Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e5, 2017

www.elsevier.com/locate/jbiosc

Detection of antibodies against hepatitis B virus surface antigen and hepatitis C

virus core antigen in plasma with a waveguide-mode sensor

Takenori Shimizu,1 Torahiko Tanaka,1 Shigeyuki Uno,1 Hiroki Ashiba,2 Makoto Fujimaki,2 Mutsuo Tanaka,3
Koichi Awazu,2 and Makoto Makishima1, *

Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan,1 Electronics and Photonics Research Institute,
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,2 and Health Research Institute, National Institute of
Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3

Received 31 October 2016; accepted 10 January 2017

Available online xxx
In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious
disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion.
However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of
the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a
waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect
antigeneantibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus
(HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant
changes in the reflectance spectra, which indicate specific antigeneantibody interaction for anti-HBs antibody and anti-
HCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl
carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for anti-
HBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an
enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be
applied to on-site blood testing in emergency settings.
Ó 2017, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Waveguide-mode sensor; Hepatitis B virus; Hepatitis C virus; Antibody; Horseradish peroxidase; Aminoethyl carbazole]

During large-scale disasters, such as significant earthquakes,
many victims need surgical treatment and/or blood transfusion. In
this setting, clinical laboratories cannot work effectively for blood
typing and screening of infectious diseases due to physical damage,
disrupted electric supply and transport system, and damage or
injury to laboratory workers (1). Although volunteer blood donors
can contribute greatly to saving the lives of victims, blood typing in
the ABO and Rh(D) systems for donors and recipients is necessary
before transfusion, and viral examinations of donor blood are
required to decrease the risk of transfusion-transmissible viral in-
fections, such as hepatitis B virus (HBV) and hepatitis C virus (HCV)
(2,3).
We have developed waveguide-mode (WM) sensors for Influ-
enza virus detection and blood typing (4,5). A WM sensor utilizes
electric field enhancement in a sensor chip, similar to a surface
plasmon resonance (SPR) sensor, and is more sensitive than a
reflectance absorption spectrometer (6,7). A WM sensor detects
target proteins on a monolithic sensing chip with a layer of silicon
sandwiched with SiO2 layers (8). Excitation of the waveguide mode
in a sensing chip decreases light reflectance at a specific wave-
length, and a change in surface conditions, such as a change in the
refractive index caused by binding of large molecules, results in
* Corresponding author. Tel./fax: þ81 3 3972 8199.
E-mail address: makishima.makoto@nihon-u.ac.jp (M. Makishima).
altered reflectance spectra (6). Biological interactions, including
antigeneantibody reactions for the detection of Influenza virus, can
be observed on a sensing chip as a change in reflectance spectra (4).
Utilization of conjugates with gold particles or dyes efficiently in-
creases detection sensitivity of biological interactions, because the
presences of such molecules on a chip surface influence reflectance
property, wavelength and depth of the change in reflectance (6,9).
A SPR sensor has been successfully used for detection of specific
antibodies in human serum samples (10e12). Although a WM
sensor is based on a principle similar to that of a SPR sensor, there
are several differences between WM and SPR sensing systems. A
WM sensor uses waveguide modes, instead of SPR (6,8). While the
material used to induce the SPR restricts the wavelength of incident
light for a SPR sensor, there is no such restriction for a WM sensor.
In addition, a stable sensing surface made of glass is available for a
WM sensor. Thus, the wavelength of a WM sensor is widely
controllable over the visible light region by adjusting the thick-
nesses of the top silicon oxide and embedded silicon layers. A WM
sensor has sensitivity similar to enzyme-liked immunosorbent
assay (ELISA) and consumes shorter experimental time than ELISA
(13). Although a WM sensor can detect leptin diluted in human
serum by immunoassay using anti-leptin antibody (14), it has been
unclear whether a WM sensor can be applied for detection of
endogenous antibodies in blood samples.

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2017.01.004

Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core
antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
2 SHIMIZU ET AL.
In this study, we evaluated the potential of a WM sensor as a
method for detection of antibodies against HBV and HCV in blood
samples. Since a WM sensor-based method can utilize a portable,
small-sized instrument and work with battery power source, it
should be useful during large-scale disasters. We increased the
detection sensitivity for anti-HBs antibody more than 300-fold by
introducing a peroxidase reaction in the detection process and
succeeded in detecting endogenous anti-hepatitis B surface (HBs)
antibody in human plasma.
MATERIALS AND METHODS
WM sensors Optical arrangement of a WM sensor device was based on the
Kretschmann configuration as reported previously (6). When light enters through a
prism at a particular angle of incidence, light of a specific wavelength is propagated
in a slab waveguide of a sensing chip, a phenomenon known as excitation of the
WM, which causes decrease in the reflectance of light (Fig. 1A). Sensing chips
were composed of a glass substrate, an embedded Si layer, and a top SiO2 layer.
The Si and SiO2 layers function as the slab waveguide. We used sensing chips for
colorless and colored targets as reported previously (8,9). When a spectrum of
reflected light from a white LED is measured by the spectrometer, a specific
wavelength appears as a dip in reflectance spectra. When target molecules
(particles) are captured on a sensing chip, the interaction influences the reflective
property of the waveguide surface and the complex refractive index near the
waveguide surface can be evaluated as a change in the depth and/or wavelength
of the dip in reflectance spectra (Fig. 1B). The interaction of colorless target
molecules on a chip preferentially induces a shift of the dip peak position (Dl) (6).
Dl levels are dependent on both the amount and size of bound molecules. For
detection of colored target molecules, a change in the depth (DR) on a chip is also
useful to obtain high sensitivity (6,15). The detection limit was defined as the
lowest concentration of antibody that showed a statistically significant difference
from control. All measurements were performed at room temperature with a WM
sensor device EVA-001 (Optex, Otsu, Japan).
Antigens and antibodies HBs adr antigen was purchased from ProsPec (East
Brunswick, NJ, USA), and HCV core domain 1 protein was generated using an
expression plasmid of core domain 1, which was made from the pON/C-5B/KE
plasmid harboring HCV strain O genome (kindly provided by Prof. Nobuyuki Kato
of Okayama University, Japan) (16). Mouse monoclonal antibodies, anti-HBs Hs33
and anti-HCV core protein H6-29, were purchased from HyTest Ltd. (Turku,
Finland) and BioAcademia Inc. (Osaka, Japan), respectively.
Immobilization of antigens on sensing chips surfaces To capture anti-
bodies, HBs antigen and HCV core antigen were immobilized on chips. For antigen
immobilization, a silica surface of a sensing chip was chemically modified with
FIG. 1. A schematic diagram of a WM sensor for detection of antigeneantibody complexes. (A) A frame format of a WM sensor. A sensing chip is composed of 3 layers of glass (SiO2
and Si). (B) Illustration of dips detected in reflectance spectra. Interaction of target molecules on a chip changes a dip shape as DR in depth and/or Dl in wavelength.
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core
antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
J. BIOSCI. BIOENG.,
triethoxysilane derivatives, C12Es (silane bearing succinimide ester moiety) and
M3EG (silane bearing methoxytriethylene glycol moiety), as described previously
(14). Chips were immersed in a mixture of C12Es and M3EG toluene solutions for
72 h at 50 C, where the molar ratio of C12Es and M3EG was 1:10. M3EG and
C12Es were used for blocking and immobilization of proteins, respectively. The
modified chip was rinsed with acetone and dried. The succinimide ester group of
C12Es was reacted to form a covalent bond with amine residues of antigens.
Detection of mouse monoclonal anti-HBs antibody and anti HCV core
antibody diluted in plasma with a WM sensor Before antigeneantibody re-
action, phosphate buffered saline (PBS) was placed on a chip immobilized with HBs
antigen or HCV core antigen at room temperature and a baseline spectrum was
measured. Each chip has a material-dependent characteristic baseline spectrum. To
develop a detection system for anti-HBs antibody on a WM sensor, human plasma
containing the mouse anti-HBs antibody Hs33 was utilized. Antibody was diluted
in plasma from HBs antibody-negative volunteers at final concentrations from
0.03 to 100 nM. Antibody containing plasma was added for antigeneantibody
reaction on a chip for 30 min. The chip surface was washed with PBS five times
and a spectrum was measured. To develop a detection system for anti-HCV core
antibody, anti-HCV core antibody H6-29 was diluted in plasma from HCV
antibody-negative persons, and was incubated on a chip immobilized with HCV
core domain 1 antigen for 30 min. The chip surface was washed with PBS five
times and a spectrum was measured.
Measurement of anti-HBs antibody with a WM sensor and signal
enhancement method For enhancement of detection sensitivity, a method
utilizing a peroxidase reaction was evaluated. For this reaction, after incubation of a
chip immobilized with HBs antigen with a test sample as described above for a
method without enhancement, HRP-conjugated anti-mouse or anti-human IgG
antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in Can
Get Signal Solution 2 (Toyobo Co., Osaka, Japan) was incubated on a chip for
30 min. The chip surface was washed with PBS-0.1% Tween 20 (PBST) instead of
PBS, and then washed with PBS. Finally, aminoethyl carbazole (AEC) solution
(Enzo Life Sciences, Farmingdale, NY, USA) was incubated as substrate of
peroxidase reaction on a chip for 5 min. HRP catalyzes the conversion of AEC to
red water-insoluble precipitates. The chip surface was washed with PBST and
with PBS to remove unreacted AEC, and a spectrum was measured. Human
plasma from healthy vaccinated and non-vaccinated volunteers were also
examined with the peroxidase method for anti-HBs antibody detection. Anti-HBs
antibody-positive blood was taken from a medical worker, who was inoculated
with recombinant hepatitis B vaccine three times and was confirmed to be
immunized by a hospital laboratory. We also utilized a polymerized secondary
antibody, MAHG-SZ (Anti-human IgG [H, g-chain specific]-Strongzyme HRP
Conjugate, Mouse-Mono; Stereospecific Detection Technologies, Baesweiler,
Germany), which harbors multiple molecules of an anti-human IgG antibody and
HRP on dextran polymers instead of the conventional HRP-conjugated anti-
human IgG. This study was approved by Research Ethics Committee of Nihon
University School of Medicine (reference number 25-6-0).
VOL. xx, 2017
RESULTS
Measurement of anti-HBs antibody with a WM sensor To
evaluate a detection system for anti-HBs antibody on a WM sensor,
we chose anti-HBs monoclonal antibody Hs33 after comparing
several commercial anti HBs antibodies for the sensitivity of
antigeneantibody interaction. First, we examined whether we
can detect interaction of anti-HBs antibody suspended in human
plasma with HBs antigen immobilized on a chip using a WM
sensor without a enhancing method. Although plasma used for
dilution of antibody contain immunoglobulin, we used control
mouse monoclonal antibody at the same concentrations to verify
specificity of anti-HBs antibody. While addition of control
antibody did not, incubation with anti-HBs antibody resulted in a
dip shape spectra (Fig. 2A). An anti-HBs antibody-dependent Dl
change was observed at 100 nM, although it was not significant
at 10 nM (Fig. 2B). A Dl value obtained from anti-HBs antibody at
100 nM was larger than that at 10 nM (Fig. 2B). The difference
between Dl values from control antibody at 10 nM and 100 nM
was not significant.
Measurement of anti-HCV antibody with a WM
sensor Similarly to detection of anti-HBs antibody, anti-HCV
core antibody H6-29 was dissolved in human plasma and was
detected with a WM sensor. We observed a dip for 10 nM anti-
HCV antibody, which was not statistically significant (Fig. 3A).
This method significantly detected anti-HCV antibody at 100 nM
in a WM sensor (Fig. 3B).
Enhanced sensitivity in detection of anti-HBs antibody We
used chips modified with M3EG-C12Es, which were reported to
show the highest nonspecific adsorption-resistant effect in
human serum (14). Although we detected antigeneantibody
interactions for anti-HBs antibody and anti-HCV antibody with a
WM sensor (Figs. 2 and 3), we investigated how to enhance
detection sensitivity and/or to reduce nonspecific signals. We
FIG. 2. Detection of anti-HBs antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control antibody. Right, anti-HBs antibody (Hs33).
(B) Anti-HBs antibody is detected with a WM sensor. *p < 0.05; **p < 0.01; n.s., not significant (one-way ANOVA followed by Tukey’s multiple comparisons). Data are presented as
means  S.D. (n ¼ 3).
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core
antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
WAVEGUIDE-MODE SENSOR FOR HEPATITIS VIRUS ANTIBODIES 3
could decrease background signals by washing a tip surface with
PBST instead of PBS. Furthermore, in order to increase detection
sensitivity, we examined a method using peroxidase-conjugated
secondary antibody with a peroxidase reaction on AEC. This
signal enhancement method successfully detected anti-HBs
antibody at 0.03 and 0.1 nM (Fig. 4A). The color change was not
recognized with naked eye. We observed a concentration-
dependent change from 0.01 nM to 10 nM, and the detection
limit was 0.03 nM (Fig. S1). Compared to the detection sensitivity
without enhancement (Fig. 2), the method using peroxidase-
conjugated secondary antibody and AEC staining increases
detection sensitivity of anti-HBs antibody in a WM sensor more
than 300-fold.
Finally, we applied a WM sensor with peroxidase enhancement
to detect anti-HBs antibody in human blood samples. Using a chip
immobilized with HBs antigen and HRP-conjugated anti-human
IgG antibody with peroxidase reaction, we observed a significant
difference in DR between plasma from non-vaccinated and vacci-
nated persons (Fig. 4B). Interestingly, when we used the polymer-
ized secondary antibody MAHG-SZ and the peroxidase reaction, the
difference in DR became larger (Fig. 4C). Therefore, a WM with
enhanced by conjugated secondary antibody and dye reaction can
be applied to detection of anti-HBs antibody in human blood
samples.
DISCUSSION
We report here a novel method to detect anti-viral antibodies in
human blood samples using a WM sensor. This is the first report
describing the use of a WM sensor for detection of endogenous
antibody in human blood. First, we evaluated a detection system by
using mouse monoclonal antibody (anti-HBs antibody or anti-HCV
core antibody) diluted in human plasma, and found that anti-HBs
antibody (100 nM) and anti-HCV core antibody (100 nM) can be
detected in a WM sensor. Next, we developed a signal enhancement
4 SHIMIZU ET AL. J. BIOSCI. BIOENG.,

FIG. 3. Detection of anti-HCV core antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control plasma. Middle and right, anti-HCV
core antibody (H6-29). (B) Anti-HCV antibody induces a concentration-dependent dip shift. **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons). Data are
presented as means  S.D. (n ¼ 3).

method and obtained improved sensitivity for detection of anti-
HBs antibody by more than 300-fold (0.03 nM) using a HRP re-
action on AEC dye. The peroxidase reaction generates red-colored
water-insoluble precipitates from AEC, which exhibit optical ab-
sorption and strongly influence light waves in a WM sensor. Thus,
the WM spectra reveal a change in the depth of a dip. Comparing
between before and after the HRP reaction, the following two fac-
tors are considered to contribute to the sensitivity enhancement: (i)
The changes in the extinction coefficient, k, caused by a colored
substance induce a larger change in the spectra than that in the
refractive index, n, caused by a colorless substance (9), and (ii) The
HRP reaction continuously generates the colored product until the
reaction is stopped by washing, and the change in k is enhanced by
the progress of the reaction. Finally, our enhanced detection
method could be applied for detection of human anti-HBs antibody
in blood samples from a vaccinated individual. These results have
encouraged us to develop a WM sensor as a portable blood test
device. The limitation of the current enhancement method is a
reaction time of about 90 min, because it needs repeated washing
to decrease nonspecific effects of plasma proteins. We are further
developing an improved detection method using conjugated sec-
ondary molecules to increase sensitivity and to shorten reaction
time.
Usual methods to detect antibodies, including anti-HBs anti-
body and anti-HCV core antibody, are hemagglutination assay,
chemiluminescent immunoassay and chemiluminescent enzyme
immunoassay. These methods are sensitive and quantitative
methods, but usually require a clinical laboratory and become
impractical during large-scale disasters. Several portable devices
for chemiluminescent reactions have been developed and may be
useful for on-site testing (17). Immunochromatography is a handy
method and can be used in cases of emergency. A method using SPR
is similar to a WM sensor in detection of molecules on a chip and is
useful in biochemical analysis (18e20). A portable SPR sensor has
been developed for immunoassay (21). We have successfully
developed microfluidic chips on a WM sensor for blood typing

FIG. 4. HRP-conjugated secondary antibody and HRP reaction increase detection sensitivity. (A) Increased sensitivity in detection of anti-HBs antibody (Hs33). HRP-conjugated anti-
mouse IgG was added after antigeneantibody interaction and HRP reacted on AEC dye. (B, C) Anti-HBs antibody in human blood samples is detected with a WM sensor in
combination with enhancing methods. While HRP-conjugated anti-human IgG was used in panels B and C, HRP reactions were performed on AEC in panel B like in panel A and on
the combination of MAHG-SZ and AEC reactions in panel C. Plasma A and B were derived from a non-vaccinated individual and a vaccinated individual, respectively. *p < 0.05;
**p < 0.01; ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons for panel A; unpaired two-group Student’s t test for panels B and C). Data are presented as
means  S.D. [(A, C) n ¼ 3; (B) n ¼ 6].

Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core
antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
VOL. xx, 2017
(5,22,23). A method using a multichannel WM will be useful for
both blood typing and screening of virus antibodies. In this study,
we used a single-channel WM sensor, which is 30 cm wide, 15 cm
tall and 20 cm deep. We previously reported a five-channel WM
sensor with a size of 27  12  7.5 cm3 (23). At present, the minimal
WM sensor is 15  7  5 cm3, equipped with four-channel chips (C
and I KK (Tsukuba, Japan), personal communications). Therefore, in
addition to immunochromatography and a portable SPR sensor, a
WM sensor could be used as an on-site portable diagnostic device
in emergency settings, particularly in large-scale disasters.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jbiosc.2017.01.004.
ACKNOWLEDGMENTS
The authors thank Ms. Risa Kono for expert technical assistance,
Dr. Mami Yamamoto, Dr. Alena Lagumdzija, Ms. Ayumi Sato, and
other members of the Makishima laboratory for helpful comments
and assistance, the Advanced Functional Materials Research Center
of Shin-Etsu Chemical Co. Ltd., for supplying sensing plates, and Dr.
Andrew I. Shulman for editorial assistance. This work was sup-
ported by SENTAN program of Japan Science and Technology
Agency (2012e2014) and Japan Agency for Medical Research and
Development (2015e2016; grant numbers 15hm0102001h0004
and 16hm0102001s0205).
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