Research Papers

Electrokinetic Behavior of Heat-Treated Mycobacterium Bacillus Calmette-Guérin Cells

[+] Author and Article Information
Hyun-Boo Lee

Department of Mechanical Engineering,
University of Washington,
P.O. Box 352600,
Seattle, WA 98195
e-mail: hyunboo1@gmail.com

Shinnosuke Inoue

Department of Mechanical Engineering,
University of Washington,
P.O. Box 352600,
Seattle, WA 98195
e-mail: nexis210@gmail.com

Jong-Hoon Kim

School of Engineering and Computer Science,
Washington State University,
Vancouver, WA 98686
e-mail: jh.kim@wsu.edu

Minjoong Jeong

National Supercomputing Center,
Korea Institute of Science and Technology
Daejeon 34141, South Korea
e-mail: jeong@kisti.re.kr

Jae-Hyun Chung

Department of Mechanical Engineering,
University of Washington,
P.O. Box 352600,
Seattle, WA 98195
e-mail: jae71@uw.edu

1Corresponding author.

Manuscript received February 28, 2018; final manuscript received June 22, 2018; published online September 21, 2018. Assoc. Editor: Yaling Liu.

J. Med. Devices 12(4), 041006 (Sep 21, 2018) (9 pages) Paper No: MED-18-1043; doi: 10.1115/1.4040677 History: Received February 28, 2018; Revised June 22, 2018

Dielectrophoresis (DEP) can be an effective tool to show the physiological change of bacterial cells. The behavior of bacterial cells under an electric field is complicated due to the combined effects of electrokinetic phenomena. This paper presents the study of the electrokinetic behavior of heat-treated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cells for a cell counting method. Through numerical and experimental study, heat-treated BCG cells are compared with control BCG cells. At various frequencies with the medium conductivity of 0.07 S/m, the equilibrium positions of both control- and heat-treated cells are analyzed in the combined fields of DEP and AC electroosmosis (ACEO). As DEP changes from negative to positive in electroosmotic flow, the equilibrium position of cells is bifurcated from the upper center between two electrodes onto the edges of both electrodes. It was found that the cells floating on electrodes should not be counted as attracted cells because the floating was resulted from the combined effect of the negative DEP and ACEO. According to the analysis, an optimum frequency is proposed to differentiate control cells from heat-treated cells using a cell counting method. The presented study will offer physical insight for the cell counting to differentiate live and dead Mycobacterium bovis BCG cells treated with heat and drugs.

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Pohl, H. A. , and Hawk, I. , 1966, “ Separation of Living and Dead Cells by Dielectrophoresis,” Science, 152(3722), pp. 647–649. [CrossRef] [PubMed]
Khoshmanesh, K. , Nahavandi, S. , Baratchi, S. , Mitchell, A. , and Kalantar-zadeh, K. , 2011, “ Dielectrophoretic Platforms for Bio-Microfluidic Systems,” Biosens. Bioelectron., 26(5), pp. 1800–1814. [CrossRef] [PubMed]
Green, N. G. , Ramos, A. , Gonzalez, A. , Morgan, H. , and Castellanos, A. , 2002, “ Fluid Flow Induced by Nonuniform AC Electric Fields in Electrolytes on Microelectrodes—III: Observation of Streamlines and Numerical Simulation,” Phys. Rev. E, 66(2 Pt. 2), p. 026305. [CrossRef]
Wu, J. , Ben, Y. X. , Battigelli, D. , and Chang, H. C. , 2005, “ Long-Range AC Electroosmotic Trapping and Detection of Bioparticles,” Ind. Eng. Chem. Res., 44(8), pp. 2815–2822. [CrossRef]
Gagnon, Z. R. , and Chang, H. C. , 2009, “ Electrothermal Ac Electro-Osmosis,” Appl. Phys. Lett., 94(2), p. 024101. [CrossRef]
Ng, W. Y. , Goh, S. , Lam, Y. C. , Yang, C. , and Rodriguez, I. , 2009, “ DC-Biased AC-Electroosmotic and AC-Electrothermal Flow Mixing in Microchannels,” Lab Chip, 9(6), pp. 802–809. [CrossRef] [PubMed]
Park, S. , and Beskok, A. , 2008, “ Alternating Current Electrokinetic Motion of Colloidal Particles on Interdigitated Microelectrodes,” Anal. Chem., 80(8), pp. 2832–2841. [CrossRef] [PubMed]
Castellanos, A. , Ramos, A. , Gonzalez, A. , Green, N. G. , and Morgan, H. , 2003, “ Electrohydrodynamics and Dielectrophoresis in Microsystems: Scaling Laws,” J. Phys. D Appl. Phys., 36(20), pp. 2584–2597. [CrossRef]
Gao, J. , Sin, M. L. Y. , Liu, T. T. , Gau, V. , Liao, J. C. , and Wong, P. K. , 2011, “ Hybrid Electrokinetic Manipulation in High-Conductivity Media,” Lab Chip, 11(10), pp. 1770–1775. [CrossRef] [PubMed]
Burg, B. R. , Bianco, V. , Schneider, J. , and Poulikakos, D. , 2010, “ Electrokinetic Framework of Dielectrophoretic Deposition Devices,” J. Appl. Phys., 107(12), p. 124308. [CrossRef]
Bahukudumbi, P. , Everett, W. N. , Beskok, A. , Bevan, M. A. , Huff, G. H. , Lagoudas, D. , and Ounaies, Z. , 2007, “ Colloidal Microstructures, Transport, and Impedance Properties Within Interfacial Microelectrodes,” Appl. Phys. Lett., 90(22), p. 224102. [CrossRef]
Castellarnau, M. , Errachid, A. , Madrid, C. , Juarez, A. , and Samitier, J. , 2006, “ Dielectrophoresis as a Tool to Characterize and Differentiate Isogenic Mutants of Escherichia Coli,” Biophys. J., 91(10), pp. 3937–3945. [CrossRef] [PubMed]
Johari, J. , Hubner, Y. , Hull, J. C. , Dale, J. W. , and Hughes, M. P. , 2003, “ Dielectrophoretic Assay of Bacterial Resistance to Antibiotics,” Phys. Med. Biol., 48(14), pp. N193–N198. [CrossRef] [PubMed]
Chung, C. C. , Cheng, I. F. , Chen, H. M. , Kan, H. C. , Yang, W. H. , and Chang, H. C. , 2012, “ Screening of Antibiotic Susceptibility to Beta-Lactam-Induced Elongation of Gram-Negative Bacteria Based on Dielectrophoresis,” Anal. Chem., 84(7), pp. 3347–3354. [CrossRef] [PubMed]
Hawkins, B. G. , Huang, C. , Arasanipalai, S. , and Kirby, B. J. , 2011, “ Automated Dielectrophoretic Characterization of Mycobacterium Smegmatis,” Anal. Chem., 83(9), pp. 3507–3515. [CrossRef] [PubMed]
Sasaki, N. , Kitamori, T. , and Kim, H. B. , 2006, “ AC Electroosmotic Micromixer for Chemical Processing in a Microchannel,” Lab Chip, 6(4), pp. 550–554. [CrossRef] [PubMed]
Huang, S. H. , Wang, S. K. , Khoo, H. S. , and Tseng, F. G. , 2007, “ AC Electroosmotic Generated In-Plane Microvortices for Stationary or Continuous Fluid Mixing,” Sensor Actuat. B-Chem., 125(1), pp. 326–336. [CrossRef]
Studer, V. , Pepin, A. , Chen, Y. , and Ajdari, A. , 2004, “ An Integrated AC Electrokinetic Pump in a Microfluidic Loop for Fast and Tunable Flow Control,” Analyst, 129(10), pp. 944–949. [CrossRef] [PubMed]
Debesset, S. , Hayden, C. J. , Dalton, C. , Eijkel, J. C. T. , and Manz, A. , 2004, “ An AC Electroosmotic Micropump for Circular Chromatographic Applications,” Lab Chip, 4(4), pp. 396–400. [CrossRef] [PubMed]
Mpholo, M. , Smith, C. G. , and Brown, A. B. D. , 2003, “ Low Voltage Plug Flow Pumping Using Anisotropic Electrode Arrays,” Sensor. Actuat. B-Chem., 92(3), pp. 262–268. [CrossRef]
Park, S. , Koklu, M. , and Beskok, A. , 2009, “ Particle Trapping in High-Conductivity Media With Electrothermally Enhanced Negative Dielectrophoresis,” Anal. Chem., 81(6), pp. 2303–2310. [CrossRef] [PubMed]
Kim, J.-H. , Yeo, W.-H. , Shu, Z. , Soelberg, S. D. , Inoue, S. , Kalyanasundaram, D. , Ludwig, J. , Furlong, C. E. , Riley, J. J. , Weigel, K. M. , Cangelosi, G. A. , Oh, K. , Lee, K.-H. , Gao, D. , and Chung, J.-H. , 2012, “ Immunosensor Towards Low-Cost, Rapid Diagnosis of Tuberculosis,” Lab Chip, 12(8), pp. 1437–1440. [CrossRef] [PubMed]
Kim, J.-H. , Hiraiwa, M. , Lee, H.-B. , Lee, K.-H. , Cangelosi, G. A. , and Chung, J.-H. , 2013, “ Electrolyte-Free Amperometric Immunosensor Using a Dendritic Nanotip,” Rsc Adv., 3(13), pp. 4281–4287. [CrossRef] [PubMed]
Inoue, S. , Shu, Z. , Kim, J.-H. , Hiraiwa, M. , Lakley, A. , Weigel, K. , Soelberg, S. , Furlong, C. , Cangelosi, G. A. , Carins, A. , Lee, H. B. , Oh, K. , Lee, K.-H. , Gao, D. , and Chung, J.-H. , 2014, “ Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum,” Plos One, 9(1), p. e86018. [CrossRef] [PubMed]
Kim, J.-H. , Inoue, S. , Cangelosi, G. A. , Lee, K.-H. , and Chung, J.-H. , 2014, “ Study of Specific Capture of Target Bacteria Onto Sensor Surface for Disease Diagnosis,” J. Micromech. Microeng., 24(4), p. 045009. [CrossRef]
Shin, I. , Lee, H.-B. , Becker, A. L. , Weigel, K. M. , Kim, J.-H. , Lee, K.-H. , Cangelosi, G. A. , and Chung, J.-H. , 2015, “ Dielectrophoretic Characterization of Antibiotic-Treated Mycobacterium Tuberculosis Complex Cells,” Anal. Bioanal. Chem., 407(25), pp. 7673–7680. [CrossRef] [PubMed]
Cangelosi, G. A. , and Meschke, J. S. , 2014, “ Dead or Alive: Molecular Assessment of Microbial Viability,” Appl. Environ. Microb., 80(19), pp. 5884–5891. [CrossRef]
Winder, F. G. , and Collins, P. B. , 1970, “ Inhibition by Isoniazid of Synthesis of Mycolic Acids in Mycobacterium tuberculosis,” J. Gen. Microbiol., 63(1), pp. 41–48. [CrossRef] [PubMed]
Takayama, K. , Wang, L. , and David, H. L. , 1972, “ Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis,” Antimicrob Agents Ch., 2(1), pp. 29–35. [CrossRef]
Yeo, W. H. , Lee, H. B. , Kim, J. H. , Lee, K. H. , and Chung, J. H. , 2013, “ Nanotip Analysis for Dielectrophoretic Concentration of Nanosized Viral Particles,” Nanotechnol., 24(18), p. 185502.
Probstein, R. F. , 1994, Physicochemical Hydrodynamics: An Introduction, Wiley, New York.
Jones, T. B. , 2005, Electromechanics of Particles, Cambridge University Press, Cambridge, New York.
Asami, K. , Hanai, T. , and Koizumi, N. , 1980, “ Dielectric Analysis of Escherichia-Coli Suspensions in the Light of the Theory of Interfacial Polarization,” Biophys. J., 31(2), pp. 215–228. [CrossRef] [PubMed]
Vilcheze, C. , Morbidoni, H. R. , Weisbrod, T. R. , Iwamoto, H. , Kuo, M. , Sacchettini, J. C. , and Jacobs, W. R. , 2000, “ Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis,” J. Bacteriol., 182(14), pp. 4059–4067. [CrossRef] [PubMed]
Shu, Z. , Weigel, K. M. , Soelberg, S. D. , Lakey, A. , Cangelosi, G. A. , Lee, K. H. , Chung, J. H. , and Gao, D. , 2012, “ Cryopreservation of Mycobacterium Tuberculosis Complex Cells,” J. Clin. Microbiol., 50(11), pp. 3575–3580. [CrossRef] [PubMed]
Lee, H. B. , Jeong, M. , and Chung, J. H. , 2017, “ Dielectrophoretic Sensitivity Analysis of Cell Characterization,” Int. J. Precis. Eng. Man., 18(5), pp. 747–754. [CrossRef]
Marr, A. G. , and Ingraham, J. L. , 1962, “ Effect of Temperature on the Composition of Fatty Acids in Escherichia Coli,” J. Bacteriol., 84(6), pp. 1260–1267. http://jb.asm.org/content/84/6/1260.short [PubMed]


Grahic Jump Location
Fig. 1

(a) Analysis domain with boundary conditions. The analysis domain for electric and fluid fields is indicated with black solid lines. The analysis domain for particle motion is the box with red-dashed lines. The electrodes are colored by orange pads. V: applied voltage (V), u: flow velocity (m/s), E: V/m (b) Double-shell ellipsoidal model of a cell with a membrane (mem) and wall. The parameters are the thickness of d (m), the conductivity of σ (S/m), and the permittivity of ε (F/m). (c) Analysis procedure to study the electrokinetic behavior of cells and the parameters. KCM: Clausius Mossotti factor.

Grahic Jump Location
Fig. 2

(a) Fabricated planar electrode with a gap size of 10 μm. (b) Illustration and a photo of the experimental setup. A microchip is placed under an objective lens of a fluorescent microscope. Electrical probes are used to apply an AC potential. The behavior of stained cells is observed by using the microscope.

Grahic Jump Location
Fig. 3

(a) Fluorescent images of the control- and the heat-killed BCG cells attracted to the planar electrodes. The images are captured at 1 min after applying frequencies of 100 kHz, 1 MHz, 5 MHz, and 10 MHz, respectively. (b) Measurement of cell speed near the electrode edges.

Grahic Jump Location
Fig. 4

Time-dependent simulation of cell movement by n-DEP and AC electroosmosis. To mimic the negative DEP case, KCM is set to −0.2 and SACEO is adjusted to make the maximum flow speed to 200 μm/s. (a) Initial distribution of cells (t = 0). ((b)(c)) The change of cell distribution with time (t = 10, 25, and 50 s); the top view (b) and the front view (c).

Grahic Jump Location
Fig. 5

(a) Contour plot of a DEP force field in x–z plane. The cell distributions at vmax = 100 μm/s and KCM = 0.5 are overlapped to the DEP force field. (b) Illustration of flow pattern by ACEO. (c) Contour plot of an ACEO flow field in x–z plane. The cell distributions at vmax = 200 μm/s and KCM = −0.2 are overlapped to the ACEO flow field. ((d)–(f)) Cell distribution computed from the time-dependent simulation of electrokinetic motion of cells. For the three cases, (umax, KCM) are (200, −0.5), (200, −0.2), and (100, 0.5), respectively. ((d')–(f')) Trajectories and equilibrium positions of cells for the cases of Figs. 5(d)5(f), respectively. The equilibrium positions are indicated by the dashed circle. (g) DEP force for the control- and the heat-killed BCG cells and the ACEO-induced drag force according to the frequencies between 100 kHz and 10 MHz. (h) Estimated KCM spectra for control- and heat-killed BCG cells.

Grahic Jump Location
Fig. 6

Sequential images for control- and heat-treated cells at 5 MHz: (a) Behavior of control cells at 5 MHz. (b) Behavior of heat-treated cells (80 °C) at 5 MHz. Since the focal plane of the microscope is adjusted on the electrode surface, a cell moving out of the focal plane is not clearly visible in the images. To show the cell location clearly, control- and heat-treated cells are expressed with circles, respectively.

Grahic Jump Location
Fig. 7

(a) Cell counting result of BCG cells at 5 MHz after heat treatment at 25, 60, and 80 °C for 10 min. The right image shows representative fluorescent images showing the attracted cells at 25, 60, and 80 °C ((b)–(d)) SEM images for the 25 °C-treated (control) (b), 60 °C-treated (c), and 80 °C-treated (d) BCG cells.



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