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Sensors and Actuators B 131 (2008) 285–289
Detection of surface immunoreactions on individual cells by
electrophoretic mobility measurement in a micro-channel
Atsushi Aki, Yusuke Nihei, Hiroto Asai, Tomofumi Ukai, Hisao Morimoto,
Yoshikata Nakajima, Tatsuro Hanajiri, Toru Maekawa ∗
Bio-Nano Electronics Research Centre, Toyo University, 2100, Kujirai, Kawagoe, Saitama 350-8585, Japan
Received 21 May 2007; received in revised form 5 November 2007; accepted 14 November 2007
Available online 4 December 2007
Abstract
The electric characteristics at the surface of cells are changed depending on the surface conditions and reactions, and electrophoresis is one of
the simplest methods of detecting the change in the electric characteristics of cells, and one which does not cause any damage to the sample cells.
In this paper, we fabricate micro-channels, attach antibodies, that is, immunoglobulin G (IgG), to the surface of sheep’s red blood cells (RBCs)
and carry out electrophoresis experiments of RBCs in the micro-channels to measure the electrophoretic mobility of each RBC. The amount of
antigen-antibody reaction on the cell surface is also measured by the well-known enzyme-linked immunosorbent assay (ELISA) method. We find
that there is a clear correlation between the electrophoretic mobility of the cells and the amount of IgG reaction on the surface of the cells, that
is, the absolute value of the mobility of the cells decreases with an increase in the amount of IgG reaction on the cells. Therefore, the amount of
surface reactions on each cell can be quickly detected by measuring the electrophoretic mobility of the cell, which is much simpler than labelling
methods such as the ELISA or flow cytometric methods. The present result suggests that simultaneous detection of different diseases and health
conditions may become possible by using multi-channel micro-fluidic electrophoresis devices.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Micro-channel; Electrophoretic mobility; Cells; Antigen–antibody reaction
1. Introduction
It is known that the surface conditions of cells and the
biochemical processes occurring on the cells influence the biochemical and pathological characteristics of living organisms
and therefore several different analytical methods of identifying the interactions between biomolecules and between the
surface of cell membranes and biomolecules have been developed in recent years [1–7]. Electrophoresis is one of the simplest
methods, and one which does not cause any damage to the
sample cells. What is more, there is no need for the labelling
of cells in the case of electrophoresis. Since the electric characteristics are affected and changed by the surface conditions
of cells in general, the measurement of the electrophoretic
mobility of cells gives important information about the surface conditions of cells [5–7]. For instance, Ambrose et al.
∗
Corresponding author. Tel.: +81 49 239 1375; fax: +81 49 234 2502.
E-mail address: [email protected] (T. Maekawa).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2007.11.046
[8] showed that the electrophoretic mobility of cancer cells
taken from a hamster’s kidney is different from that of normal
cells. Walter and Widen [9] detected the difference in the electrophoretic mobilities between red blood cells (RBCs) from a
patient with Alzheimer’s disease and normal cells after having added polyethylene glycol to the buffer solution, in which
the above cells were dispersed. Electrophoresis measurement
systems have been developed for the accurate evaluation of
the mobilities and the separation of cells [5–7,10–13], but they
are, in general, large-scale and quite complicated. Micro-fluidic
devices such as micro-total analysis systems (␮-TASs), on the
other hand, are being intensively developed for the quick detection of diseases and health conditions [14–18]. Li and Harrison
[19] carried out electrophoresis of yeast, Escherichia coli and
dog’s RBCs in a micro-channel for the first time. Ichiki et al.
[20] demonstrated that the migration velocity of sheep’s RBCs,
in which both the electrophoretic and electroosmotic effects
are included, is reduced by adding immunoglobulin G (IgG) to
the cells’ dispersed buffer solution in a micro-channel. Omasu
et al. [21] measured the electrophoretic mobility of sheep’s
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A. Aki et al. / Sensors and Actuators B 131 (2008) 285–289
RBCs in a micro-channel, the inner surfaces of which were
coated with a polymer film so that the RBCs did not adhere
to the channel surfaces. However, the relationship between
the amount of molecular reaction on the surface of cells and
the electrophoretic mobility has not yet been studied quantitatively.
In this paper, we fabricate micro-channels, carry out
electrophoresis experiments using sheep’s RBCs in the microchannels and investigate the effect of the amount of IgG reaction
on the surface of individual RBCs on the electrophoretic mobility of each cell. The amount of IgG attached to the cell surface is
measured by the enzyme-linked immunosorbent assay (ELISA)
method. We show a clear correlation between the electrophoretic
mobility of the cells and the amount of IgG reaction on the surface of the cells. We explain the experimental details in Section
2 and show the results of the electrophoresis and ELISA experiments in Section 3. After discussing the relationship between
the mobility of the cells and the amount of IgG reaction on the
cells, we summarize the result obtained in this study in the final
section.
2. Experimental details
2.1. Micro-fluidic electrophoresis system
The outline of a micro-electrophoresis system is shown in
Fig. 1. Micro-channels were fabricated on polydimethylsiloxane (PDMS) substrates (TSE3450; GE Toshiba Silicones, Japan)
by the conventional softlithography technique [22]. The width
of the micro-channels was changed as follows; 20 ␮m, 40 ␮m
and 60 ␮m, to investigate the effect of the channel width on
the mobility measurement. The depth of the channels was
36 ␮m. The surface of the PDMS, where the micro-channels
were fabricated, was stuck to a glass plate (Matsunami Glass,
Japan). Holes of 3.0 mm in diameter were fabricated at the
ends of the micro-channels and conical electrodes were installed
in the holes. The distance between the two electrodes was
13.5 mm. The surfaces of the micro-channel and the holes
were coated with 2-methacryloyloxyethylphosphorylcholine
(MPC) (Lipidure CM5206E; NOF, Japan) in order to prevent cells adhering to the channel surfaces [21,23]. The
movement of RBCs in the micro-channel was observed by
inverted microscope, recorded on hard disc and analysed by
computer.
2.2. Electroosmotic effect
We checked the electroosmotic mobility of phosphate
buffered saline (PBS) of pH 6.9, which was used for the later
electrophoresis experiments, using reference latex particles of
0.99 ␮m in diameter (Sekisui Chemical, Japan), the surfaces of
which were coated with hydroxypropylcellulose. The mobility
of the reference particles was measured in advance by a conventional electrophoretic machine (ELS-6000; Otsuka Electronics,
Japan) [24]. We then measured the electroosmotic mobility of
the PBS in the micro-channels, using the above reference particles.
Fig. 1. Outline of a micro-fluidic electrophoresis system. (a) Micro-fluidic electrophoresis device. A micro-channel is fabricated on PDMS, which is stuck
to a glass substrate. The surface of the channel is coated with MPC so that
RBCs do not adhere to the surface of the channel. A dc electric field is applied
along the channel. (b) Cross-section of a micro-channel of 20 ␮m in width. The
scale bar represents 30 ␮m. The channel width was changed; 20 ␮m, 40 ␮m and
60 ␮m. (c) Measurement system. The movement of RBCs in the micro-channel
was observed by inverted microscope, recorded on hard disc and analysed by
computer.
2.3. Measurement of the amount of antibodies attached to
RBCs by the ELISA method
The method of attaching antibodies to the surface of RBCs
and the method of measuring the amount of antibodies attached
to the RBCs are summarized in this section. We prepared
a PBS solution of pH 6.9, in which sheep’s RBCs (Nippon
Bio-Supp. Centre, Japan) were dispersed. The hematocrit was
0.5%. Primary antibodies, that is, anti-sheep red blood cells IgG
(Rockland, USA) were added to the RBCs/PBS solution (see
Fig. 2). The concentration of the antibodies was changed as follows; 0.5 ␮g ml−1 , 2.0 ␮g ml−1 , 5.0 ␮g ml−1 , 20 ␮g ml−1 and
50 ␮g ml−1 . We introduced a certain amount of the RBCs/PBS
solution into the micro-channels to measure the electrophoretic
mobility of each RBC, to which antibodies were attached (see
Section 2.4.). The rest of the solution was introduced into an
ELISA system to measure the amount of antibodies attached
to the RBCs. Secondary antibodies, that is, anti-rabbit IgG
F(c) alkaline phosphatase (AP) conjugated polyclonal antibodies (Rockland, USA) and substrates, that is, the p-nitrophenyl
phosphate liquid substrate system (Sigma, USA), were used for
the ELISA method (see Fig. 2). After each process, the RBCs
were washed to eliminate non-attached antibodies and then the
hematocrit of each sample was adjusted to 1.5%. Three samples were prepared for the ELISA measurement for the same
A. Aki et al. / Sensors and Actuators B 131 (2008) 285–289
Fig. 2. Outline of the ELISA method. Primary antibodies react with the antigens
on the surface of a RBC. Secondary antibodies and substrates are added to the
solution. A xenon light is introduced in the solution and the absorbance of the
incident light of 405 nm in wavelength is measured to evaluate the amount of
the primary antibodies attached to the surface of the RBCs.
concentration of IgG. 3 M of sodium hydroxide solution was
added to the solution to stop any reactions between the enzymes
attached to the secondary antibodies and substrates. We irradiated a light beam from a xenon lamp into the RBCs/PBS solution
and measured the absorbance of the incident light of 405 nm in
wavelength to evaluate the amount of antibodies attached to the
surface of the RBCs. Note that we also added secondary antibodies to the RBCs/PBS solution, in which there were no primary
antibodies included, and measured the absorbance of the incident light in order to evaluate the nonspecific cross-reactions
between the RBCs and the secondary antibodies.
2.4. Measurement of electrophoretic mobilities of RBCs in
micro-channels
We measured the electrophoretic mobility of each RBC dispersed in the PBS in the micro-channels by observing the motion
of each RBC with an inverted microscope. The intensity of the
electric field was set at 2.2 × 103 V m−1 . The measurement was
carried out using 100 cells, which were located at least 5 ␮m
away from the sidewalls of the channels. In each experiment,
the effect of the electroosmotic mobility of the PBS was cancelled out in order to obtain the true electrophoretic mobility of
the RBCs.
3. Results and discussion
First of all, let us show the result of the measurement
of the electroosmotic mobility of the PBS, which was used
for the electrophoresis experiments. As we explained in Section 2.2, we measured the electrophoretic mobility of the
reference particles by a conventional method and it was
−1.9 × 10−9 m2 V−1 s−1 . The electroosmotic mobility of the
PBS, which was measured using the above reference particles, was +0.2 × 10−9 m2 V−1 s−1 , which coincides with that
obtained by Omasu et al. [21]. This preliminary experiment
shows that electroosmotic flow is remarkably reduced thanks
to the MPC coating.
287
Fig. 3. Dependence of the absorbance of an incident light of 405 nm in wavelength on the concentration of antibodies in phosphate buffered saline. The
hematocrit is 1.5%. The dashed line represents the absorbance without any
primary antibodies.
Fig. 3 shows the result of the ELISA experiment; that is, the
dependence of the absorbance of the incident light of 405 nm
in wavelength on the initial concentration of the primary antibodies dispersed in the RBCs/PBS solution. It clearly shows
that the absorbance of the incident light of 405 nm in wavelength increases with an increase in the concentration of the
antibodies. In other words, the amount of antibodies reacting
with the RBCs increases as the concentration of the antibodies
increases.
Snapshots of RBCs in micro-channels of 20 ␮m in width are
shown in Fig. 4, where an electric field of 2.2 × 103 V m−1 is
applied along the channels. The electrophoretic velocity of a
RBC without any reactions on the cell surface is higher than
that of one with antigen–antibody reactions. As we explained
in Section 2.4, the true electrophoretic mobility of RBCs
was measured by cancelling out the electroosmotic mobility
of the PBS, which was +0.2 × 10−9 m2 V−1 s−1 . The difference in the channel width; that is, 20 ␮m, 40 ␮m and 60 ␮m,
had no effect on the measured mobility. The dependence of
the electrophoretic mobilitiy of RBCs on the concentration
of antibodies in PBS is shown in Fig. 5. The mean value
of the electrophoretic mobility of RBCs without any surface reactions was −17.9 × 10−9 (±0.6 × 10−9 ) m2 V−1 s−1 ,
which agrees well with the result obtained by a conventional machine (ELS-6000; Otsuka Electronics, Japan), that
is, −19.0 × 10−9 (±0.8 × 10−9 ) m2 V−1 s−1 . It is clear that
the absolute value of the electrophoretic mobility of RBCs
decreases with an increase in the concentration of the antibodies.
Finally, we analyse the dependence of the electrophoretic
mobility of RBCs on the amount of antigen–antibody reaction on the surface of RBCs combining Figs. 3 and 5, the
result of which is shown in Fig. 6. There is a clear correlation between the electrophoretic mobility and the amount of
antigen–antibody reaction on the surface of RBCs. Therefore,
the amount of surface reaction on individual cells, in general,
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A. Aki et al. / Sensors and Actuators B 131 (2008) 285–289
Fig. 6. Dependence of the electrophoretic mobility of RBCs on the amount of
antibodies attached to them.
Fig. 4. Snapshots of red blood cells in a micro-channel. The width of the channel
is 20 ␮m and a dc electric field of 2.2 × 103 V m−1 is applied along the channel.
The time interval between two RBCs in the channel is 0.3 s. The scale bar
is 10 ␮m. (a) Movement of a RBC without any antigen–antibody reactions. (b)
Movement of a red blood cell with antigen–antibody reaction. The concentration
of antibodies in the PBS is 50 ␮g ml−1 .
can be quickly detected by measuring the electrophoretic mobility of the cells by the present method, in which case there
is no need for the labelling of the cells. Even when the concentration of the antibodies was as low as 0.5 ␮g ml−1 , which
is much lower than that usually employed by the conventional agglutination method, the difference in the electrophoretic
mobility of RBCs with or without reactions was clearly
detected.
In summary, we demonstrated that the amount of antigen–
antibody reaction on the surface of individual RBCs can be
detected easily and quickly using the present micro-fluidic
electrophoresis device. We suppose that different types
of reaction occurring on the surfaces of cells can also be
detected quickly and that the detection of diseases such
as cancer and Alzheimer’s disease may become possible
eventually using micro-fluidic electrophoresis devices. Simultaneous detection of different diseases and health conditions
may also become possible by using multi-channel microfluidic electrophoresis devices. Bacterial toxins and viruses
may be detected by combining the membrane-derivatized
beads technique [4] with micro-fluidic electrophoresis
devices.
4. Conclusions
Fig. 5. Dependence of the electrophoretic mobility of RBCs on the concentration
of antibodies in the PBS. The dashed line represents the absorbance without any
primary antibodies.
We fabricated micro-channels and measured the electrophoretic mobility of individual RBCs by preventing the
attachment of the RBCs to the surface of the micro-channels
and reducing the electroosmotic flow. We found that the difference in the electrophoretic mobility of RBCs with or without
antigen–antibody reactions on the cell surface can be accurately detected using the present micro-fluidic electrophoresis
device even when the concentration of the antibodies is as low
as 0.5 ␮g ml−1 , which is much lower than that usually employed
by the conventional agglutination method. There is a clear correlation between the electrophoretic mobility of each cell and
the amount of reaction on the cell, that is, the absolute value of
the electrophoretic mobility of RBCs decreases as the amount of
antibodies reacting on the cell surface increases. We suppose that
simultaneous detection of different diseases, health conditions,
bacterial toxins and viruses may eventually become possible by
using multi-channel micro-fluidic electrophoresis devices.
A. Aki et al. / Sensors and Actuators B 131 (2008) 285–289
Acknowledgments
Part of this study has been supported by a Grant for the 21st
Century’s Centre of Excellence Programme organised by the
Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan, since 2003. A. Aki and Y. Nakajima would like
to thank MEXT for their financial support.
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Biographies
A. Aki is a PhD candidate at the Graduate School of Engineering, Toyo University. He is developing fundamental technologies and methodologies such as
manipulations of particles and cells for the design of micro-total analysis systems
at the Bio-Nano Electronics Research Centre, Toyo University.
Y. Nihei is a master course student at the Graduate School of Engineering,
Toyo University. Although his major was computational science during the
undergraduate course, he is now interested in the development of micro-bio
analytical systems based at the Bio-Nano Electronics Research Centre, Toyo
University.
H. Asai was a master course student at the Graduate School of Engineering,
Toyo University when this research project was carried out. He then joined a
private company called Toshiba Medical Systems Co., Japan. He is interested
in the fusion research between microelectronics and medical engineering.
T. Ukai is a post-doctoral researcher at the Bio-Nano Electronics Research
Centre, Toyo University. He received his PhD in 2003 and has been involved in
the development of micro-total analysis systems.
H. Morimoto received his PhD in 1998 and is an associate professor of the
Graduate School of Engineering, Toyo University. He has been studying complexity in nano/micro systems at the Bio-Nano Electronics Research Centre,
Toyo University.
Y. Nakaijima received his PhD in 2005 and is a post-doctoral researcher at
the Bio-Nano Electronics Research Centre, Toyo University. He is interested
in nanoelectronics and bio-nano fusion such as the interaction between carbon
nanotubes and biomolecules.
T. Hanajiri received his PhD in 1994 and is an associate professor of the
Graduate School of Interdisciplinary New Science, Toyo University. He is also
the vice-director of the Bio-Nano Electronics Research Centre, Toyo University. He is interested in the design and creation of new nano/micro electron
devices.
T. Maekawa received his PhD in 1983. He is a professor of the Graduate
School of Interdisciplinary New Science, Toyo University, and the director
of the Bio-Nano Electronics Research Centre, Toyo University. The research
centre was selected as a 21st Century’s Centre of Excellence in the field
of Interdisciplinary New Science by the Ministry of Education, Culture,
Sports, Science and Technology, Japan. He has been studying bio-nano fusion
research.