. Lashkaryov Institute of Semiconductor Physics, NAS of Ukraine,
41, prospect Nauky, 03028 Kyiv, Ukraine
E-mail: khristosenko@ukr.net
Abstract. The influence of the sensitive element fabrication technology and noise
performance of surface plasmon resonance (SPR) immunosensor on sensitivity and
stability of operation inherent to the “Plasmon” series instrument has been investigated to
improve reliability of diagnosis and treatment of the Epstein–Barr herpes virus disease.
To minimize the effect of the temperature change induced noise, compensation
(introduction of the reference channel) and stabilization (active thermal control) methods
were applied, allowing to substantially enhance resolution of the used SPR sensor
system.
Keywords: Epstein–Barr virus (EBV), antibodies, surface plasmon resonance (SPR),
sensitive element, noise effect.
Manuscript received 10.11.15; revised version received 21.01.16; accepted for
publication 16.03.16; published online 08.04.16.
1. Introduction
For several recent decades, there observed is a growing
interest to the miniature, cheap and sensitive optical
transducers for direct detection of molecular interactions
in a real time scale without molecular labeling, and
application of these transducers in the fields such as
environment and industrial waste monitoring, production
quality assessment, discovery of new drugs and clinical
diagnosis [1]. In these sensors, one type of interacting
molecules is immobilized on the transducer surface,
forming a sensitive element, and the binding of a com-
plementary molecule to it is monitored measuring the
changes in optical density at the sensor surface. The sur-
face plasmon resonance (SPR) method is one of the most
advanced and well-developed optical sensing techniques
and is widely applied for detection of chemical and
biological substances [2]. However, up to this date the
SPR-based instruments suffer from insufficient
performance, sensitivity, and high production cost [3].
Surface plasmons (SP) are the normal modes of
charge density existing at the interface between
dielectric and metal [4]. Conditions for the resonant
coupling of SP and electromagnetic field of exciting
light are extremely sensitive to the changes in optical
properties of dielectric medium near the metal surface.
These conditions are characterized by the surface
plasmon resonance phenomenon and can be registered
using various configurations for surface electromagnetic
wave excitation (prism or grid) and various methods for
measuring the metal/dielectric interface reflectance
(scanning the angle of incidence at the fixed wavelength,
or wavelength scanning for the fixed angle of incidence,
or combining both of them).
Since the spreading of viral infections grows
inexorably, adversely affecting human health, one of the
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2016. V. 19, N 1. P. 84-89.
doi: 10.15407/spqeo19.01.084
© 2016, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
85
important fields of SPR method application is rapid and
accurate detection of viral agents and antibodies in blood
serum [5-7].
The Epstein–Barr virus (EBV, 4th type human
herpevirus) is one of the most widespread and dangerous
human viruses, named after the English virologist prof.
Michael Anthony Epstein and his graduate student
Yvonne Barr, who described the virus in 1964 [8].
A clinical form of initial EBV infection is the infectious
mononucleosis that affects lymphatic and nervous
systems [9]. The EBV facilitates development of chronic
fatigue, autoimmune disorders and cancer [10]. In
majority of cases, the disease is asymptomatic, however
like to the other herpevirae EBV activates during
immunodeficiencies of various origins (HIV infection,
cancer, hard radiation or stress) and can by itself become
a cause for immunosuppression.
The purpose of this work was to investigate the
influence of sensitive elements preparation technology
and noise characteristics of the SPR immunosensor on
sensitivity and stability of “Plasmon” series instrument
operation for reliable detection of EBV antibodies in the
human blood serum.
2. Methods and materials
Methods. In clinical practice, molecular biology
methods such as polymerase chain reaction (PCR),
immunofluorescence, and enzyme-linked immune-
sorbent assay (ELISA) are used for EBV infection
diagnosis. The latter method is the most suitable for
large-scale analysis, however, it is expensive and time-
consuming [11]. All these methods are based on a
specific antigen-antibody interaction. Antibodies are
soluble proteins (immunoglobulins) produced by B-lym-
phocytes in response to the presence of external or
intrasystem antigens. They selectively bind to the
antigens with formation of an inert antigen-antibody
complex, thus blocking the antigen interaction with the
other host molecules. Presence of the specific antibodies
in circulating system can serve as a marker of various
diseases, such as microbial or viral infection, allergy,
autoimmune disorder or damaged tissue.
In this work, the surface plasmon resonance
spectroscopy method with prismatic excitation in the
thin gold film in Kretschmann configuration with
mechanical angular scanning was used for detection of
EBV antibodies in the human blood serum. The method
allows to investigate kinetics of molecular interaction
without labeling by means of the measurement of SPR
resonant angle in real time with high sensitivity.
The gold films were deposited on glass substrates
without heating by thermal evaporation in vacuum
(the VUP-4 setup with the residual pressure close to
4⋅10–4 Pa, deposition rate of 40…50 Ǻ/s), with the thin
chromium layer (~2 nm) used to improve adhesion.
Sensitive materials. The Epstein–Barr virus
(antigen) was accumulated and extracted from the B95-8
cell culture using the Walls and Crawford method [12] at
the D. Zabolotny Institute of Microbiology and Virology
of NAS of Ukraine. The B95-8 cell culture consists of
the B-lymphocytes from peripheral blood of marmoset
monkeys transformed with EBV and continuously
producing the virus. Lysates of the lymphoblast B95-8
cells were used as the antigen, containing the full
spectrum of EBV proteins, namely EBNA, EA and
VCA. This way, a specific antigen for the detection of
Epstein–Barr virus antibodies (IgG and IgM) was
obtained, having high specificity and sensitivity
properties. To evaluate the sensitivity and specificity of
the obtained antigen, the reproducibility of positive and
negative analysis results for the selected panels of
respectively positive and negative sera was compared
using our test system and several foreign test systems
utilizing PCR (“АмплиСенс-100-R”, Russia) and
ELISA (“Platelia EBV EBNA IgG” from “Sanofi
diagnostics Pasteur”, France; “SIATM Epstein–Barr
EBNA IgG” from “Sigma diagnostics”, USA).
Analytes. The blood sera from the infected donors
were supplied by “ДНК-лаборатория”, Ltd. (Kiev), and
sera taken from healthy donors were supplied by Kiev
city blood transfusion station. All sera were tested for
the presence of EBV antibodies by means of PCR and
ELISA reference methods.
3. Results and discussion
In the case when SPR method is used for detection of
biomolecules in liquid medium, sensitivity can be
enhanced by improving the sensitive element preparation
technology and by minimizing the effect of the noise.
Preparation of the SPR sensitive element.
Enhancement of the method sensitivity is based, first of
all, on the specific interaction of EBV antibodies,
circulating in the blood serum, with the antigen (EBV
itself) immobilized on the gold film surface. The use of
immune molecular reaction of the antigen-antibody
interaction as the basis for biosensor design allows to
obtain uniquely specific responses and to perform
analyses of the complex biological samples such as
urine, saliva, blood or serum [13].
However, adsorption of biomolecules onto the
raw gold surface can lead to undesirable optical and
structural effects of metallic surface that distort the
SPR instrument response. On the other hand, in order
to obtain reliable analysis results the EBV protein
molecule has to be immobilized on the gold surface
while preserving its natural state and accessible spatial
orientation, via an intermediate transition layer. The
maximum sensitivity of transducer can be achieved, if
the transition layer thickness is substantially smaller
than the distance of surface plasmon wave penetration
to the external environment (100…200 nm for visible
light).
To form a protective layer on the sensitive element,
self-organized monolayers (SOM) with controlled
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2016. V. 19, N 1. P. 84-89.
doi: 10.15407/spqeo19.01.084
© 2016, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
86
composition, structure and thickness can be used. The
most widespread are layers based on functionalized
mercaptans, particularly aliphatic thiols (HS-(CH2)n-X)
due to simplicity of preparation, stability and functional
diversity. The primary idea of application of these linear
molecules consists in usage of gold affinity to sulfur
atoms. Organization of the modifying layer on the noble
metal surface happens as a result of prolonged sponta-
neous chemisorption from solution, predominantly in
ethanol, with the concentration (0.1…1.0)·10–3 M and
includes two stages. Initially the HS group of thiol
interacts with the gold atoms on the surface, forming the
gold thiolate. After this, microstructural reorganization
of the bound monolayer takes place, controlled by
hydrophobic interaction of the aliphatic chains and
accompanied by reconstruction of the noble metal
surface. This way, a densely packed molecular coating is
formed, providing a significant barrier for electron and
ion transfer, and possessing high kinetic and
thermodynamic stability. The terminal functional groups
determine physical and chemical properties of the
modified metal surface, and hydrocarbon chains provide
spatial shielding up to tens of angstroms (characteristic
length of a C-H bond is ∼1.1 Å), which does not
significantly affects the transducer sensitivity.
Protective properties of the dodecanthiol film
(HS(CH2)11CH3) are illustrated in Fig. 1. It can be seen
that the baseline of the SPR instrument with the applied
modifier coating is more stable, monotonous and shows
the trend of slight increase with time (~32 angular
seconds per hour), which may indicate the presence of
small quantity of adsorbate substances in water. For the
unprotected gold film, the shift of SPR minimum
position toward the lower angles can be observed
(~88 angular seconds per hour), likely related to the
water pervasion through the pores and microcrystal
boundaries in the gold film.
Based on the above, the following method was
developed for preparation of the sensitive element (chip)
for detection of the Epstein–Barr virus antibodies in
human blood serum [14]. Immobilization of EBV
proteins was performed within the protective-orienting
coating (three-dimensional structure of a polysaccharide
hydrogel – Dextran 17000 from Sigma) deposited onto
the gold film surface on glass substrate from the solution
in 0.05% citrate buffer at pH 5.0–5.2 with the
concentration 2 mg/ml for 5 hours at the temperature
20…25 °C. After washing in the citrate buffer, solution
of the antigen in citrate buffer was left on the surface at
the temperature of 4…8 °C for 18…24 hours. To prevent
nonspecific interaction, after immobilization of the EBV
proteins, remaining free sites were blocked using the 1%
solution of bovine serum albumin (BSA) in citrate buffer
for 1 hour at the temperature 20…25 °C. After that, the
chips were rinsed with citrate buffer, dried with the clean
air flow and stored at 4…8 °C in the small sterile sealed
containers.
0 1000 2000 3000 4000
150
200
250
300
350
400
Au with thiol film
Au
R
es
on
an
t a
ng
le
sh
ift
, a
ng
ul
ar
se
co
nd
s
Time, s
Fig. 1. Temporal drift of the “Plasmon” series instrument
baseline in water for the sensitive elements with the raw gold
surface and surface modified with dodecanthiol.
Influence of the noise effects. The total noise of a
SPR based sensor, determining the lower limit for the
measurable changes in the refraction index [15],
includes: the photodetector noise; light source
fluctuations, largely conditioned by the electronic noise
and the quality of controlling circuitry; thermal noise,
caused by temperature fluctuations; mechanical
fluctuations noise that is a result of internal and external
forces applied to the sensor chip and the optical system;
the interphase fluctuations noise caused by random
sorption-desorption processes at the interface, including
the effects from the microscopic air bubbles at the gold
film surface, flow/pressure fluctuations caused by the
sample supplying pump operation, and the electro-
magnetic interference from the external sources,
affecting the instrument electronics.
The most significant are the effects of temperature
fluctuations of different origins [16]. First of all, thermal
changes in the gold layer can significantly influence on
SP generation. As the temperature increases, SP
oscillations substantially weaken due to the increased
electron-phonon scattering in metal. It leads to widening
the SPR curve and decreasing the instrument sensitivity.
The temperature change influences operation of
important components of the sensory system (light
source output signal, detector response and sensor
geometry). However, the parameter most sensitive to the
change in ambient temperature is the effective refractive
index of the sample. For instance, the water refractive
index changes by 10–5, when the temperature changes by
0.1 °C, which is close to the SPR instruments detection
limit [17].
To eliminate the noise caused by temperature
fluctuations, stabilization (active temperature control)
and compensation (reference channel introduction)
principles are used.
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2016. V. 19, N 1. P. 84-89.
doi: 10.15407/spqeo19.01.084
© 2016, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
87
For the implementation of temperature stabilization
principle, the thermal stabilization box completely
enclosing the operating “Plasmon” series instrument was
used (Fig. 2). The box controls the temperature within 2
to 40 °C range with the accuracy close to 0.1 °C, and
uses the developed at V. Lashkaryov Institute of Semi-
conductor Physics of NAS of Ukraine thermostat with
optimized parameters of the proportional differential-
integral controller that modulates the input power to the
Peltier-effect based thermoelectric module.
Fig. 3 demonstrates that temperature stabilization
of the entire instrument facilitates minimization of the
noise level and substantially increases resolution of the
used SPR sensor system. However, this approach results
in a bulky setup, degrades convenience for performing
the experiment, and thus is used, as a rule, only for
detection of small molecules at low concentrations.
Fig. 2. Exterior view of the thermal stabilization chamber with
the “Plasmon” series instrument during operation.
0 1000 2000 3000 4000
65.32
65.34
65.36
65.38
65.40
65.42
65.44
65.46
65.48
65.50
2
1
R
es
on
an
t a
ng
le
s
hi
ft,
d
eg
.
Time, s
Fig. 3. Long-term drift of the SPR signal in water, obtained
with the “Plasmon” series instrument using the described
sensor chip: 1 – operating in a non-isolated environment; 2 –
enclosed within the thermal stabilization box.
For clinical detection of EBV antibodies in human
blood serum, the beforehand prepared biochips with
EBV proteins immobilized on the gold film surface were
used with the “Plasmon” series instrument equipped
with an additional reference channel, which readings are
registered simultaneously with the primary working
channel [18]. Using the reference channel, the changes
in the instrument response that are not related to
adsorption or molecular interaction (e.g., the temperature
changes) are registered, which leads to the improvement
of accuracy and reliability of measurements.
Optical layout of the dual channel version of the
“Plasmon” series instrument is shown in Fig. 4. The in-
strument uses a single light source, with the beam being
split into two with the specially designed beam-splitting
prism. The distance between the axes of beams is
7.5 mm. The beam-splitting prism is the main element of
the layout, responsible for formation of the two identi-
cally polarized light beams, without refraction and with
the spatial shift relative to the light source axis. Only un-
der these conditions, identical SPR curves can be obtai-
ned in two channels. The detailed description of “Plas-
mon” series instrument operation can be found in [19].
In the experiment, an optimal dilution ratio of
1:100 was used for the anti-VBE sera in citrate buffer at
pH 5.0…5.5. The channel 1 was used directly for mea-
suring the antibodies content in serum, while the channel
2 was used as the reference one to improve accuracy and
reliability of these measurements. To prevent nonspe-
cific binding of antibodies to EBV, negative serum from
a healthy donor was supplied to the sample cell first. The
unbound material was then washed out, and the serum
from an EBV infected donor was supplied to the cell.
Quantification of the antigen-antibody interaction results
was performed by quantitative determination of the reso-
nant angle shift (in angular seconds) in time (seconds).
The sensogram of the measurements is shown in Fig. 5.
Fig. 4. Optical layout of the dual channel “Plasmon” series
instrument.
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2016. V. 19, N 1. P. 84-89.
doi: 10.15407/spqeo19.01.084
© 2016, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
88
0 1000 2000 3000 4000 5000
0
500
1000
1500
2000
2500
response indicating
the disease
2
1
buffer
pH 5.5
EBV infected
serum
buffer
pH 5.5
negative
serum
buffer
pH 5.5
R
es
on
an
t a
ng
le
sh
ift
, a
ng
ul
ar
se
co
nd
s
Time, s
Fig. 5. Sensogram of interaction of the specific antigen (EBV) immobilized on the gold surface of the SPR chip with antibodies in
the human blood serum: 1 – operation channel, 2 – reference channel.
The routine analysis is performed as follows. From
the average value obtained over 25 negative sera with
three measurements for each serum, the threshold value
(ThV) is calculated. The serum is considered positive if
the resonant angle shift exceeds ThV by 10%, and nega-
tive, if the shift is by 10% lower than ThV. The obtained
data were compared with the ELISA results. Reproduci-
bility of the proposed method outcomes was about 95%.
4. Conclusion
Analysis of the influence of SPR sensitive element
preparation technology and noise effects of the
“Plasmon” series instruments has been performed in
order to reliably detect specific antibodies against the
Epstein–Barr herpevirus in the human blood serum.
To minimize the influence of temperature
fluctuations, the instrument with an additional reference
channel was used, the reference channel data being
registered simultaneously with the working channel.
Immobilization of the EBV proteins was performed
within the protective-orienting coating – three-dimen-
sional structure of the polysaccharide dextran hydrogel
(2 mg/ml of Dextran 17000 from Sigma in 0.05% citrate
buffer at pH 5.0…5.2) deposited onto the gold film
surface on glass substrate, and the remaining free sites
were blocked with bovine serum albumin molecules (1%
BSA in citrate buffer at pH 5.0…5.2) to prevent non-
specific interaction.
It has been shown that the developed SPR method
in dual channel version can be used for rapid automated
diagnosis of herpeviral disease caused by Epstein–Barr
virus in population, on a par with linked immunosorbent
assay.
Acknowledgments
The author would like to thank the employees of
D. Zabolotny Institute of Microbiology and Virology of
NAS of Ukraine: N.V. Nesterova, S.D. Zagorodnya,
G.V. Baranova, A.V. Golovan, and the employees of
V. Lashkaryov Institute of Semiconductor Physics of
NAS of Ukraine Yu.V. Ushenin and A.V. Samoylov for
their assistance in performing this work.
This work was supported by the National Academy
of Sciences of Ukraine through the project “Pilot
operation and metrological support diagnostic
instrument based on surface plasmon resonance for the
diagnosis of herpes virus. Design and manufacture of
sensing elements, electronic, mechanical and software
modules devices based on surface plasmon resonance”.
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