Journal of Fluorescence

, Volume 14, Issue 5, pp 561–568 | Cite as

Time-Resolved Fluorescent Imaging of Glucose



A method for the fluorescent imaging of glucose is described that is based on the detection of enzymatically produced hydrogen peroxide, using the europium(III) tetracycline complex as the fluorescent probe incorporated into a hydrophilic polymer layer. Coadsorption of glucose oxidase (GOx) makes these sensor layers respond to the hydrogen peroxide produced by the GOx-assisted oxidation of glucose. The hydrogel layers are integrated into a 96-microwell plate for a parallel and simultaneous detection of various samples. Glucose is visualized by means of time resolved luminescence lifetime imaging. Unlike in previous methods, the determination of H2O2 does not require the addition of peroxidase or a catalyst to form a fluorescent product. The lifetime-based images obtained are compared with conventional fluorescence intensity-based methods with respect to sensitivity and the dynamic range of the sensor layer. The main advantages provided by this sensing scheme for H2O2 include reversibility, applicability at neutral pH, and the straightforwardness of the transducer system and the imaging device.

Optical sensor time-resolved fluorescence imaging europium tetracycline glucose sensor 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    E. A. Hall (1990). Biosensors, Open University Press, Buckingham, pp. 253–282.Google Scholar
  2. 2.
    K. Habermüller, A. Ramanavicius, V. Laurinavicius, and W. Schuhmann (2000). An oxygen-insensitive reagentless glucose biosensor based on osmium-complex modified polypyrrole. Elec-troanalysis 12, 1383–1389.Google Scholar
  3. 3.
    D. B. Papkovsky (1995). New oxygen sensors and their application to biosensing. Sens. Actuat. B 29, 213–218.Google Scholar
  4. 4.
    J. Kulys (1999). The carbon paste electrode encrusted with a mi-croreactor as glucose biosensor. Biosens. Bioelectron. 14, 473–479.Google Scholar
  5. 5.
    A. Poscia, M. Mascini, D. Moscone, M. Luzzana, G. Caramenti, P. Cremonesi, F. Valgimigli, C. Bongiovanni, and M. Varalli (2003). A microdialysis technique for continuous subcutaneous glucose mon-itoring in diabetic patients. Biosens. Bioelectron. 18, 891–898.Google Scholar
  6. 6.
    F. Ricci, C. Goncalves, A. Amine, L. Gorton, G. Palleschi, and D. Moscone (2003). Electroanalytical study of prussian blue modified glassy carbon paste electrodes. Electroanalysis 15, 1204–1211.Google Scholar
  7. 7.
    S. F. White, A. P. F. Turner, U. Biltewski, J. Bradley, and R. D. Schmid (1995). On-line monitoring of glucose, glutamate and glu-tamine during mammalian cell cultivations. Biosens. Bioelectron. 10, 543–551.Google Scholar
  8. 8.
    R. Narayanaswamy and F. Sevilla (1988). Anal. Lett. 21, 1165–1175.Google Scholar
  9. 9.
    B. P. H. Schaffar and O. S. Wolfbeis (1990). A fast responding fibre optic glucose biosensor based on an oxygen optrode. Biosens. Bioelectron. 5, 137–148.Google Scholar
  10. 10.
    O. S. Wolfbeis, I. Oehme, N. Papkovskaya, and I. Klimant (2000). Sol-gel based glucose biosensors employing optical oxygen trans-ducers, and a method for compensating for variable oxygen back-ground. Biosens. Bioelectron. 15,69–76.Google Scholar
  11. 11.
    H. M. Heise (2000) in R. A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, Wiley, New York, pp. 56–83.Google Scholar
  12. 12.
    K. Kellner, G. Liebsch, I. Klimant, O. S. Wolfbeis, T. Blunk, M. B. Schulz,and A. Göpferich (2002). Determination of oxygen gradients in engineered tissue using a fluorescent sensor. Biotechnol. Bioeng. 80,73–83.PubMedGoogle Scholar
  13. 13.
    G. Liebsch, I. Klimant, B. Frank, G. Holst, and O. S. Wolfbeis (2000). Luminescence lifetime imaging of oxygen, pH, and carbon dioxide distribztion using optical sensors. Appl. Spectrosc. 54, 548–559.Google Scholar
  14. 14.
    P. Babilas, V. Schacht, G. Liebsch, O. S. Wolfbeis, M. Landthaler, M. Szeimies, and C. Abels (2003). Effect of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo. Br. J. Cancer 88, 1462–1469.Google Scholar
  15. 15.
    A. Shiino, M. Haida, and B. Beauvoit, and B. Chance (1999). Three-dimensional redox image of the normal gerbil brain. Neu-roscience 91, 1581–1585.Google Scholar
  16. 16.
    R. E. Anderson and F. B. Meyer (2002). In C. K. Sen and L. Packer (Eds.), Methods in Enzymology, Vol. 352, Academic Press, San Diego, pp. 482–494.Google Scholar
  17. 17.
    M. Hashimoto, Y. Takeda, T. Sato, H. Kawahara, O. Nagano, and M. Hirakawa (2000). Dynamic changes of NADH fluorescence im-ages and NADH content during spreading depression in the cerebral cortex of gerbils. Brain Res. 872, 294–300.Google Scholar
  18. 18.
    A. V. Kuznetsov, O. Mayboroda, D. Kunz, K. Winkler, W. Schubert, and W. S. Kunz (1998). Functional imaging of mitochon-dria in saponin-permeabilized mice muscle fibers. J. Cell Biol. 140, 1091–1099.CrossRefPubMedGoogle Scholar
  19. 19.
    M. Weinlich and H. Acker (1990). Flavoprotein-fluorescence imag-ing for metabolic studies in multicellular spheroids by means of confocal scanning laser microscopy. J. Microsc. 160, RP1–RP2.Google Scholar
  20. 20.
    S. Van Stedum and W. King (2002). Basic FISH techniques and troubleshooting. Methods Mol. Biol. 204,51–63.PubMedGoogle Scholar
  21. 21.
    T. Liehr and U. Claussen (2002). Multicolor-FISH approaches for the characterization of human chromosomes in clinical genetics and tumor cytogenetics. Curr. Genom. 3, 213–235.Google Scholar
  22. 22.
    B. Rautenstrauss and T. Liehr (2002). FISH Technology, Springer-Verlag, Berlin, Germany, 494 pp.Google Scholar
  23. 23.
    M. Andreeff and D. Pinkel (1999). Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, Wiley-Liss, New York, 455 pp.Google Scholar
  24. 24.
    G. Liebsch, I. Klimant, C. Krause, and O. S. Wolfbeis (2001). Fluo-rescent imaging of pH with optical sensors using time domain dual lifetime referencing. Anal. Chem. 73, 4354–4363.Google Scholar
  25. 25.
    G. Liebsch, I. Klimant, and O. S. Wolfbeis, (1999). Cross-reactive metal ion sensor array in a micro titer plate format. Adv. Mat. 11, 1296–1299.CrossRefGoogle Scholar
  26. 26.
    K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg (2002). Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient. Biophys. J. 83, 1682–1690.PubMedGoogle Scholar
  27. 27.
    P. C. Schneider and R. M. Clegg (1997). Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications. Rev. Sci. Instr. 68, 4107–4119.Google Scholar
  28. 28.
    M. C. Moreno-Bondi, O. S. Wolfbeis, M. J. P. Leiner, and B. P. H. Schaffar (1990). Oxygen optrode for use in a fiber-optic glucose biosensor. Anal. Chem. 62, 2377–2380.PubMedGoogle Scholar
  29. 29.
    O. S. Wolfbeis, I. Oehme, N. Papkovskaya, and I. Klimant (2000). Sol-gel based glucose biosensors employing optical oxygen trans-ducers, and a method for compensating for variable oxygen back-ground. Biosens. Bioelectron. 15,69–76.Google Scholar
  30. 30.
    J. S. Schultz, S. Mansouri, and I. J. Goldstein (1982). Affinity sensor: A new technique for developing implantable sensors for glucose and other metabolites. Diabetes Care 5, 245–254.Google Scholar
  31. 31.
    D. Meadows and J. S. Schultz (1988). Fiber-optic biosensors based on fluorescence energy transfer. Talanta 35, 145–153.CrossRefGoogle Scholar
  32. 32.
    L. Tolosa, H. Szmacinski, G. Rao, and J. R. Lakowicz (1997). Lifetime-based sensing of glucose using energy transfer with a long lifetime donor. Anal. Biochem. 250, 102–108.CrossRefGoogle Scholar
  33. 33.
    N. DiCesare and J. R. Lakowicz (2001). Evaluation of two syn-thetic glucose probes for fluorescence-lifetime-based sensing. Anal. Biochem . 294, 154–160.CrossRefGoogle Scholar
  34. 34.
    L. L. E. Salins, R. A. Ware, C. M. Ensor, and S. Daunert (2001). A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein. Anal. Biochem. 294,19–26.CrossRefGoogle Scholar
  35. 35.
    O. S. Wolfbeis, A. Dürkop, M. Wu, and Z. Lin, (2002). A europium-ion-based luminescent sensing probe for hydrogen peroxide. Angew. Chem. 114, 4681–4684; Angew. Chem. Int. Ed. 41, 4495-4498.CrossRefGoogle Scholar
  36. 36.
    O. S. Wolfbeis, M. Schäferling, and A. Dürkop, (2003). Reversible optical sensor membrane for hydrogen peroxide using an immobi-lized fluorescent probe, and its application to a glucose biosensor. Microchim. Acta 143, 221–227Google Scholar
  37. 37.
    M. Schäferling, M. Wu, J. Enderlein, H. Bauer, and O. S. Wolfbeis (2003). Time-resolved luminescence imaging of hydrogen peroxide using sensor membranes in a microwell format. Appl. Spectrosc. 57, 1386–1392.Google Scholar
  38. 38.
    P. Hartmann and W. Ziegler (1996). Lifetime imaging of luminescent oxygen sensors based on all-solid-state technology. Anal. Chem. 68, 4512–4514.CrossRefGoogle Scholar
  39. 39.
    P. Hartmann, W. Ziegler, G. Holst, and D.W. Lübbers (1997). Oxygen flux fluorescence lifetime imaging. Sens. Actuat. B 38, 110–115.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2004

Authors and Affiliations

  • Michael Schäferling
    • 1
  • Meng Wu
    • 1
  • Otto S. Wolfbeis
    • 1
  1. 1.Institute of Analytical Chemistry, Chemo- and BiosensorsUniversity of RegensburgRegensburgGermany

Personalised recommendations