Applied Biochemistry and Biotechnology

, Volume 121, Issue 1–3, pp 361–371 | Cite as

A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures

  • Eliana M. Alhadeff
  • Andréa M. Salgado
  • Nei PereiraJr.
  • Belkis Valdman


A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 ± 2.3% and 63.2 ± 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1–30 % v/v (10.0–238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034–0.087 g ethanol/L working with the on-line dilution integrated to the biosensor—FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Index Entries

Biosensors ethanol flow injection analysis alcohol oxidase horseradish peroxidase 


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  1. 1.
    Taniai, T., Sukurragawa, A, and Okitani, T (2001) J. AOAC Internat. 84, 1475–1483.Google Scholar
  2. 2.
    Castillo, J., Gaspar, S., Sakharov, I., and Csoregi, E. (2003) Biosenso. Bioeletron. 18, 705–714.CrossRefGoogle Scholar
  3. 3.
    Prada, A. G-V., Peña, N., Mena, M. L., Reviejo, A. J., and Pingarrón, J. M. (2003) Biosens. Bioelectron. 18, 1279–1288.CrossRefGoogle Scholar
  4. 4.
    Prada, A. G-V., Peña, N., Parrado, C., Reviejo, A. J., and Pingarrón, J. M. (2004) Talanta 62, 896–903.CrossRefGoogle Scholar
  5. 5.
    Niculescu, M., Mieliauskiene, R., Laurinavicius, V., and Csoregi, E. (2003) Food Chem. 82, 481–489.CrossRefGoogle Scholar
  6. 6.
    Alhadeff,, E.A., Salgado, A. M., Pereira Jr., N., and Valdman, B. (2004) Appl. Biochem. Biotechnol., 113–116, 125–136.CrossRefGoogle Scholar
  7. 7.
    Tkac, J., Vostiar, I., Gorton, L., Gemeiner, P., and Sturdik, E. (2003) Biosen. Bioelectron. 18, 1125–1134.CrossRefGoogle Scholar
  8. 8.
    Schugerl, K. (2001) J. Biotechnol. 85, 49–173.CrossRefGoogle Scholar
  9. 9.
    Gué, A.M, Tap, H, Gros, P, and Maury, F. (2002) Sensor Actuators B 82, 227–232.CrossRefGoogle Scholar
  10. 10.
    Thévenot, D. R, Toth, K. Durst, R. A., and Wilson, G. S. (2001) Biosen. Bioelectron. 16, 121–131.CrossRefGoogle Scholar
  11. 11.
    Akyilmaz, E., and Dinçkaya, E. (2003) Talanta, 61, 113–118.CrossRefGoogle Scholar
  12. 12.
    Setkus, A., Razumiene, J., Galdikas, A., Laurinavicius, V., Meskys, R., and Mironas, A. (2003) Sensors and Actuators B Chemical 95, 344–351.CrossRefGoogle Scholar
  13. 13.
    Razumiene, J., Vilkanauskyte, A., Gureviciene, V., et al. (2003) J. Organometallic Chem. 668, 83–90.CrossRefGoogle Scholar
  14. 14.
    Razumiene, J., Gureviciene, V., Vilkanauskyte, A. et al. (2003) Sensors and Actuators B Chemical 95, 378–383.CrossRefGoogle Scholar
  15. 15.
    Santos, A. S., Freire, R.S., and Kubota, L. T. (2003) J. Electroanal. Chem. 547, 135–142.CrossRefGoogle Scholar
  16. 16.
    Rotariu, L., Bala, C., and Megaru, V.(2004) Anal. Chim. Acta in press.Google Scholar
  17. 17.
    Salgado, A. M., Folly, R. O. M., Valdman, B., Cós, O., and Valero F. (2000) Biotechn. Lett. 22, 327–330.CrossRefGoogle Scholar
  18. 18.
    Azevedo, A. M., Cabral, J. M. S., Prazeres, D. M. F., Gibson, T. D., and Fonseca, L. P. (2004) J. Molec. Catal. B: Enzymatic 27, 37–45.CrossRefGoogle Scholar
  19. 19.
    De Matinis, B. S., and Martin, C. C. S. (2002), Forensic Sci. Inter. 128, 115–119.CrossRefGoogle Scholar
  20. 20.
    Ukeda, H., Ohira, M., and Sawamura, M. (1999) Anal. Sci. 15, 447–450.CrossRefGoogle Scholar
  21. 21.
    Mello, L. D., Sotomayor, M. D. P. T., and Kubota, L. T. (2003) Sensors and Actuators B Chemical 96, 636–645.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Eliana M. Alhadeff
    • 2
  • Andréa M. Salgado
    • 2
  • Nei PereiraJr.
    • 2
  • Belkis Valdman
    • 1
  1. 1.Departamento de Engenharia Química, Escola de QuímicaUniversidade Federal do Rio de Janeiro, C.T.Rio de JaneiroBrasil
  2. 2.Departamento de Engenharia Bioquímica, Escola de QuímicaUniversidade Federal do Rio de Janeiro, C.T.Rio de JaneiroBrasil

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