Blood plasma separation and extraction from a minute amount of blood using dielectrophoretic and capillary forces

Sensors and Actuators B 145 (2010) 561–569

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Blood plasma separation and extraction from a minute amount of blood using dielectrophoretic and capillary forces Yuta Nakashima 1 , Sakiko Hata 1 , Takashi Yasuda ∗,1 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan

a r t i c l e

i n f o

Article history: Received 2 April 2009 Received in revised form 19 September 2009 Accepted 30 November 2009 Available online 5 December 2009 Keywords: Dielectrophoresis Blood separation Blood plasma extraction Capillary force

a b s t r a c t This paper presents a microfluidic device that can separate and extract blood plasma from several microliters of blood without external mechanical driving sources such as a centrifugal machine and a syringe pump. This device consists of a main-channel, many side-channels, a reservoir, and two electrodes. After blood was automatically injected into the main-channel by capillary force, blood cells blocked the sidechannel entrances and prevented blood plasma from entering them. Next, when AC voltage application between the two electrodes separated blood into cells and plasma by dielectrophoresis (DEP), cells were removed from the side-channel entrances. This permitted plasma to be injected into the side-channels connecting to the reservoir. Experiments using diluted blood samples (dilution 1:9) showed that blood cell removal and blood plasma extraction were affected by channel geometry and magnitude/frequency of applied AC voltage. In case of applied AC voltage of 10 V and 1 MHz, blood cells were removed by about 97%, and blood plasma of about 300 nL was extracted from 5 ␮L blood. Moreover, we found that optimal dimension of the side-channels for blood plasma extraction is 5 ␮m wide and 2 ␮m deep. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The concern for healthcare and preventive medical care is rising with increase of lifestyle-related diseases and aging of the population. There is going to be a greater and greater demand for periodical and ongoing blood tests which are carried out at home by nonprofessional people. Hence, a stress-free, quick, and simple blood test method should be developed for that purpose. Generally, only the blood plasma which is separated and extracted from whole blood is used for diagnoses such as the measurement of cholesterol level, blood glucose level, hepatic function, etc. For separating blood into blood cells and blood plasma, external mechanical driving sources such as a centrifugal machine or a pressure source for blood filtration are usually required. Also, pipetting is necessary to extract the separated blood plasma for diagnoses. However, these apparatuses for blood plasma separation and extraction are expensive and use large space, and their operations are difficult for nonprofessional people. This makes it impossible to perform at-home health checkup using the conventional methods. Therefore, development of an easy-to-use microfluidic device for at-home blood test is awaited.

∗ Corresponding author. Tel.: +81 93 695 6047; fax: +81 93 695 6047. E-mail addresses: [email protected] (Y. Nakashima), [email protected] (T. Yasuda). 1 Tel: +81 93 695 6057; fax: +81 93 695 6047. 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.11.070

Many microfluidic devices for blood separation and plasma extraction have already been developed. For example, blood plasma was extracted from blood using sedimentation of blood cells in a microchannel [1,2]. Also, blood cells were separated by centrifugal force [3–6], and red blood cells, white blood cells, and platelets were differentiated using hydrodynamic separation [7–11]. Other devices separated blood into blood cells and blood plasma by capturing blood cells using microfilters or microchannels with taper toward the tip [12–15]. Moreover, the microdevices which extract blood plasma from whole blood using the Zweifach–Fung effect and allow plasma protein measurements were developed [16]. The Zweifach–Fung effect is a phenomenon where particles at branching points of microchannels tend to flow into the wider channel having a faster flow rate [17,18]. However, these devices require external mechanical driving sources such as a syringe pump and tube connections between devices and mechanical driving sources. The need of these peripheral equipments will highly increase the overall dimension of a microfluidic system, and also make device setting and replacement very cumbersome and complicated. Moreover, tube connections will increase dead volume in a microfluidic system and need a large volume of redundant blood sample. One of the effective solutions for those problems is to combine a cell handling technique using dielectrophoresis (DEP) [19–28] and a liquid transportation technique using capillary force because both of them do not require any external mechanical driving source. The DEP technique allows blood separation and blood cell handling using an electrostatic force that is caused by an inhomogeneous

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electric field between two electrodes [29–32]. This requires an electric power supply unit and control circuits outside a microfluidic device, but these peripheral devices can be miniaturized easily using LSI technology. Also, electric connections between a microfluidic device and circuit units will make setting and replacement of microfluidic devices much easier than tube connections. Moreover, no tube connections will reduce dead volume dramatically, and lead to a highly significant decrease in blood sample. Therefore, blood separation and blood plasma extraction using DEP and capillary forces is suitable for at-home blood tests where a compact device, easy operation, and a minute amount of testing sample are strongly required. In this paper, we present a microfluidic device for separating blood into blood cells and blood plasma by DEP, and extracting blood plasma by transporting it from the separation channel into a reservoir through many other narrower channels by capillary force. Also, we evaluated blood cell removal efficiency and extracted blood plasma volume. Moreover, we investigated optimal design of the microchannels for blood plasma extraction.

Fig. 1. Real part of the Clausius–Mossotti factor as the function of the logarithmic frequency of an electric field.

2. Theory

3. Materials and methods

Dielectrophoresis (DEP) is a technique to handle neutral particles in liquid medium using polarization of particles and medium in an inhomogeneous electric field. The DEP force on a particle is given by the following equation [33,34]:

3.1. Device design



2

FDEP = 2εm r 3 Re(fCM )∇ Erms 

(1)

where εm is the permittivity of the medium, r is the radius of the particle, Erms is the RMS electric field, and Re(fCM ) is the real part of the Clausius–Mossotti (CM) factor which determines whether the DEP is positive or negative. The CM factor is given by fCM =

ε∗p − ε∗m ε∗p + 2ε∗m

(2)

where ε∗p and ε∗m are the complex permittivities of the particle and medium, respectively, which depend on their electric conductivities and frequency of the AC electric field. The complex permittivity is given by  (3) ω √ where j = −1, ε is the real permittivity,  is the electric conductivity, and ω is the angular frequency of the AC electric field. The sign of the CM factor varies depending on whether particles are more or less polarizable than their surrounding medium. When the particle permittivity is higher than that of the medium, Re(fCM ) is positive, and the particles are gravitated toward regions of high electric field. This behavior is known as positive DEP (pDEP). On the other hand, when the particle permittivity is lower than that of the medium, Re(fCM ) is negative, and the particles move toward the weaker electric field. This behavior is known as negative DEP (nDEP). The electric permittivity of particles is highly dependent on the frequency of the applied electric field [35]. We calculated the real part of the CM factor of diluted blood. The relative permittivity and electric conductivity of diluted blood plasma can be assumed to be 80 and 55 mS/m, and those of a blood cell are known to be 63 and 1.0 ␮S/m, respectively [24,31]. Substituting these values into Eqs. (2) and (3), we obtained the CM factor dependency on the frequency of electric field as shown in Fig. 1. The real part of the CM factor is consistently negative over a wide range from low frequency to high frequency. Therefore, we used nDEP in order to separate blood into blood cells and blood plasma on a device. ε∗ = ε − j

Fig. 2 shows the schematic of representation of the present microfluidic device. The device consists of a main-channel, many side-channels that were fabricated along the sidewall of the mainchannel, a plasma reservoir, and two electrodes. The microchannels and the plasma reservoir were made of PDMS (polydimethylsiloxane, Dow Corning Corp., Sylgard 184) and their mold was fabricated with double layered SU-8 photoresist films. The two electrodes were designed like a framed rectangle shape (Fig. 2(A)(a)) and a pin shape (Fig. 2(A)(b)), and were fabricated on the glass plate. The rectangular cross-section of each side-channel was designed to have a shorter side than the diameter of blood cells: red blood cells, white blood cells, and blood platelets measuring 7–8 ␮m, 8–12 ␮m, and 2–3 ␮m in diameter, respectively. This prevents their infiltration into side-channels. The photographs of the fabricated device are shown in Fig. 3. The main-channel measures 500 ␮m in width, 20 mm in length, and 100 ␮m in depth. The side-channels measure 5 ␮m in width, 1 mm in length, and 2 ␮m in depth. Note that this cross-sectional dimension of the side-channel was obtained from the experimental results as discussed later. The plasma reservoir measures 3 mm in width, 3 mm in length, and 100 ␮m in depth. The width of each electrode is about 60 ␮m, and the interelectrode distance is 30 ␮m. 3.2. Principle of blood separation and blood plasma extraction Fig. 4 shows the blood separation and blood plasma extraction method. When we drop a 5 ␮L blood droplet at the inlet of the mainchannel using a micropipette (Gilson, Inc., Pipetman 10), about 1 ␮L blood automatically enters the main-channel by capillary force (Fig. 5), and blood cells block the entrances of the side-channels (Fig. 4(a)). This prevents blood plasma from entering the sidechannels. When we apply an AC voltage with a high frequency between the two electrodes using a function generator (Agilent Technologies, Inc., 33120A), inhomogeneous electric field generated between the two electrodes will give a nDEP force to blood cells, that will move them toward the weaker electric field direction [35–38]. As a result, the blood cells that blocked the side-channel entrances will be repelled from the pin-electrode, and trapped in the framed-rectangle-electrode, i.e. blood cells are removed from the side-channel entrances (Fig. 4(b)). This permits blood plasma to be injected into the side-channels by capillary force.

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Fig. 2. Schematic of representation of the present microfluidic device.

Fig. 4. Principle of blood separation and blood plasma extraction.

Fig. 3. Photographs of the fabricated device. (a) Main components of the device and (b) geometry of the two electrodes.

Fig. 5. Schematic illustration of the device holding diluted blood.

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4.1. Blood plasma separation test

Fig. 6. Fabrication process of the device.

3.3. Device fabrication Fig. 6 shows the fabrication process of the microfluidic device. The microchannels were made of PDMS and bonded with a glass plate that has two electrodes. The device was fabricated as follows. First, a 2 ␮m thick diluted SU-8 3050 photoresist (Kayaku MicroChem Co., Ltd) layer for a mold of side-channels was patterned on a Si wafer (Fig. 6(a)). Secondly, a 100 ␮m thick SU-8 3050 photoresist layer for a mold of a main-channel and reservoir was patterned on the Si wafer that has the side-channel mold structure (b). Next, we casted PDMS over the SU-8 mold and cured it in a furnace (c). On the other hand, two electrodes were fabricated on the glass plate as follows. First, the glass plate was spin-coated with ZPN-1150 photoresist (ZEON Corp.) and patterned (e). Next, an about 100 nm thick Au layer with an about 30 nm thick adhesive Cr layer was deposited by vacuum evaporation (ULVAC Kiko, Inc., VPC-260) (f), and the two electrodes were patterned by lift-off process (g). Finally, the PDMS device was peeled from the mold Si wafer (d), and bonded to the glass plate over the two electrodes (h). 4. Results and discussion We carried out several experiments in order to evaluate blood separation efficiency and blood plasma extraction volume using two kinds of devices that has and does not have side-channels and a reservoir. Whole blood was extracted from a healthy adult donor through venipuncture. The extracted whole blood was mixed with anticoagulant EDTA (Wako Pure Chemical Industries, Ltd., 34301883) so that its concentration was 1 mg/mL. The sample was then diluted in the PBS (phosphate buffer solution) in a volume ratio of 1:9 (sample:PBS). The performance of the device was checked using this 5 ␮L blood sample. We observed motion of blood cells and blood plasma with a stereoscopic microscope (Nikon Co., SMZ 1500), an inverted microscope (Nikon Co., TS 100) and a CCD camera (Tokyo Electronic Industry Co., Ltd., cs5270) while AC voltage was applied between the two electrodes.

We carried out the blood plasma separation test using the microfluidic device that does not have the side-channels and the reservoir. Blood plasma separation in case of applied AC voltage of 10 V and 1 MHz was observed under the stereoscopic microscope as shown in Fig. 7. When we applied AC voltage, blood cells on and in between the two electrodes moved toward the weaker electric field by nDEP, i.e. blood cells moved toward the framed rectangle shape electrode (Fig. 7(b)(c)). Eventually, blood cells were trapped on the center of the framed rectangle shape electrode, and blood plasma separated from cells was stored in between the two electrodes (Fig. 7(d)). The separated plasma areas where no cells exist between the two electrodes were measured from the freeze-frames of the video images. Then, we defined the blood separation efficiency as the separated plasma area divided by the measurement area that is the rectangular area between the two electrodes as shown in Fig. 8. The blood separation efficiency when a voltage of 1 MHz or 10 MHz was applied for 90 s was plotted against the voltage magnitude in Fig. 8. As a result, the blood separation efficiency increased with the voltage magnitude. Also, the efficiency in case of 1 MHz was about twice as large as that in case of 10 MHz. This is because the nDEP force acting on blood cells in case of 1 MHz is two times larger than that in case of 10 MHz. This result is consistent with the theory indicating that the real part of the CM factor in case of 1 MHz is about −0.5 and that in case of 10 MHz is about −0.25 as shown in Fig. 1. Moreover, to figure out the effective shape of the electrodes for blood separation, we tested three designs of electrodes as shown in Fig. 9. The differences of the three designs are whether the right pin electrode is sharp or not and whether the left electrode has an enclosure frame or not. The former difference will vary the gradient of electric field, and the latter will affect the effectiveness of cell trapping in the left electrode. Consequently, they will seriously affect the blood separation efficiency. The 1st design has a framed rectangular electrode and a rectangular pin electrode. The 2nd design has a framed rectangular electrode and a sharp-pointed pin electrode. The 3rd design has an unframed electrode and a rectangular pin electrode. The blood separation efficiencies obtained from these electrodes were plotted in Fig. 9. Note that these efficiencies were derived when the blood separation was completely finished after a long-time voltage application. The blood separation efficiency in case of 1 MHz was larger than that in case of 10 MHz in every case. However, unlike the result in Fig. 8, the former was smaller than twice the latter because the separation in case of 10 MHz proceeded more slowly but continued for a longer time than in case of 1 MHz. We obtained the highest efficiency, 98%, in case of the 1st design. It’s not known exactly why the efficiency of the 2nd design was lower than that of the 1st one. The detailed numerical analysis will be required to clarify the reason in the near future. Also, in case of the 3rd design, blood cells repelled from the right side electrode could not be trapped effectively in the left side electrode because an enclosure frame was not fabricated in the left side electrode, thus this decreased the blood separation efficiency. We cannot claim that the optimal design will be one of the three designs. In order to obtain the truly optimal design, the detailed numerical analysis of the electric field and cell motion generated by designed electrodes will be strongly required. 4.2. Blood plasma extraction test We carried out blood plasma extraction from the main-channel to the plasma reservoir through the side-channels. Blood plasma extraction in case of applied AC voltage of 10 V and 1 MHz is shown in Fig. 10. First, when a blood droplet of 5 ␮L was placed at the inlet of the main-channel using a micropipette, the blood droplet

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Fig. 7. Sequential photographs of blood separation into cells and plasma by nDEP.

was injected into the main-channel by capillary force (Fig. 10(a)). In this case, blood plasma could not enter the side-channels because blood cells blocked the entrances of the side-channels. Note that in Fig. 10(a) it seems as if there were no blood cells on the electrodes because the device was observed from below using an inverted microscope, but actually a large number of cells exist on the electrodes at the same density as on the other part of the main-channel. Next, when we applied AC voltage between the two electrodes, the blood was separated into blood cells and blood plasma by nDEP (Fig. 10(b)). Then, the blood cells that blocked the side-channel entrances were repelled from the pin-electrodes. In Fig. 10(b), it seems as if many cells had stayed at the side-channel entrances, but actually all of them were levitated and trapped at the upper corner of the main-channel as shown in Fig. 10(b) because the electric field is weak there. This permitted blood plasma to be injected

into the side-channels by capillary force (Fig. 10(c)). Because the plasma was injected into all of the 120 side-channels in all three trials, the efficiency of cell removal from the side-channel entrances is presumed to be almost 100% under this experimental condition. Finally, blood plasma was transported to the plasma reservoir (Fig. 10(d)). We carried out the experiments using various devices that have different channel geometries: different side-channel width, w, and different main-channel depth, d, which is/is not terraced near the side-channel entrances. Fig. 11 shows the blood cell removal efficiency in various devices with different geometries. The blood cell removal efficiency, , is given by the following equation: =1−

x1 x0

(4)

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Fig. 8. Relationship between applied voltage and blood separation efficiency.

where x0 is the number of blood cells per unit volume injected into the main-channel, and x1 is the number of blood cells per unit volume transported to the reservoir. These blood cells were counted using a blood cell counting chamber (ERMA INC., Burkerturk KN3318781). Note that blood cells counted here include white blood cells and blood platelets as well as red blood cells. The 2nd type device having a 100 ␮m deep main-channel and 5 ␮m wide side-channels gave the highest efficiency of blood cell removal because the narrow inlets of the side-channels prevented blood cells from breaking into the side-channels. In this case, even blood platelets which are smaller than red/white blood cells and have the diameter close to the side-channel depth hardly entered the side-channels. This is probably because the small spherical shape of blood platelets were not deformed easily by a small hydrodynamic force that was induced by capillary phenomenon, and so the inlets of side-channels were shallow enough to prevent blood platelets from entering the side-channels. As a result, we could remove about

Fig. 9. Blood separation efficiencies in cases of three devices having different electrode designs.

97% of blood cells using the 2nd type device in case of applied AC voltage of 10 V and 1 MHz. This blood cell removal efficiency was more than twice the 1st type and 3rd type channel geometries. In case of the 1st type channel, a large amount of blood cells were injected easily to the side-channels because the side-channel width was larger than the diameters of red blood cell and white blood cell. Unlike the other channel types, when we applied AC voltage of 10 MHz, blood cell removal efficiency turned out to be larger than that of 1 MHz because blood cells could not move easily by DEP and thus blocked the entrances of the side-channels even after apply-

Fig. 10. Sequential photographs from blood injection to plasma extraction.

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Fig. 12. Variation in extracted plasma volume in four different conditions. Fig. 11. Blood cell removal efficiencies in cases of three devices having different channel geometries.

ing voltage. In other words, blood cell injection to the side-channels was unintentionally inhibited by blood cells that blocked the sidechannel entrances, and only a small amount of blood plasma that included few blood cells was transported to the reservoir. In case of the 3rd type channel, the length of the terraced part was much larger than the diameters of typical red blood cell, and its depth was nearly equal to the thickness of typical red blood cell. Therefore, due to a hydrodynamic force induced by capillary phenomenon, blood cells were drawn into the terraced part without major deformation. However, they were of small amount because the hydrodynamic force was small at the entrance of the terraced part. A small amount of blood cells were not enough to completely block the side-channel entrances, and this allowed plasma to flow into the side-channels by capillary force even without voltage application. Subsequently, the blood cells at the terraced part were deformed and drawn into the side-channels due to a large hydrodynamic force generated by the plasma flow. For this reason, the blood cell removal efficiency of the 3rd type channel geometry was lower than that of the 2nd type. From the above discussions, in order to obtain high efficiency of blood cell removal, we should block the side-channel entrances with blood cells completely before applying voltage, and remove the cells from the entrances efficiently after applying voltage. Variation in extracted blood plasma volume in four different conditions is shown in Fig. 12. We succeeded in transporting blood plasma of about 300 nL to the reservoir using the device that has 5 ␮m wide side-channels in case of applied AC voltage of 10 V and 1 MHz. This extracted blood plasma volume was about three times larger than that in case of applied AC voltage of 10 V and 10 MHz.

the side-channel depth. When the side-channel depth was 2 ␮m, blood plasma of about 300 nL separated from 5 ␮L blood was transported to the reservoir, and this condition gave the largest amount of extracted plasma volume. When the side-channels were deeper than 2 ␮m, the extracted plasma volume became larger with an increase of the side-channel depth. This is because blood cells entered and clogged the side-channels, preventing blood plasma extraction. In case of the 3 ␮m deep side-channel, blood plasma was almost never transported to the reservoir probably because red blood cells fitted the side-channel snugly. It is remarkable that the difference of just 1 ␮m in depth of the side-channels generated so dramatically different effects. When the side-channels were deeper than 3.5 ␮m, blood plasma of more than 300 nL was extracted, but it included many blood cells, i.e. blood cells did not block the sidechannel entrances and were transported to the reservoir because the side-channel depth was larger than the typical blood cell thickness (about 2–3 ␮m). On the other hand, the relationship between the side-channel depth and the blood cell removal efficiency in this experiment is shown in Fig. 14. When the side-channel depth was smaller than 2.5 ␮m, blood cells were removed by almost 100%. However, when the side-channels were deeper than 3.5 ␮m, blood cells were not removed at all. From the above discussions, it was found that optimal dimension of the side-channel depth. This is

4.3. Optimal design of side-channel depth We investigated optimal design of the side-channel depth using various devices that have different side-channel depths and the same side-channel width of 5 ␮m. Fig. 13 shows the relationship between the side-channel depth and the extracted blood plasma volume. In this experiment, we applied the AC voltage of 10 V and 1 MHz. When the side-channel depth was shallower than 1.5 ␮m, blood plasma entered the side-channels, but did not go out into the reservoir because the shallower side-channels generated larger capillary force for keeping blood plasma stored in the side-channels. When the side-channel depth was 1.5–2 ␮m, the extracted plasma volume decreased larger with an increase in

Fig. 13. Relationship between side-channel depth and extracted plasma volume.

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[5]

[6]

[7]

[8]

[9] [10]

[11] Fig. 14. Relationship between side-channel depth and blood cell removal efficiency. [12]

because blood large volume of extracted blood plasma and high efficiency of blood cell removal is 5 ␮m wide and 2 ␮m deep. The amount of blood plasma, 300 nL, extracted using the present device is much less than that of several tens of microliters required for a conventional blood test. However, Fan et al. have recently succeeded in detecting the multiple plasma proteins from about 100 nL blood plasma in individual narrow channels [16]. Because we can fabricate detecting microelectrodes inside the side-channels or reservoir using the MEMS technology, the amount of extracted blood plasma will be large enough for the blood test. 5. Conclusions

[13]

[14]

[15]

[16]

[17] [18]

We succeeded in blood separation and blood plasma extraction from a minute amount of blood without any external mechanical driving source. An electrode having an enclosure frame shape was required for separating blood cells and blood plasma, and its efficiency depended on the magnitude and frequency of the applied AC voltage. The blood separation efficiency became larger with an increase in applied voltage, and that in case of voltage frequency of 1 MHz was larger than in case of 10 MHz. When AC voltage of 10 V and 1 MHz was applied, blood cells were removed by about 97%, and blood plasma of about 300 nL separated from 5 ␮L blood was transported to the reservoir. Moreover, the optimal dimensions of a side-channel for blood plasma extraction were found to be 5 ␮m in width and 2 ␮m in depth. In the near future, blood plasma will be extracted more efficiently without need of blood dilution by adding side-channels and optimizing arrangement and shape, etc. of device components. Acknowledgments

[19] [20]

[21] [22] [23] [24]

[25]

[26] [27] [28] [29]

This work was partly supported by a fund of the Knowledge Cluster Initiative implemented by MEXT (Ministry of Education, Culture, Sports, Science and Technology) and the Grant-in-Aid for Strategic Research Advancement, Kyutech 2009.

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fabrication, and application of microfluidic systems such as cell handling devices and liquid handling devices.

Biographies

Takashi Yasuda received the BE, ME, and PhD degrees in Mechanical Engineering from the University of Tokyo in 1989, 1991, and 1994, respectively. From 1994 to 2001, he worked as a research associate and a lecturer at the Department of Mechano-Informatics, the University of Tokyo. In 2001, he joined the Kyushu Institute of Technology, and is currently an associate professor of the Department of Biological Functions and Engineering, the Graduate School of Life Science and Systems Engineering. His research interests include liquid handling, cell handling, bio-sensing, etc. using MEMS technology.

Yuta Nakashima received the ME and PhD degree in Life Science and Systems Engineering from the Kyushu Institute of Technology, Japan, in 2004 and 2007, respectively. He is currently a postdoctoral fellow of the Department of Biological Functions and Engineering, the Graduate School of Life Science and Systems Engineering, the Kyushu Institute of Technology. His research interests include design,

Sakiko Hata received the BE degree in Electrical Engineering and Electronics from the Kyushu Institute of Technology in 2009.