Amperometric enzyme electrodes for the determination of volatile alcohols in the headspace above fruit and vegetable juices

Abstract

We have investigated two amperometric biosensors for the determination of volatile alcohols in the headspace of fruit juices and vegetable juices. One type of sensor is based on the use of alcohol dehydrogenase (ADH) and the detection of NADH (at +300 mV vs. Ag/AgCl with phenothiazine as redox mediator), and the other on the use of alcohol oxidase (AOx) and the detection of hydrogen peroxide (at +600 mV vs. Ag/AgCl). Samples were analyzed with the AOx-based biosensor by measuring the alcohol concentration in the headspace above the liquid without prior sorption or pre-concentration. The sensor has a linear response in the range 0.1–20.0 mM of alcohol (referred to the concentration in the liquid sample). It has excellent stability in that the signal decreases by 4.5 % only over a 60 h operational period. However, a comparison of the AOx-based biosensor with HPLC and an enzyme test kit revealed an overestimation of ethanol levels in juices by the biosensor due to the simultaneous detection of methanol present in the samples. A flow-through version of the biosensor placed at the exit of a HPLC system proved this assumption. In order to improve the specificity for ethanol, the ADH-based sensor was studied. While showing no cross sensitivity to methanol, its stability was rather limited, this making it not suitable for practical applications. Headspace analysis offers advantages such as high selectivity (because it can be interfered by volatile substances only) and the lack of contamination of the sensor by species in the liquid juice.

Headspace analysis of alcohol in juices (tomato, grape, currant, apple) with an amperometric enzyme electrode: sensor set-up (left), calibration with ethanol samples (right)

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References

  1. 1.

    Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (2002) Leitsätze für Fruchtsäfte. http://bmelv.de/cae/servlet/contentblob/379766/publicationFile/22011/LeitsaetzeFruchtsaefte.pdf. Accessed 28 February 2012

  2. 2.

    Wucherpfennig K, Dietrich H, Bechtel J (1983) Actual, total and potential methyl alcohol content of fruit juices. Flüssiges Obst 50:348

    CAS  Google Scholar 

  3. 3.

    Braun G, Zur K (2009) Methanol: Wie sicher sind unsere Getränke. http://ua-bw.de/pub/beitrag.asp?subid=1&Thema_ID=2&ID=1244&Pdf=No. Accessed 28 February 2012

  4. 4.

    Rodionov YV, Keppen OI, Sukhacheva MV (2002) A photometric assay for ethanol. Appl Biochem Microbiol 38:395

    Article  CAS  Google Scholar 

  5. 5.

    Gessei T, Sato H, Kazawa E, Kudo H, Saito H, Mitsubayashi K (2009) Bio-sniffers for ethanol and acetaldehyde using carbon and Ag/AgCl coated electrodes. Microchim Acta 165:179

    Article  CAS  Google Scholar 

  6. 6.

    Mitsubayashi K, Matsunaga H, Nishio G, Toda S, Nakanishi Y (2005) Bio-sniffer sticks for breath analysis after drinking. Sens Actuators B 108:660

    Article  Google Scholar 

  7. 7.

    Mitsubayashi K, Matsunaga H, Nishio G, Toda S, Nakanishi Y (2005) Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking. Biosens Bioelectron 20:1573

    Article  CAS  Google Scholar 

  8. 8.

    Kudo H, Goto T, Saito T, Saito H, Otsuka K, Mitsubayashi K (2008) Biochemical sniffer with choline oxidase for measurement of choline vapour. Microchim Acta 160:421

    Article  CAS  Google Scholar 

  9. 9.

    Dennison MJ, Hall JM, Turner APF (1996) Direct monitoring of formaldehyde vapour and detection of ethanol vapour using dehydrogenase-based biosensors. Analyst 121:1769

    Article  CAS  Google Scholar 

  10. 10.

    Hämmerle M, Hilgert K, Achmann S, Moos R (2010) Direct monitoring of organic vapours with amperometric enzyme gas sensors. Biosens Bioelectron 25:1521

    Article  Google Scholar 

  11. 11.

    Park J, Yee H, Kim S (1995) Amperometric biosensor for determination of ethanol vapor. Biosens Bioelectron 10:587

    Article  CAS  Google Scholar 

  12. 12.

    Mitsubayashi K, Nishio G, Sawai M, Saito T, Kudo H, Saito H, Otsuka K, Noguer T, Marty J (2008) A bio-sniffer stick with FALDH (formaldehyde dehydrogenase) for convenient analysis of gaseous formaldehyde. Sens Actuators B 130:32

    Article  Google Scholar 

  13. 13.

    Hämmerle M, Achmann S, Moos R (2008) Gas diffusion electrodes for use in an amperometric enzyme biosensor. Electroanal 20:2279

    Article  Google Scholar 

  14. 14.

    Achmann S, Hermann M, Hilbrig F, Jérôme V, Hämmerle M, Freitag R, Moos R (2008) Direct detection of formaldehyde in air by a novel NAD+- and glutathione-independent formaldehyde dehydrogenase-based biosensor. Talanta 75:786

    Article  CAS  Google Scholar 

  15. 15.

    Sandström KJM, Newman J, Sunesson A, Levin J, Turner APF (2000) Amperometric biosensor for formic acid in air. Sens Actuators B 70:182

    Article  Google Scholar 

  16. 16.

    Sandström KJM, Sunesson AL, Levin JO, Turner APF (2003) A gas-phase biosensor for environmental monitoring of formic acid: laboratory and field validation. J Environ Monit 5:477

    Article  Google Scholar 

  17. 17.

    Minamide T, Mitsubayashi K, Saito H (2005) Bioelectronic sniffer with monoamine oxidase for methyl mercaptan vapor. Sens Actuators B 108:639

    Article  Google Scholar 

  18. 18.

    Mitsubayashi K, Nakayama K, Taniguchi M, Saito H, Otsuka K, Kudo H (2006) Bioelectronic sniffer for nicotine using enzyme inhibition. Anal Chim Acta 573–574:69

    Article  Google Scholar 

  19. 19.

    Dennison MJ, Hall JM, Turner APF (1995) Gas-phase microbiosensor for monitoring phenol vapor at ppb levels. Anal Chem 67:3922

    Article  CAS  Google Scholar 

  20. 20.

    Kaisheva A, Iliev I, Christov S, Kazareva R (1997) Electrochemical gas biosensor for phenol. Sens Actuators B 44:571

    Article  Google Scholar 

  21. 21.

    Hämmerle M, Hilgert K, Horn MA, Moos R (2011) Analysis of volatile alcohols in apple juices by an electrochemical biosensor measuring in the headspace above the liquid. Sens Actuators B 158:313

    Article  Google Scholar 

  22. 22.

    Leskovac V, Trivic S, Pericin D (2002) The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast Res 2:481

    CAS  Google Scholar 

  23. 23.

    Rebelo MJF, Compagnone D, Guibault GG, Lubranod GJ (1994) Alcohol electrodes in beverage measurements. Anal Lett 27:3027

    Article  CAS  Google Scholar 

  24. 24.

    Santos AS, Pereira AC, Durán N, Kubota LT (2006) Amperometric biosensor for ethanol based on co-immobilization of alcohol dehydrogenase and Meldola’s Blue on multi-wall carbon nanotube. Electrochim Acta 52:215

    Article  CAS  Google Scholar 

  25. 25.

    Sander R (1999) Compilation of Henry’s law constants for inorganic and organic species of potential importance in environmental chemistry. http://henrys-law.org. Accessed 28 February 2012

  26. 26.

    Dennison MJ, Turner APF (2000) Gas-phase enzyme electrodes. In: Bilitewski U, Turner APF (eds) Biosensors for environmental monitoring. Harwood Academic Publishers, Amsterdam, pp 285–308

    Google Scholar 

  27. 27.

    Kühne J, Hener U, Jung J, Münch A, Dietrich H, Patz C, Mosandl A (2007) Zur Qualitätsbewertung von Apfelsaft – 3-Methylbutanol, 2-Methylbutanol und Ethanol als Kenngrößen. Dtsch Lebensm Rundsch 103:247

    Google Scholar 

  28. 28.

    Bucur B, Radu GL, Toader CN (2008) Analysis of methanol–ethanol mixtures from falsified beverages using a dual biosensors amperometric system based on alcohol dehydrogenase and alcohol oxidase. Eur Food Res Technol 226:1335

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Dr. Marcus A. Horn (Lehrstuhl für Ökologische Mikrobiologie, University of Bayreuth) for his kind support in the HPLC measurements.

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Correspondence to Martin Hämmerle.

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Schlangen, C., Hämmerle, M. & Moos, R. Amperometric enzyme electrodes for the determination of volatile alcohols in the headspace above fruit and vegetable juices. Microchim Acta 179, 115–121 (2012). https://doi.org/10.1007/s00604-012-0867-5

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Keywords

  • Alcohol oxidase
  • Alcohol dehydrogenase
  • Ethanol
  • Methanol
  • Fruit juices