Non-invasive Flux Measurements Using Microsensors: Theory, Limitations, and Systems

  • Ian NewmanEmail author
  • Shao-Liang Chen
  • D. Marshall Porterfield
  • Jian Sun
Part of the Methods in Molecular Biology book series (MIMB, volume 913)


Knowledge of the fluxes of ions and neutral molecules across the outer membrane or boundary of living tissues and cells is an important strand of applied molecular biology. Such fluxes can be measured non-invasively with good resolution in time and space. Two systems (MIFE™ and SIET) have been developed and have become widely used to implement this technique, and they are commercially available. This Chapter is the first comparative description of these two systems. It gives the context, the basic underlying theory, practical limitations inherent in the technique, theoretical developments, guidance on the practicalities of the technique, and the functionality of the two systems. Although the technique is strongly relevant to plant salt tolerance and other plant stresses (drought, temperature, pollutants, waterlogging), it also has rich relevance throughout biomedical studies and the molecular genetics of transport proteins.

Key words

ISE MIFE SIET Salinity Ion flux Electrophysiology Membrane transport Ion selective microelectrodes Microsensors Self-referencing 



We thank Mr. Wenjun Wang for providing equipment information of SIET and NMT:SIET system. We thank Mr. Yue (Jeff) Xu and Professor Sergey Shabala for information, advice and critical reading of the manuscript.


  1. 1.
    Lucas WJ, Kochian LV (1986) Ion transport processes in corn roots: an approach utilizing microelectrode techniques. In: Gensler WG (ed) Advanced agricultural instrumentation: design and use 402-425. Martinus Nijhoff, DordrechtGoogle Scholar
  2. 2.
    Newman IA, Kochian LV, Grusak MA et al (1987) Fluxes of H+ and K+ in corn roots—characterization and stoichiometries using ion selective microelectrodes. Plant Physiol 84:1177–1184PubMedCrossRefGoogle Scholar
  3. 3.
    Kochian LV, Shaff JE, Lucas WJ (1989) High affinity K+ uptake in maize roots. A lack of coupling with H+ efflux. Plant Physiol 91:1202–1211PubMedCrossRefGoogle Scholar
  4. 4.
    Jaffe LF, Levy S (1987) Calcium gradients measured with a vibrating calcium-selective electrode. Proc IEEE/EMBS Conf 9:779–781Google Scholar
  5. 5.
    Shabala SN (2006) Non-invasive microelectrode ion flux measurements in plant stress physiology. In: Volkov AG (ed) Plant electrophysiology—theory and methods. Springer, BerlinGoogle Scholar
  6. 6.
    Chen Z, Pottosin II, Cuin TA et al (2007) Root plasma membrane transporters controlling K+/Na+ homeostasis in salt-stressed barley. Plant Physiol 145:1714–1725PubMedCrossRefGoogle Scholar
  7. 7.
    Shirihai D, Smith P, Hammar K et al (1998) Microglia generate external proton and potassium gradients utilizing a member of the H/K ATPase family. Glia 23:339–348PubMedCrossRefGoogle Scholar
  8. 8.
    Tyerman SD, Beilby M, Whittington J et al (2001) Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp. Aust J Plant Physiol 28:591–604Google Scholar
  9. 9.
    Ryan PR, Newman IA, Shields B (1990) Ion fluxes in corn roots measured by microelectrodes with ion-specific liquid membranes. J Membr Sci 53:59–69CrossRefGoogle Scholar
  10. 10.
    Smith PJS, Hammar K, Porterfield DM et al (1999) Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux. Microsc Res Tech 46:398–417PubMedCrossRefGoogle Scholar
  11. 11.
    Newman IA (2001) Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function. Plant Cell Environ 24:1–14PubMedCrossRefGoogle Scholar
  12. 12.
    Kunkel JG, Cordeiro S, Xu Y et al (2006) Use of non-invasive ion-selective microelectrode techniques for the study of plant development. In: Volkov AG (ed) Plant electrophysiology—theory and methods. Springer, BerlinGoogle Scholar
  13. 13.
    Messerli MA, Robinson KR, Smith PJS (2006) Electrochemical sensor applications to the study of molecular physiology and analyte flux in plants. In: Volkov AG (ed) Plant electrophysiology—theory and methods. Springer, BerlinGoogle Scholar
  14. 14.
    Porterfield DM (2007) Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: recent developments in self-referencing amperometry for physiological sensing. Biosens Bioelectron 22:1186–1196PubMedCrossRefGoogle Scholar
  15. 15.
    McLamore ES, Porterfield DM (2011) Non-invasive tools for measuring metabolism and biophysical analyte transport: self-referencing physiological sensing. Chem Soc Rev 40(11):5308–5320PubMedCrossRefGoogle Scholar
  16. 16.
    Henriksen GH, Bloom AJ, Spanswick RM (1990) Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion selective microelectrodes. Plant Physiol 93:271–280PubMedCrossRefGoogle Scholar
  17. 17.
    Henriksen GH, Raman DR, Walker LP et al (1992) Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique. Plant Physiol 99:734–747PubMedCrossRefGoogle Scholar
  18. 18.
    Sun J, Chen S, Dai S et al (2009) Ion flux profiles and plant ion homeostasis control under salt stress. Plant Signal Behav 4:261–264PubMedCrossRefGoogle Scholar
  19. 19.
    Jaffe LF, Nuccitelli R (1974) An ultrasensitive vibrating probe for measuring steady state extracellular currents. J Cell Biol 63:614–628PubMedCrossRefGoogle Scholar
  20. 20.
    Xu Y, Sun T, Yin L (2006) Application of non-invasive microsensing system to simultaneously measure both H+ and O2 fluxes around the pollen tube. J Integr Plant Biol 48:823–831CrossRefGoogle Scholar
  21. 21.
    Porterfield DM, Trimarchi JR, Keefe DL et al (1998) Metabolism and calcium homeostasis during development of the mouse embryo to the blastocyst stage in M2 culture medium. Biol Bull 195:208–209PubMedCrossRefGoogle Scholar
  22. 22.
    Land SC, Porterfield DM, Sanger RH et al (1999) The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells. J Exp Biol 202:211–218PubMedGoogle Scholar
  23. 23.
    Porterfield DM, Smith PJS (2000) Charac­terization of trans-cellular oxygen and proton fluxes from Spirogyra grevilleana using self-referencing microelectrodes. Protoplasma 212:80–88CrossRefGoogle Scholar
  24. 24.
    McLamore ES, Diggs A, Marzal PC et al (2010) Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor technique. Plant J 63:1004–1016PubMedCrossRefGoogle Scholar
  25. 25.
    Porterfield DM, Laskin JD, Jung S-K et al (2001) Proteins and lipids define the diffusional field of nitric oxide. Measurement of nitric oxide fluxes from macrophages using a self-referencing electrode. Am J Physiol 281:L904–L912Google Scholar
  26. 26.
    McLamore ES, Shi J, Jaroch D et al (2011) A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport. Biosens Bioelectron 26:2237–2245PubMedCrossRefGoogle Scholar
  27. 27.
    Shi J, McLamore ES, Jaroch D et al (2011) Oscillatory glucose flux in INS 1 pancreatic β cells: a self-referencing microbiosensor study. Anal Biochem 411:185–193PubMedCrossRefGoogle Scholar
  28. 28.
    McLamore ES, Mohanty S, Shi J et al (2010) A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux. J Neurosci Methods 189:14–22PubMedCrossRefGoogle Scholar
  29. 29.
    Pang JY, Newman I, Mendham N et al (2006) Microelectrode ion and O2 fluxes measurements reveal differential sensitivity of barley root tissues to hypoxia. Plant Cell Environ 29:1107–1121PubMedCrossRefGoogle Scholar
  30. 30.
    Chatni MR, Porterfield DM (2009) Self-referencing optrode technology for non-invasive real-time measurement of biophysical flux and physiological sensing. Analyst 134:2224–2232PubMedCrossRefGoogle Scholar
  31. 31.
    Chatni MR, Maier DE, Porterfield DM (2009) Optimization of oxygen sensitive optical dye membrane polymers for fluorescent lifetime based physiological biosensing. Sens Actuators B 141:471–477CrossRefGoogle Scholar
  32. 32.
    Chatni MR, Li G, Porterfield DM (2009) Frequency domain fluorescence lifetime optrode system design and instrumentation without a concurrent reference LED. Appl Opt 48:5528–5536PubMedCrossRefGoogle Scholar
  33. 33.
    McLamore ES, Jaroch D, Chatni R et al (2010) Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems. Planta 211:384–389Google Scholar
  34. 34.
    Jayakannan M, Babourina O, Rengel Z (2011) Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes. J Plant Physiol 168:1045–1051PubMedCrossRefGoogle Scholar
  35. 35.
    Demarest JR, Morgan JLM (1995) Effect of pH buffers on proton secretion from gastric xyntic cells measured with vibrating ion-selective microelectrodes. Biol Bull 189:219–220PubMedGoogle Scholar
  36. 36.
    Arif I, Newman IA, Keenlyside N (1995) Proton flux measurements from tissues in buffered solution. Plant Cell Environ 18:1319–1324CrossRefGoogle Scholar
  37. 37.
    Porterfield DM, McLamore ES, Banks MK (2009) Microsensor technology for measuring H+ flux in buffered media. Sens Actuators B 136:383–387CrossRefGoogle Scholar
  38. 38.
    Ryan PR, Newman IA, Arif I (1992) Rapid calcium exchange for protons and potassium in cell walls of Chara. Plant Cell Environ 15:675–683CrossRefGoogle Scholar
  39. 39.
    Arif I, Newman IA (1993) Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment. Planta 189:377–383CrossRefGoogle Scholar
  40. 40.
    Shabala S, Newman I (2000) Salinity effects on the activity of plasma membrane H+ and Ca2+ transporters in bean leaf mesophyll: masking role of the cell wall. Ann Bot 85:681–686CrossRefGoogle Scholar
  41. 41.
    Shipley AM, Feijó JA (1999) The use of the vibrating probe technique to study steady extracellular currents during pollen germination and tube growth. In: Cresti M, Cai G, Moscatelli S (eds) Fertilization in higher plants: molecular and cytological aspects. Springer, Berlin Heidelberg New York, pp 235–252CrossRefGoogle Scholar
  42. 42.
    Faszewski EE, Kunkel JG (2001) Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology. J Exp Zool 290:652–661PubMedCrossRefGoogle Scholar
  43. 43.
    Kunkel JG, Lin L-Y, Xu Y et al (2001) The strategic use of good buffers to measure proton gradients around growing pollen tubes. In: Geitmann A, Cresti M, Heath IB (eds) Cell biology of plant and fungal tip growth. Ios Press, Amsterdam, pp 81–94Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ian Newman
    • 1
    Email author
  • Shao-Liang Chen
    • 2
  • D. Marshall Porterfield
    • 3
  • Jian Sun
    • 2
  1. 1.School of Mathematics and PhysicsUniversity of TasmaniaHobartAustralia
  2. 2.College of Biological Sciences and TechnologyBeijing Forestry UniversityBeijingChina
  3. 3.Agricultural and Biological EngineeringPurdue UniversityWest LafayetteUSA

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