A microsensing system for the in vivo real-time detection of local drug kinetics

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 μm in diameter. We show that, in the guinea pig cochlea, the system can measure—simultaneously and in real time—changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.

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Fig. 1: Measurement of bumetanide using a BDD microsensor in vitro.
Fig. 2: Electrochemical properties of native perilymph in the guinea pig cochlea.
Fig. 3: In vivo detection of bumetanide and measurement of EP in the guinea pig cochlea.
Fig. 4: Quantification of the bumetanide concentration in the cochlea.
Fig. 5: Simultaneous recording of bumetanide and neuronal activity in the rat brain.
Fig. 6: Simultaneous recording of lamotrigine and neuronal activity in the rat brain.
Fig. 7: Simultaneous in vivo measurement of doxorubicin and EP in the guinea pig.


  1. 1.

    Li, Y. et al. Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 998–1002 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Patel, D. S. et al. Application of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of bumetanide in human plasma for a bioequivalence study. J. Pharm. Biomed. Anal. 66, 365–370 (2012).

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Chaurasia, C. S. et al. AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives. Pharm. Res. 24, 1014–1025 (2007).

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Bito, L., Davson, H., Levin, E., Murray, M. & Snider, N. The concentrations of free amino acids and other electrolytes in cerebrospinal fluid, in vivo dialysate of brain, and blood plasma of the dog. J. Neurochem. 13, 1057–1067 (1966).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Massoud, T. F. & Gambhir, S. S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 17, 545–580 (2003).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Clark, J. J. et al. Chronic microsensors for longitudinal, subsecond dopamine detection in behaving animals. Nat. Methods 7, 126–129 (2010).

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Wang, J. Electrochemical glucose biosensors. Chem. Rev. 108, 814–825 (2008).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Ivandini, T. A., Rao, T. N., Fujishima, A. & Einaga, Y. Electrochemical oxidation of oxalic acid at highly boron-doped diamond electrodes. Anal. Chem. 78, 3467–3471 (2006).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Fujishima, A., Einaga, Y., Rao, T. N. & Tryk, D. A. Diamond Electrochemistry (Elsevier–BKC, Tokyo, Japan, 2005).

    Google Scholar 

  10. 10.

    Härtl, A. et al. Protein-modified nanocrystalline diamond thin films for biosensor applications. Nat. Mater. 3, 736–742 (2004).

    Article  PubMed  Google Scholar 

  11. 11.

    Koppang, M. D., Witek, M., Blau, J. & Swain, G. M. Electrochemical oxidation of polyamines at diamond thin-film electrodes. Anal. Chem. 71, 1188–1195 (1999).

    CAS  Article  Google Scholar 

  12. 12.

    Yano, T., Tryk, D. A., Hashimoto, K. & Fujishima, A. Electrochemical behavior of highly conductive boron-doped diamond electrodes for oxygen reduction in alkaline solution. J. Electrochem. Soc. 145, 1870–1876 (1998).

    CAS  Article  Google Scholar 

  13. 13.

    Fierro, S. et al. In vivo assessment of cancerous tumors using boron doped diamond microelectrode. Sci. Rep. 2, 901 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Suzuki, A. et al. Fabrication, characterization, and application of boron-doped diamond microelectrodes for in vivo dopamine detection. Anal. Chem. 79, 8608–8615 (2007).

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Nin, F. et al. The endocochlear potential depends on two K+ diffusion potentials and an electrical barrier in the stria vascularis of the inner ear. Proc. Natl Acad. Sci. USA 105, 1751–1756 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Green, D. G. & Kellerth, J. O. Postsynaptic versus presynaptic inhibition in antagonistic stretch reflexes. Science 152, 1097–1099 (1966).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Giebisch, G. Measurements of electrical potential differences on single nephrons of the perfused Necturus kidney. J. Gen. Physiol. 44, 659–678 (1961).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Bakan, A., Lazo, J. S., Wipf, P., Brummond, K. M. & Bahar, I. Toward a molecular understanding of the interaction of dual specificity phosphatases with substrates: insights from structure-based modeling and high throughput screening. Curr. Med. Chem. 15, 2536–2544 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Hibino, H. & Kurachi, Y. Molecular and physiological bases of the K+ circulation in the mammalian inner ear. Physiology (Bethesda) 21, 336–345 (2006).

    CAS  Google Scholar 

  20. 20.

    Hibino, H., Nin, F., Tsuzuki, C. & Kurachi, Y. How is the highly positive endocochlear potential formed? The specific architecture of the stria vascularis and the roles of the ion-transport apparatus. Pflugers Arch. 459, 521–533 (2010).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Yoshida, T. et al. NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K+ transport that controls the electrochemical properties in the mammalian cochlea. Pflugers Arch. 467, 1577–1589 (2015).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Schmiedt, R. A., Lang, H., Okamura, H. O. & Schulte, B. A. Effects of furosemide applied chronically to the round window: a model of metabolic presbyacusis. J. Neurosci. 22, 9643–9650 (2002).

    CAS  PubMed  Google Scholar 

  23. 23.

    Crouch, J. J., Sakaguchi, N., Lytle, C. & Schulte, B. A. Immunohistochemical localization of the Na–K–Cl co-transporter (NKCC1) in the gerbil inner ear. J. Histochem. Cytochem. 45, 773–778 (1997).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Xu, J. C. et al. Molecular cloning and functional expression of the bumetanide-sensitive Na–K–Cl cotransporter. Proc. Natl Acad. Sci. USA 91, 2201–2205 (1994).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Flamenbaum, W. & Friedman, R. Pharmacology, therapeutic efficacy, and adverse effects of bumetanide, a new “loop” diuretic. Pharmacotherapy 2, 213–222 (1982).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Kahle, K. T., Barnett, S. M., Sassower, K. C. & Staley, K. J. Decreased seizure activity in a human neonate treated with bumetanide, an inhibitor of the Na+–K+–2Cl cotransporter NKCC1. J. Child Neurol. 24, 572–576 (2009).

    Article  PubMed  Google Scholar 

  27. 27.

    Halstenson, C. E. & Matzke, G. R. Bumetanide: a new loop diuretic (Bumex, Roche Laboratories). Drug Intell. Clin. Pharm. 17, 786–797 (1983).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Flamenbaum, W. Diuretic use in the elderly: potential for diuretic-induced hypokalemia. Am. J. Cardiol. 57, 38A–43A (1986).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Rybak, L. P. Ototoxicity of loop diuretics. Otolaryngol. Clin. North Am. 26, 829–844 (1993).

    CAS  PubMed  Google Scholar 

  30. 30.

    Thorne, M. et al. Cochlear fluid space dimensions for six species derived from reconstructions of three-dimensional magnetic resonance images. Laryngoscope 109, 1661–1668 (1999).

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Wangemann, P. Supporting sensory transduction: cochlear fluid homeostasis and the endocochlear potential. J. Physiol. 576, 11–21 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Salt, A. N., Melichar, I. & Thalmann, R. Mechanisms of endocochlear potential generation by stria vascularis. Laryngoscope 97, 984–991 (1987).

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Armbruster, D. A., Tillman, M. D. & Hubbs, L. M. Limit of detection (LQD)/limit of quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC–MS assays of abused drugs. Clin. Chem. 40, 1233–1238 (1994).

    CAS  PubMed  Google Scholar 

  34. 34.

    Kusakari, J., Kambayashi, J., Ise, I. & Kawamoto, K. Reduction of the endocochlear potential by the new “loop” diuretic, bumetanide. Acta Otolaryngol. 86, 336–341 (1978).

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Sewell, W. F. The effects of furosemide on the endocochlear potential and auditory-nerve fiber tuning curves in cats. Hear. Res. 14, 305–314 (1984).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Bland, J. M. & Altman, D. G. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310 (1986).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Frelin, C., Chassande, O. & Lazdunski, M. Biochemical characterization of the Na+/K+/Cl co-transport in chick cardiac cells. Biochem. Biophys. Res. Commun. 134, 326–331 (1986).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Van Mil, H. G. J., Geukes Foppen, R. J. & Siegenbeek van Heukelom, J. The influence of bumetanide on the membrane potential of mouse skeletal muscle cells in isotonic and hypertonic media. Br. J. Pharmacol. 120, 39–44 (1997).

    Article  PubMed  Google Scholar 

  39. 39.

    Ben-Ari, Y., Tyzio, R. & Nehlig, A. Excitatory action of GABA on immature neurons is not due to absence of ketone bodies metabolites or other energy substrates. Epilepsia 52, 1544–1558 (2011).

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Sipilä, S. T., Schuchmann, S., Voipio, J., Yamada, J. & Kaila, K. The cation–chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus. J. Physiol. 573, 765–773 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Brown, P. D., Davies, S. L., Speake, T. & Millar, I. D. Molecular mechanisms of cerebrospinal fluid production. Neuroscience 129, 957–970 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Gross, J. Analytical methods and experimental approaches for electrophysiological studies of brain oscillations. J. Neurosci. Methods 228, 57–66 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Lenoir, M., Tang, J. S., Woods, A. S. & Kiyatkin, E. A. Rapid sensitization of physiological, neuronal, and locomotor effects of nicotine: critical role of peripheral drug actions. J. Neurosci. 33, 9937–9949 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Kim, E. J. & Lee, M. G. Pharmacokinetics and pharmacodynamics of intravenous bumetanide in mutant Nagase analbuminemic rats: importance of globulin binding for the pharmacodynamic effects. Biopharm. Drug Dispos. 22, 147–156 (2001).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Töpfer, M. et al. Consequences of inhibition of bumetanide metabolism in rodents on brain penetration and effects of bumetanide in chronic models of epilepsy. Eur. J. Neurosci. 39, 673–687 (2014).

    Article  PubMed  Google Scholar 

  46. 46.

    Walker, M. C., Tong, X., Perry, H., Alavijeh, M. S. & Patsalos, P. N. Comparison of serum, cerebrospinal fluid and brain extracellular fluid pharmacokinetics of lamotrigine. Br. J. Pharmacol. 130, 242–248 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Hunt, M. J., Garcia, R., Large, C. H. & Kasicki, S. Modulation of high-frequency oscillations associated with NMDA receptor hypofunction in the rodent nucleus accumbens by lamotrigine. Prog. Neuropsychopharmacol. Biol. Psychiatry 32, 1312–1319 (2008).

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Brandt, C., Nozadze, M., Heuchert, N., Rattka, M. & Löscher, W. Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy. J. Neurosci. 30, 8602–8612 (2010).

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Cleary, R. T. et al. Bumetanide enhances phenobarbital efficacy in a rat model of hypoxic neonatal seizures. PLoS ONE 8, e57148 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Ward, A. & Heel, R. C. Bumetanide. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use. Drugs 28, 426–464 (1984).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Lee, S. H., Lee, M. G. & Kim, N. D. Pharmacokinetics and pharmacodynamics of bumetanide after intravenous and oral administration to rats: absorption from various GI segments. J. Pharmacokinet. Biopharm. 22, 1–17 (1994).

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Kissinger, P. T., Hart, J. B. & Adams, R. N. Voltammetry in brain tissue—a new neurophysiological measurement. Brain Res. 55, 209–213 (1973).

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Robinson, D. L., Venton, B. J., Heien, M. L. & Wightman, R. M. Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo. Clin. Chem. 49, 1763–1773 (2003).

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Hashemi, P. et al. Brain dopamine and serotonin differ in regulation and its consequences. Proc. Natl Acad. Sci. USA 109, 11510–11515 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Liu, J. et al. In vivo electrochemical monitoring of the change of cochlear perilymph ascorbate during salicylate-induced tinnitus. Anal. Chem. 84, 5433–5438 (2012).

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Gonon, F., Buda, M., Cespuglio, R., Jouvet, M. & Pujol, J. F. In vivo electrochemical detection of catechols in the neostriatum of anaesthetized rats: dopamine or DOPAC? Nature 286, 902–904 (1980).

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Marsden, C. A., Bennett, G. W., Brazell, M., Sharp, T. & Stolz, J. F. Electrochemical monitoring of 5-hydroxytryptamine release in vitro and related in vivo measurements of indoleamines. J. Physiol. Paris 77, 333–337 (1981).

    CAS  PubMed  Google Scholar 

  58. 58.

    Nakazato, T. & Akiyama, A. In vivo electrochemical measurement of the long-lasting release of dopamine and serotonin induced by intrastriatal kainic acid. J. Neurochem. 69, 2039–2047 (1997).

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Sceniak, M. P. & Maciver, M. B. Cellular actions of urethane on rat visual cortical neurons in vitro. J. Neurophysiol. 95, 3865–3874 (2006).

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Pagliardini, S., Funk, G. D. & Dickson, C. T. Breathing and brain state: urethane anesthesia as a model for natural sleep. Respir. Physiol. Neurobiol. 188, 324–332 (2013).

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Gad, S. C. & Gad, S. C. Safety Pharmacology in Pharmaceutical Development: Approval and Post Marketing Surveillance 2nd edn (CRC Press, Boca Raton, FL, 2012).

    Google Scholar 

  62. 62.

    Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Hibino, H. et al. An ATP-dependent inwardly rectifying potassium channel, KAB-2 (Kir4.1), in cochlear stria vascularis of inner ear: its specific subcellular localization and correlation with the formation of endocochlear potential. J. Neurosci. 17, 4711–4721 (1997).

    CAS  PubMed  Google Scholar 

  64. 64.

    Natsume, K., Hallworth, N. E., Szgatti, T. L. & Bland, B. H. Hippocampal theta-related cellular activity in the superior colliculus of the urethane-anesthetized rat. Hippocampus 9, 500–509 (1999).

    CAS  Article  PubMed  Google Scholar 

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We thank P. Bredeloux for comments on the experimental results, and Y. Takahashi and H. Shiku for technical advice. This study was partially supported by the following research grants: Grant-in-Aid for Scientific Research B 25293058 (to H.H.); Grant-in-Aid for Scientific Research C 15K10770 (to K.D.); and Grants-in-Aid for Young Scientists B 25870248 (to F.N.) and 26870210 (to G.O.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and JST-ACCEL (to Y.E.). In addition, funds were provided by the Nakatani Foundation (to H.H.), Takeda Science Foundation (to F.N.), Uehara Memorial Foundation (to F.N.) and Astellas Foundation for Research on Metabolic Disorders (to F.N.).

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G.O., F.N., H.K., T.Y., Y.E. and H.H. designed the experiments. G.O. and K.D. developed the experimental setup. F.N., T.Y. and S.K. were involved in developing the surgical procedures. Y.I., K.A. and Y.E. prepared the BDD electrodes. Y.I., K.A., Y.E. and M.T. contributed to establishing the protocol for the electrochemical experiments. G.O. performed the electrochemical experiments. T.H., T.O. and K.H. supported the data collection. Y.S., K.M. and H.K designed the LC–MS/MS experiments. Y.S. performed the LC–MS/MS experiments. G.O., S.S., T.O. and I.F. analysed the results. G.O., T.O., I.F. and H.H. wrote the paper. All authors edited the paper.

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Correspondence to Yasuaki Einaga or Hiroshi Hibino.

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Ogata, G., Ishii, Y., Asai, K. et al. A microsensing system for the in vivo real-time detection of local drug kinetics. Nat Biomed Eng 1, 654–666 (2017). https://doi.org/10.1038/s41551-017-0118-5

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