Login | Register

Electrical Lab-on-a-Chip for Ex Vivo Study on the Influence of Electric Fields on Pollen Cells


Electrical Lab-on-a-Chip for Ex Vivo Study on the Influence of Electric Fields on Pollen Cells

Agudelo, Carlos G. (2015) Electrical Lab-on-a-Chip for Ex Vivo Study on the Influence of Electric Fields on Pollen Cells. PhD thesis, Concordia University.

[thumbnail of Agudelo_PhD_F2015.pdf]
Text (application/pdf)
Agudelo_PhD_F2015.pdf - Accepted Version


Pollen tubes are polarly growing plant cells that are able to respond to a combination of chemical, mechanical, and electrical cues during their journey through the flower pistil in order to accomplish fertilization. How signals are perceived and processed in the pollen tube is still poorly understood and evidence for electrical guidance, in particular, is vague and highly contradictory. To generate reproducible experimental conditions for ex vivo pollen cell cultures, here we present a low-cost, reusable Electrical Lab-on-a-Chip (ELoC) for investigating the influence of electric fields on growing cells. Viability of pollen growth using a structured microfluidic network is first investigated and validated. Then the integration of microelectrodes into the device is addressed in detail. Characterization of the pollen growth medium conductivity and simulation of the ELoC electrical configuration were carried out to define the experimental conditions. Reusability of the microdevice is achieved by structuring the design into two separate rebondable modules: a microfluidic module and a microelectrode module. Two experimental approaches were realized: a batch design for exposing simultaneously a large number of cells to a global electric field, and a single-cell design in which a localized electric field is applied to individual cells. Extensive batch results indicate that DC fields were inhibitory above 6 V/cm. However, switching to AC fields re-established pollen tube growth at frequencies above 100 mHz, suggesting a significant role of the medium conductivity in controlling the cellular response. Unlike macroscopic open-assay experimental setups, single-cell tests further indicate no reorientation of pollen tube growth, suggesting that previously reported tropic behavior was caused by ion movement in the substrate rather than by a direct effect of the electric field on the cell.

Divisions:Concordia University > Gina Cody School of Engineering and Computer Science > Mechanical and Industrial Engineering
Item Type:Thesis (PhD)
Authors:Agudelo, Carlos G.
Institution:Concordia University
Degree Name:Ph. D.
Program:Mechanical Engineering
Date:19 April 2015
Thesis Supervisor(s):Packirisamy, Muthukumaran
Keywords:pollen, mems, microfluidics, cell growth, electric field
ID Code:980378
Deposited On:16 Jun 2016 15:59
Last Modified:18 Jan 2018 17:51


Agudelo, C.G., Sanati Nezhad, A., Ghanbari, M., Packirisamy, M., and Geitmann, A. (2012). A microfluidic platform for the investigation of elongation growth in pollen tubes. J Micromechanics Microengineering. 22, doi:10.1088/0960-1317/22/11/115009.
Agudelo, C.G., Packirisamy, M., and Geitmann, A. (2013a). Lab-on-a-Chip for studying growing pollen tubes. In: Plant Cell Morphogenesis: Methods and Protocols, Series "Methods in Molecular Biology", eds. Žárský V, Cvrčková F, Springer, pp 237-248.
Agudelo, C.G., Sanati Nezhad, A., Ghanbari, M., Naghavi, M., Packirisamy, M., and Geitmann, A. (2013b). TipChip: a modular, MEMS-based platform for experimentation and phenotyping of tip-growing cells. Plant J Cell Mol Biol. 73, 1057–1068.
Benkert, R., Obermeyer, G., and Bentrup, FW. (1997). The turgor pressure of growing lily pollen tubes. Protoplasma. 198, 1–8.
Blasiak, J., Mulcahy, D.L., and Musgrave, M. (2001). Oxytropism: a new twist in pollen tube orientation. Planta. 213, 318–322.
Boedicker, J.Q., Vincent, M.E., and Ismagilov, R.F. (2009). Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability. Angew Chem Int Ed. 48, 5908–5911.
Bove, J., Vaillancourt, B., Kroeger, J., Hepler, P.K., Wiseman, P.W., and Geitmann, A. (2008). Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching. Plant Physiol. 147, 1646–1658.
Brewbaker, J.L., and Kwack, B.H. (1963). The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot. 50, 859–865.
Brower, D.L., and Giddings, T.H. (1980). The effects of applied electric fields on Micrasterias. II. The distributions of cytoplasmic and plasma membrane components. J Cell Sci 42, 279–290.
Burgess, J., and Linstead, P.J. (1982). Cell-wall differentiation during growth of electrically polarised protoplasts of Physcomitrella. Planta. 156, 241–248.
Campetelli, A., Bonazzi, D., and Minc, N. (2012). Electrochemical regulation of cell polarity and the cytoskeleton. Cytoskeleton. 69, 601–612.
Chandra, A., and Bagchi, B. (2000). Frequency dependence of ionic conductivity of electrolyte solutions. J Chem Phys. 112, 1876–1886.
Chebli, Y., and Geitmann, A. (2007). Mechanical principles governing pollen tube growth. Funct Plant Sci Biotechnol. 1, 232–245.
Chen, T.-H., and Jaffe, L.F. (1979). Forced calcium entry and polarized growth of Funaria spores. Planta. 144, 401–406.
Cheung, A.Y., Wang, H., and Wu, H.M. (1995). A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth. Cell. 82, 383–393.
Cooper, J.R., Qin, Y., Jiang, L., Palanivelu, R., and Zohar, Y. (2009). Microsystem-based study of pollen-tube attractants secreted by ovules. In: IEEE 22nd International Conference on MEMS 2009, pp. 208–211.
Crombie, C., Gow, N.A., and Gooday, G.W. (1990). Influence of applied electrical fields on yeast and hyphal growth of Candida albicans. J Gen Microbiol. 136, 311–317.
Bou Daher, F., and Geitmann, A. (2011). Actin is involved in pollen tube tropism through redefining the spatial targeting of secretory vesicles. Traffic. 12, 1537–1551.
Fayant, P., Girlanda, O., Chebli, Y., Aubin, C.-É., Villemure, I., and Geitmann, A. (2010). Finite element model of polar growth in pollen tubes. Plant Cell. 22, 2579–2593.
Feijó, J.A., Malhó, R., and Obermeyer, G. (1995). Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma. 187, 155–167.
Feijó, J.A., Sainhas, J., Hackett, G.R., Kunkel, J.G., and Hepler, P.K. (1999). Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J Cell Biol. 144, 483–496.
Geitmann, A., and Palanivelu, R. (2007). Fertilization requires communication: signal generation and perception during pollen tube guidance. Floric Ornam Biotechnol. 1, 77–89.
Gossot, O., and Geitmann, A. (2007). Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton. Planta. 226, 405–416.
Gray, J., Chaloner, W.G., and Westoll, T.S. (1985). The microfossil record of early land plants: advances in understanding of early terrestrialization. Philos Trans R Soc Lond B Biol Sci. 309, 167–195.
Holdaway-Clarke, T.L., and Hepler, P.K. (2003). Control of pollen tube growth: role of ion gradients and fluxes. New Phytol. 159, 539–563.
Ishikawa, H., and Evans, M.L. (1990). Electrotropism of maize roots role of the root cap and relationship to gravitropism. Plant Physiol. 94, 913–918.
Jaffe, L.F., and Nuccitelli, R. (1977). Electrical controls of development. Annu Rev Biophys Bioeng. 6, 445–476.
Jones, T. (1995). Electromechanics of particles. Cambridge University Press. 288p.
Kristen, U., and Kappler, R. (1995). The pollen tube growth test. In: In Vitro Toxicity Testing Protocols, Series "Methods in Molecular Biology”, Springer Protocols, 43, pp. 189–198.
Kühtreiber, W.M., and Jaffe, L.F. (1990). Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol. 110, 1565–1573.
Langdon, P.G., Barber, K.E., and Morriss, S.H.L.-C. (2004). Reconstructing climate and environmental change in northern England through chironomid and pollen analyses: evidence from Talkin Tarn, Cumbria. J Paleolimnol. 32, 197–213.
Li, J., and Lin, F. (2011). Microfluidic devices for studying chemotaxis and electrotaxis. Trends Cell Biol. 21, 489–497.
Lush, W.M. (1999). Whither chemotropism and pollen tube guidance?. Trends Plant Sci. 4, 413–418.
Malhó, R. (1998). Pollen tube guidance–the long and winding road. Sex Plant Reprod. 11, 242–244.
Malhó, R. (2006). The Pollen Tube: a cellular and molecular perspective. Springer-Verlag Berlin Heidelberg. 295p.
Malhó, R., Feijó, J.A., and Pais, M.S.S. (1992). Effect of electrical fields and external ionic currents on pollen-tube orientation. Sex Plant Reprod. 5, 57–63.
Marsh, G., and Beams, H.W. (1945). The orientation of pollen tubes of Vinca in the electric current. J Cell Comp Physiol. 25, 195–204.
Mascarenhas, J.P., and Machlis, L. (1964). Chemotropic response of the pollen of Antirrhinum majus to Calcium. Plant Physiol. 39, 70–77.
McCaig, C.D., Sangster, L., and Stewart, R. (2000). Neurotrophins enhance electric field-directed growth cone guidance and directed nerve branching. Dev Dyn. 217, 299–308.
McCaig, C.D., Rajnicek, A.M., Song, B., and Zhao, M. (2005). Controlling cell behavior electrically: current views and future potential. Physiol Rev. 85, 943–978.
McGillivray, A.M., and Gow, N.A.R. (1986). Applied electrical fields polarize the growth of mycelial fungi. J Gen Microbiol. 132, 2515–2525.
Melling, A. (1997). Tracer particles and seeding for particle image velocimetry. Meas Sci Technol. 8, 1406–1416.
Messerli, M.A., Danuser, G., and Robinson, K.R. (1999). Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes. J Cell Sci. 112, 1497–1509.
Messerli, M.A., Créton, R., Jaffe, L.F., and Robinson, K.R. (2000). Periodic increases in elongation rate precede increases in cytosolic Ca2+ during pollen tube growth. Dev Biol. 222, 84–98.
Michard, E., Alves, F., and Feijó, J.A. (2009). The role of ion fluxes in polarized cell growth and morphogenesis: the pollen tube as an experimental paradigm. Int J Dev Biol. 53, 1609–1622.
Morgan, H., and Green, N. (2003). AC electrokinetics: colloids and nanoparticles. Research Studies Press. 360p.
Nahmias, Y. (2009). Methods in bioengineering: microdevices in biology and medicine. Artech House.
Nakamura, N., Fukushima, A., Iwayama, H., and Suzuki, H. (1991). Electrotropism of pollen tubes of camellia and other plants. Sex Plant Reprod. 4. 138–143.
Nozue, K., and Wada, M. (1993). Electrotropism of Nicotiana pollen tubes. Plant Cell Physiol. 34, 1291–1296.
Okuda, S., and Higashiyama, T. (2010). Pollen tube guidance by attractant molecules: LUREs. Cell Struct Funct. 35, 45–52.
Palanivelu, R., and Preuss, D. (2000). Pollen tube targeting and axon guidance: parallels in tip growth mechanisms. Trends Cell Biol. 10, 517–524.
Parre, E., and Geitmann, A. (2005). Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense. Planta. 220, 582–592.
Peng, H.B., and Jaffe, L.F. (1976). Polarization of fucoid eggs by steady electrical fields. Dev Biol. 53, 277–284.
Pierson, E.S., Miller, D.D., Callaham, D.A., Shipley, A.M., Rivers, B.A., Cresti, M., and Hepler, P.K. (1994). Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell. 6, 1815–1828.
Platzer, K., Obermeyer, G., and Bentrup, F.-W. (1997). AC fields of low frequency and amplitude stimulate pollen tube growth possibly via stimulation of the plasma membrane proton pump. Bioelectrochem Bioenerg. 44, 95–102.
Prado, A.M., Porterfield, D.M., and Feijó, J.A. (2004). Nitric oxide is involved in growth regulation and re-orientation of pollen tubes. Development. 131, 2707–2714.
Pu, J., and Zhao, M. (2005). Golgi polarization in a strong electric field. J Cell Sci. 118, 1117–1128.
Rajnicek, A.M., McCaig, C.D., and Gow, N.A. (1994). Electric fields induce curved growth of Enterobacter cloacae, Escherichia coli, and Bacillus subtilis cells: implications for mechanisms of galvanotropism and bacterial growth. J Bacteriol. 176, 702–713.
Robinson, D.A. (1968). The electrical properties of metal microelectrodes. Proc. IEEE. 56, 1065–1071.
Robinson, K.R. (1985). The responses of cells to electrical fields: a review. J Cell Biol. 101, 2023–2027.
Robinson, K.R., and Messerli, M.A. (2003). Left/right, up/down: the role of endogenous electrical fields as directional signals in development, repair and invasion. BioEssays. 25, 759–766.
Sanati Nezhad, A., and Geitmann, A. (2013). The cellular mechanics of an invasive lifestyle. J Exp Bot. 64, 4709–4728.
Sanati Nezhad, A., Naghavi, M., Packirisamy, M., Bhat, R., and Geitmann, A. (2013a). Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling. Proc Natl Acad Sci. 110, 8093–8098.
Sanati Nezhad, A., Naghavi, M., Packirisamy, M., Bhat, R., and Geitmann, A. (2013b). Quantification of the Young’s modulus of the primary plant cell wall using Bending-Lab-On-Chip (BLOC). Lab Chip. 13, 2599–2608.
Sanati Nezhad, A., Ghanbari, M., Agudelo, C.G., Packirisamy, M., Bhat, R.B., and Geitmann, A. (2013c). PDMS microcantilever-based flow sensor integration for Lab-on-a-Chip. IEEE Sens J. 13, 601–609.
Sanati Nezhad, A., Ghanbari, M., Agudelo, C.G., Naghavi, M., Packirisamy, M., Bhat, R.B., and Geitmann, A. (2013d). Optimization of flow assisted entrapment of pollen grains in a microfluidic platform for tip growth analysis. Biomed Microdevices. 16, 23–33.
Sanati Nezhad, A., Packirisamy, M., Bhat, R., and Geitmann, A. (2013e). In vitro study of oscillatory growth dynamics of Camellia pollen tubes in microfluidic environment. IEEE Trans Biomed Eng. 60, 3185–3193.
Sawidis, T., and Reiss, H.-. D. (1995). Effects of heavy metals on pollen tube growth and ultrastructure. Protoplasma. 185, 113–122.
Spanjers, A.W. (1981). Bioelectric potential changes in the style of Lilium longiflorum Thumb. after self- and cross-pollination of the stigma. Planta. 153, 1–5.
Sperber, D. (1984). Das Wachstum pflanzlicher Zellen und Organe im magnetischen und elektrischen Feld. Konstanzer Dissertationen. Hartung-Gorre.
Sperber, D., Dransfeld, K., Maret, G., and Weisenseel, M.H. (1981). Oriented growth of pollen tubes in strong magnetic fields. Naturwissenschaften. 68, 40–41.
Stahlberg, R. (2006). Historical overview on plant neurobiology. Plant Signaling and Behavior. 1, 6–8.
Stenz, H.-G., and Weisenseel, M.H. (1993). Electrotropism of maize (Zea mays L.) roots. Plant Physiol. 101, 1107–1111.
Vanapalli, S.A., Duits, M.H.G., and Mugele, F. (2009). Microfluidics as a functional tool for cell mechanics. Biomicrofluidics. 3. doi:10.1063/1.3067820.
Velve-Casquillas, G., Le Berre, M., Piel, M., and Tran, P.T. (2010). Microfluidic tools for cell biological research. Nano Today. 5, 28–47.
Wang, P., and Liu, Q. (2010). Cell-based biosensors: principles and applications. Artech House. 271p.
Wang, C., Rathore, K.S., and Robinson, K.R. (1989). The responses of pollen to applied electrical fields. Dev Biol. 136, 405–410.
Weisenseel, M.H., and Jaffe, L.F. (1976). The major growth current through Lily pollen tubes enters as K+ and leaves as H+. Planta. 133, 1–7.
Weisenseel, M.H., Nuccitelli, R., and Jaffe, L.F. (1975). Large electrical currents traverse growing pollen tubes. J Cell Biol. 66, 556–567.
Weisenseel, M.H., Dorn, A., and Jaffe, L.F. (1979). Natural H+ currents traverse growing roots and root hairs of barley (Hordeum vulgare L.). Plant Physiol. 64, 512–518.
White, R.G., Hyde, G.J., and Overall, R.L. (1990). Microtubule arrays in regenerating Mougeotia protoplasts may be oriented by electric fields. Protoplasma. 158, 73–85.
Whitesides, G.M. (2011). What Comes Next?. Lab Chip. 11, 191–193.
Witters, D., Sun, B., Begolo, S., Rodriguez-Manzano, J., Robles, W., and Ismagilov, R.F. (2014). Digital biology and chemistry. Lab Chip. 14, 3225–3232.
Wulff, H.D. (1935). Galvanotropismus bei Pollenschläuchen. Planta. 24, 602–608.
Yetisen, A.K., Jiang, L., Cooper, J.R., Qin, Y., Palanivelu, R., and Zohar, Y. (2011). A microsystem-based assay for studying pollen tube guidance in plant reproduction. J Micromechanics Microengineering. 21. doi:10.1088/0960-1317/21/5/054018
Zeijlemaker, F.C.J. (1956). Growth of pollen tubes in vitro and their reaction on potential differences. Acta Bot Neerlandica. 5, 179–186.
Ziaie, B., Baldi, A., Lei, M., Gu, Y., and Siegel, R.A. (2004). Hard and soft micromachining for BioMEMS: review of techniques and examples of applications in microfluidics and drug delivery. Adv Drug Deliv Rev. 56, 145–172.
Zonia, L., Cordeiro, S., Tupy, J., and Feijó, J.A. (2002). Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-Tetrakisphosphate. Plant Cell. 14, 2233–2249.
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Research related to the current document (at the CORE website)
- Research related to the current document (at the CORE website)
Back to top Back to top