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Analytica Chimica Acta

Manuscript Draft

Manuscript Number: ACA-17-1155

Title: Microfluidic device coupled with a microfabricated oxygen


electrode for the measurement of bactericidal activity of neutrophil-like
cells

Article Type: Full Length Article

Section/Category: ELECTROCHEMISTRY

Keywords: Neutrophil-like cells; Bactericidal activity; Escherichia coli;


Oxygen electrode; Microfluidics; Psychological stresses.

Manuscript Region of Origin: JAPAN


*Graphical Abstract

Graphical abstract
*Highlights

Highlights

■ A microfabricated Clark-type oxygen electrode was coupled with a flow channel structure.
■ Neutrophil-like cells were co-incubated with E. coli cells.
■ Changes in the consumption of oxygen by E. coli cells was measured using the oxygen
electrode.
■ The activity of the neutrophil-like cells could be measured using the device.
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(Yamagishi

Microfluidic device coupled with a microfabricated oxygen electrode


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3 for the measurement of bactericidal activity of neutrophil-like cells
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8 Anna Yamagishia, Koji Tanabea, Masatoshi Yokokawaa, Yuji Morimotob, Manabu Kinoshitac,
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10 and Hiroaki Suzukia,*
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15 a
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai,
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18 Tsukuba, Ibaraki 305-8573, Japan
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Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical
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23 College, Namiki 3-2, Tokorozawa 359-8513, Japan
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25 Department of Immunology and Microbiology, National Defense Medical College, Namiki
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3-2, Tokorozawa 359-8513, Japan
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32 Corresponding author. Tel.: +81 29 853 5598; fax: +81 29 853 4490.
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35 E-mail addresses: hsuzuki@ims.tsukuba.ac.jp (H. Suzuki)
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ABSTRACT
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2 A microfluidic device coupled with a microfabricated Clark-type oxygen electrode was
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5 used to measure the bactericidal activity of neutrophil-like cells differentiated from HL-60
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7 cells. The neutrophil-like cells and Escherichia coli cells were cultured in the same medium,
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10 which was introduced into the flow channel of the device. Changes in the respiratory activity
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12 of E. coli were measured as changes in the consumption of dissolved oxygen. As the activity
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of the neutrophil-like cells increased, the rate of elimination of E. coli increased. The
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17 accompanying decrease in the number of E. coli reduced the consumption of dissolved
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19 oxygen. The changes were actually observed as changes in generated current. A distinct
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22 difference in changes in dissolved oxygen concentrations was observed between E. coli cells
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24 co-incubated with IFN-γ-activated or non-activated neutrophil-like cells. The required sample
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27 volume was less than 10 μL, and results could be obtained within 1–2 h. The device may be
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29 useful for the assessment of psychological stresses that affect the activity of neutrophils.
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34 Keywords:
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36 Neutrophil-like cells, Bactericidal activity, E. coli, Oxygen electrode, Microfluidics,
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39 Psychological stresses.
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1. Introduction
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2 In clinical research fields related to infectious diseases, allergic reactions, tumors, and
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5 organ transplantation, there is a rapidly increasing demand for the evaluation of individual
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7 host immune responses. Neutrophils play a critical role in host defense against external
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10 harmful substances.[1-3] Although neutrophils effectively eradicate infectious pathogens
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12 under healthy host conditions, their activity can be seriously affected by external stimuli
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including surgical stresses such as burn injury, multiple trauma, or hemorrhagic shock.[4, 5]
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17 Similarly, psychological stresses, e.g. depressive disorder, bereavement, or undue anxiety,
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19 also reportedly induce functional alterations in neutrophils.[6-8] However, neutrophils
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22 sometimes exhibit equivocal behaviors under stress conditions. For example, burn injuries
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24 enhance the superoxide production activity of neutrophils, although they potently reduce
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27 neutrophil bactericidal activity, which is the most important and essential role of neutrophils
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29 in host defense against invading bacteria.[4] In addition, superoxide production from
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neutrophils may not precisely indicate host defense conditions, as severely damaged
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34 neutrophils cannot effectively produce superoxide.[9]
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36 The HL-60 cell line is a human promyelocytic leukemia cell line that has been widely
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39 used to study various leukocyte functions.[10] HL-60 cells differentiate into neutrophil-,
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41 monocyte-, or eosinophil-like cells depending on the method of differentiation. HL-60 cells
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44 can be differentiated into neutrophil-like cells with all-trans-retinoic acid treatment,[11]
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46 allowing for the evaluation of host defense capabilities by assessment of their bactericidal
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49 activity.
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51 The bactericidal activity of neutrophils is usually evaluated by co-culturing neutrophils
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with target bacteria for a certain time period (1–6 h).[4, 9] Decreases in the number of bacteria
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56 are measured by counting the number of colonies. However, this method requires at least half
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58 a day for the evaluation and optimization of co-culture conditions. Therefore, a more rapid
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and simple on-site measurement technique would be preferable.
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2 The respiratory activity of aerobic microorganisms can be evaluated by measuring their
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5 oxygen consumption. Based on this principle, various microorganism-based biosensors were
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7 developed, particularly in the 1980s, to detect analytes that can act as nutrients for
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10 microorganisms.[12] In sensor applications, a sufficient number of microorganisms are
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12 immobilized on the sensitive area of a transducer (typically an oxygen electrode), and the
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concentration of an analyte is measured. Conversely, by fixing the concentrations of nutrients
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17 for the microorganisms, the number of live cells can also be estimated from the consumption
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19 of oxygen. This method is also applicable to measuring the bactericidal activity of neutrophils.
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22 If the number of aerobic bacteria decreases due to bacterial killing by neutrophils, a change in
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24 the bacterial count may be measured from the change in the dissolved oxygen concentration.
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27 In general, it is not difficult to obtain a large number of microorganisms for implementing
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29 this method. However, this is not the case for animal cells obtained from physiological fluids
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or obtained after culturing. Therefore, the volume of solution containing the animal cells to be
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34 analyzed should ideally be small. In addition, the consumption of expensive reagents, such as
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36 IFN-γ, should be minimized.
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39 To measure the consumption of oxygen, microfabricated electrodes can be directly
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41 immersed in a cell solution.[13, 14] However, the electrodes should preferably be separated
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44 from the solution to be handled to minimize the influence of interferents. For this purpose, we
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46 used a microfabricated Clark-type oxygen electrode coupled with a microfluidic system.
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49 Miniaturization of the Clark-type oxygen electrode as an independent probe was actively
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51 performed, particularly in the 1980s and 1990s.[15-20] However, to our knowledge, oxygen
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electrodes have not previously been integrated into microfluidic systems.
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56 To address this issue, we developed a microdevice to measure the activity of neutrophils.
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58 We used a microfabricated Clark-type oxygen electrode coupled with a microfluidic channel
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and bacteria (E. coli). Using this method, we measured changes in the consumption of oxygen
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2 following a decrease in the number of bacteria resulting from phagocytosis by neutrophils.
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5 Using this technique, the consumption of oxygen can be measured rapidly (3 min) with a trace
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7 amount of sample solution (less than 10 μL).
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12 2. Experimental section
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2.1 Materials
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17 Reagents and materials used for fabrication and characterization of the device were
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19 obtained from the following commercial sources: glass wafers (no. 7740, 3 inch, 500 μm
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22 thick) from Corning (Corning, NY, USA); a thick-film photoresist, SU-8 25, from
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24 MicroChem (Newton, MA, USA); poly(dimethylsiloxane) (PDMS), KE-1300T and
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27 CAT-1300, from Shin-Etsu Chemical (Tokyo, Japan); a silicone rubber film (30 μm thick)
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29 from Mitsubishi Plastics (Tokyo, Japan). Luria-Bertani (LB) medium and human IFN-γ were
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purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise noted, all other
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34 chemicals were reagent grade or higher. Deionized Milli-Q water (18.2 MΩ cm; Millipore,
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36 Billerica, MA, USA) was used for all solutions.
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41 2.2 Fabrication of the device
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44 The Clark-type oxygen electrode consists of a cathode and an anode immersed in an
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46 electrolyte solution in a container with an oxygen-permeable membrane at one end. With an
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49 appropriate potential applied to the cathode (working electrode) with respect to the anode,
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51 oxygen is reduced on the cathode. Under diffusion-limiting conditions, the generated current
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changes in proportion to the dissolved oxygen concentration. The oxygen electrode structure
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56 was miniaturized and incorporated into the device shown in Fig. 1. The device was
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58 constructed from a glass substrate with cathode (platinum) and anode (Ag/AgCl) patterns and
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a PDMS substrate with a flow channel structure. The platinum and silver patterns for the
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2 cathode and anode were formed stepwise by a thin-film process including the formation of
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5 photoresist patterns, sputtering, and lift-off. The thicknesses of these layers were 300 nm and
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7 550 nm, respectively. A chromium intermediate layer (30 nm thick) was used under the
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10 platinum layer to promote adhesion. The active areas of the electrodes were delineated with a
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12 polyimide layer. In the oxygen electrode, the state of AgCl in the Ag/AgCl anode influences
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the stability and determines the lifespan. To form the AgCl layer during the fabrication
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17 process is not efficient, as the layer is easily damaged by the dissolution of AgCl
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19 accompanying the formation of silver complexes.[21] Therefore, AgCl was grown during the
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22 operation of the oxygen electrode. Ideally, to achieve a long lifespan, the anode area should
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24 be large; however, with a large anode area, it takes longer to grow the AgCl and stabilize the
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27 anode potential. To solve this conflict, we used an Ag/AgCl structure covered with a
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29 protective layer, and AgCl was grown from pinholes formed in the protective layer.[19, 22]
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Because the Ag/AgCl layer is anodically polarized while using the oxygen electrode, AgCl is
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34 constantly and spontaneously formed from the pinholes onto the silver layer. Thus, the anode
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36 potential is automatically stabilized. In the fabricated device, 16 pinholes were formed in the
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39 insulating layer on the anode silver pattern. Compartments to accommodate an electrolyte
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41 solution and flow channels to connect the compartments and introduce the electrolyte solution
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44 were formed with a thick-film photoresist (SU-8). The height of the structures was 60 μm.
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46 The sensitive area (compartment for the cathode) was circular with a diameter of 1.35 mm.
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49 A flow channel structure for the transport of solutions containing E. coli and/or
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51 neutrophil-like cells was formed with PDMS by replica molding using the pattern of the
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thick-film photoresist as a mold. The flow channels were 500-μm wide and 230-μm high. The
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56 volume of the sensing region to accommodate the cells was approximately 850 nL. A silicone
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58 rubber oxygen-permeable membrane with a thickness of 30 μm was bonded to the PDMS
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substrate with the flow channel structure. For this purpose, the PDMS substrate and the
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2 oxygen-permeable membrane were exposed to an oxygen plasma (10 W, 20 Pa) for 10 s to
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5 activate their surfaces (BP-1; Samco, Kyoto, Japan). By placing the membrane onto the
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7 PDMS substrate immediately after activation, they were bonded spontaneously. To complete
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10 the device, the PDMS substrate with the oxygen-permeable membrane was placed on the
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12 SU-8 structure of the oxygen electrode after carefully aligning the patterns of the flow
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channel and the electrodes. The substrates were inserted between two poly(methyl
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17 methacrylate) holders, which were fixed with bolts and nuts.
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22 2.3 E. coli and neutrophil-like cells
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24 E. coli K-12 strain MG1655 was grown overnight by shaking in LB medium (20 mL) at
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27 37°C. After centrifugation for 10 min at 3,000 ×g and 4°C, the pellet was resuspended in
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29 ice-cold LB medium, and the number of E. coli cells was counted by optical density
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measurement (OD600) (UV-1800; Shimadzu, Kyoto, Japan). After dilution with ice-cold LB
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34 medium (OD: 1.0), the bacterial solution was aliquoted and stored at −80°C as individual
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36 standard stocks in a deep freezer until use.
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39 Neutrophil-like cells were differentiated from human promyelocytic leukemia cells (HL-60,
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41 Riken Cell Bank) as described previously.[23-25] HL-60 cells were first cultured in
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44 RPMI1640 medium supplemented with 10% fetal bovine serum in a humidified 5% CO2
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46 atmosphere at 37°C. For differentiation, HL-60 cells were cultured with 2 µM all-trans
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49 retinoic acid for 5 days.
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2.4 Measurement procedures
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56 For use of the oxygen electrode, a 0.1 M Tris-HCl buffer solution (pH 8.5) containing 0.1
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58 M KCl was used as the internal filling solution and was introduced into the oxygen electrode
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compartment using a microsyringe. The electrodes on the chip were connected to a
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2 potentiostat (Autolab PGSTAT12; Eco Chemie, Utrecht, Netherlands). The potential applied
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5 to the cathode was −0.8 V with respect to the Ag/AgCl anode unless otherwise noted.
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7 Response of the oxygen electrode was checked by changing the medium in the flow channel
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10 from air to a saturated Na2SO3 solution and back to air.
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2.5 Measurement of the consumption of dissolved oxygen by E. coli
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17 A stock of frozen E. coli was thawed by incubating it for 5 min at 37°C. After
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19 centrifugation for 15 min at 1,500 ×g and 4°C, the pellet of E. coli was resuspended in
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22 phosphate-buffered saline (PBS). The concentration of E. coli was checked using a bacterial
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24 counter (#A161; Sunlead Glass, Saitama, Japan) under a microscope (BX51; Olympus, Tokyo,
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27 Japan) and was adjusted to 1 × 109 cells mL−1. Although the suspension contained both live
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29 and dead cells, it was expected that the neutrophil-like cells eliminated both of these and that
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the oxygen consumption of live cells could be measured. Serial dilutions of E. coli suspension
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34 were made with PBS supplemented with 10 mM glucose followed by incubation for 1 h at
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36 37°C. Then, the dilutions were immediately introduced into the flow channel of the device to
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39 measure the oxygen concentration. All measurements were carried out at room temperature.
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43 2.6 Measurement of E. coli killing by neutrophil-like cells using the microfabricated oxygen
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46 electrode
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48 After differentiation, neutrophil-like cells were centrifuged for 3 min at 1,500 ×g and 37°C
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51 and were resuspended in PBS containing 50 mM glucose (4 × 107 cells mL−1). Thereafter, the
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53 cells were incubated with or without human IFN-γ (150 units mL−1) for 2 h at 37°C. An E.
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56 coli culture (1 × 109 cells mL−1 in PBS containing 50 mM glucose) was prepared in parallel
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58 and incubated for 1 h at 37°C. Before co-incubation, the IFN-γ-stimulated HL-60 cells were
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centrifuged for 3 min at 1,500 ×g and 37°C and were resuspended in PBS of the same volume
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2 containing 50 mM glucose (without IFN-. Then, 500 L of the solution of the
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5 neutrophil-like cells (4 × 107 cells mL−1) and 100 L of the E. coli suspension (1 × 109 cells
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mL−1) were mixed. The ratio of E. coli cells to neutrophil-like cells was 5:1. The cells were
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10 co-incubated in the absence of IFN- for 1 h at 37°C. To measure the elimination of E. coli,
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the mixture was immediately introduced into flow channel A of the device, and the current of
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15 the oxygen electrode was measured on a hotplate maintained at 25°C.
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17 For comparison, the number of E. coli in the mixture was also measured by transferring 20
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20 µL of the suspension to the bacterial counter and manually counting the number of cells under
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22 an optical microscope.
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27 2.7 On-chip co-incubation of E. coli and neutrophil-like cells and measurement of the
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30 elimination of E. coli
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32 In situ co-incubation of the neutrophil-like cells and E. coli in the flow channel and
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subsequent measurement of the oxygen consumption were also conducted. An E. coli culture
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37 of 2 × 109 cells mL−1 (100 µL) was mixed with a suspension of HL-60 cells at 8 × 107 cells
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39 mL−1 (100 µL). Then, the mixture was diluted twice with PBS containing glucose and IFN-.
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42 The final concentrations of glucose and IFN- were 50 mM and 150 units mL−1, respectively.
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The suspension was immediately introduced into the flow channel of the device, and the
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47 change in dissolved oxygen was measured thereafter. During the measurement, the
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49 temperature of the device was maintained at 37°C on a hotplate.
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54 2.8 Statistical analyses
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57 Statistical analyses were performed using the Stat View 4.02J software package (Abacus
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59 Concepts, Berkeley, CA, USA). Statistical evaluations were compared using one-way analysis
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of variance, followed by Bonferroni post-hoc test. Data are presented as means  standard
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deviations (SDs), with p < 0.05 considered to be statistically significant.
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7 3. Results and discussion
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10 3.1 Basic properties of the oxygen electrode
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12 In the Clark-type oxygen electrode, the hydrophobic oxygen-permeable membrane
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15 separates the internal electrolyte solution from the external solution to be analyzed. This
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17 avoids the influence of electroactive compounds in the external solution and contamination of
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the cathode, which is advantageous in analyzing a solution whose contents are unknown. Fig.
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22 2 shows a typical response curve of the oxygen electrode when the medium in the sensing
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24 region was changed from air to a saturated Na2SO3 solution. Na2SO3 was used to remove
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27 dissolved oxygen. Although the oxygen electrode we fabricated was much smaller than
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29 commercialized conventional oxygen electrodes, it showed clear, stable, reproducible
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32 responses. The 90% response time was less than 5 s for increasing changes and 20 s for
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34 decreasing changes. While conducting the experiments, however, a non-negligible residual
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37 current was detected. The residual current was 5% of that of the current for air. It is possible
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39 that the influx of oxygen from the surrounding PDMS walls was rapid and that even the
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saturated Na2SO3 solution moving through the flow channel could not maintain a zero-oxygen
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44 state before reaching the cathode region. However, a degree of oxygen influx is necessary to
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46 maintain the dissolved oxygen concentration in the liquid plug under the steady state.
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51 3.2 Measurement of oxygen consumption by E. coli
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54 In the microorganism-based biosensors mentioned earlier, the respiratory activity of
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56 microorganisms immobilized in a restricted space can be measured clearly. In addition, the
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response to the corresponding analyte (nutrient) can be very large depending on the
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concentration. This alternatively suggests that the current of the oxygen electrode in the
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2 presence of the microorganisms can be adjusted by changing the analyte concentration. In the
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5 experiments that follow, we fixed the concentration of glucose at 50 mM. We first examined
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7 the response of the oxygen electrode with E. coli suspensions of different concentrations. Fig.
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10 3A shows time courses of the current recorded for E. coli concentrations ranging from 6.5 ×
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12 105 to 1.0 × 109 cells mL−1. It should be noted that the suspensions contained both live and
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dead cells, as their separation is not realistic for practical use. Detectable changes could be
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17 observed when the concentration of E. coli was higher than 108 cells mL−1. A linear
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19 relationship was observed between the current decrease and the concentration of E. coli in the
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22 range of 105 to 108 cells mL−1 (Fig. 3B).
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24 Oxygen consumption by E. coli depends on experimental conditions. In particular, the
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27 respiratory activity depends significantly on nutrient concentrations, which suggests that the
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29 relationship between the current of the oxygen electrode and the respiratory activity of E. coli
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can be adjusted to make the observed changes larger. To check this, changes in current were
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34 measured after changing the glucose concentration. In the range of concentrations tested, the
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36 magnitude of the decrease in current increased with increasing concentrations of glucose, with
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39 the maximum activity obtained at 50 mM glucose. Based on this result, we decided to use 50
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41 mM glucose for subsequent experiments.
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46 3.3 Measurement of the activity of E. coli using a bacterial counter
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49 After co-incubating neutrophil-like cells and E. coli cells for 1 h, the number of E. coli cells
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51 was counted manually using the bacterial counter. The number of E. coli cells did not
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decrease during co-incubation without IFN-γ stimulation (#1 in Fig. 4). In contrast, the
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56 number of E. coli cells decreased significantly in the co-incubation of E. coli and
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IFN-γ-activated neutrophil-like cells (#0 in Fig. 4). Based on these results, IFN-γ, the
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2 strongest inflammatory cytokine, elevated the bactericidal activity of HL-60 cells.[26, 27]
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7 3.4 Measurement of bactericidal activity in the device
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10 Fig. 5 shows the currents recorded at 180 s with various combinations of E. coli,
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12 neutrophil-like cells, and IFN-γ. We first examined the respiratory activity of neutrophil-like
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cells. Current decreases over time were observed with both activated (IFN-γ-stimulated) and
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17 non-activated neutrophil-like cells, though the current recorded with neutrophil-like cells
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19 activated with IFN-γ (#2 in Fig. 5) was markedly lower than that of non-activated cells (#1 in
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22 Fig. 5). The current recorded with only E. coli was comparable with that of activated
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24 neutrophil-like cells (#3 in Fig. 5). Interestingly, co-incubation with IFN-γ-activated
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27 neutrophil-like cells decreased E. coli oxygen consumption levels, although co-incubation
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29 with non-activated cells did not result in a decrease in oxygen consumption. Activated
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neutrophil-like cells may therefore potently eliminate E. coli, resulting in a decrease in total
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34 oxygen consumption. These observations were consistent with those using the bacterial
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36 counter (Fig. 4). The results demonstrate that our device can effectively evaluate bactericidal
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39 activity by neutrophil-like cells.
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44 3.5 In situ co-incubation and measurement of oxygen consumption
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46 In the experiment mentioned in section 3.4, suspensions of neutrophil-like cells and E. coli
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49 cells were mixed and co-incubated in a centrifuge tube. Thereafter, the mixture was
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51 introduced into flow channel A, and the changes in current were measured using the oxygen
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electrode. However, in a subsequent experiment, the cells were co-cultured with or without
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56 IFN-in the device itself at 37°C. Fig. 6 shows typical time courses of the current for this
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59 experiment. The current increased in the presence of IFN-γas time elapsed, suggesting that
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the number of living E. coli cells decreased as a result of the elevated activity of the
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2 neutrophil-like cells. In contrast, the current decreased in the absence of IFN-γ, suggesting
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5 that the activity of the non-activated neutrophil-like cells was low and that the number of E.
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12 4. Conclusions
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The bactericidal activity of neutrophil-like cells can be measured by co-incubation with E.
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17 coli and assessment of the respiratory activity of E. coli cells in terms of the consumption of
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19 dissolved oxygen. For this purpose, our proposed microfluidic device coupled with a
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22 Clark-type oxygen electrode is useful. With a small volume of sample solution containing
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24 neutrophil-like cells, distinct differences can be obtained in the current of the Clark-type
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27 oxygen electrode. This technique was used to demonstrate that IFN-γ is effective at activating
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29 neutrophil-like cells and enhancing changes in the current. While we used neutrophil-like
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cells in this study, the activity of real neutrophils could also be measured in the same manner
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34 for the assessment of psychological stress.
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39 Acknowledgements
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41 We thank Prof. J. Fukuda of Yokohama National University, Japan, for providing the
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44 HL-60 cell line. This study was supported by the Tsukuba University Nanofabrication
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46 Platform of the Nanotechnology Platform Project sponsored by the Ministry of Education,
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49 Culture, Sports, Science and Technology (MEXT), Japan.
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Figure captions
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5 Fig. 1. Construction of the device. (A) Exploded view of the device. (B) Top view of the
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7 device showing the relationships among electrodes, compartments, and flow channels.
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12 Fig. 2. Response curve of the oxygen electrode when the medium in the sensing region was
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14 changed from air to a solution saturated with Na2SO3 and back to air.
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19 Fig. 3. E. coli quantification by the micro-oxygen sensor. (A) Response curves of the oxygen
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22 electrode obtained with difference densities of E. coli. (B) Correlation between the current
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24 decrease and the density of E. coli (n = 5).
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29 Fig. 4. Bacterial counts after incubation with E. coli and HL-60 cells. E. coli, which were
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31 incubated with or without HL-60 cells for 1 h at room temperature, were used to assess
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34 residual bacterial viability. The number of E. coli was determined by manual counting under
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36 an optical microscope. Data are means ± SD (n = 5). * p < 0.01 vs. #1 and 2.
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41 Fig. 5. Currents recorded after 180 s with mixtures of E. coli and HL-60 cells. E. coli cultures,
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43 which were incubated with or without HL-60 cells and IFN-for 1 h at room temperature,
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46 were assessed. Data are means ± SD (n = 7 in each group). * p < 0.01 vs. #2–5 and † p < 0.05
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48 vs. #4.
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52 Fig. 6. Changes in bacterial counts in on-chip bacterial killing assay. A culture of E. coli was
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54 diluted in PBS containing HL-60 cells either with (red) or without (black) IFN- and
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57 incubated in the device at 37°C. Currents were measured with the on-chip oxygen sensor at
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the indicated times. The variation in the observed current in a series of experiments was
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Figure
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