Optical biochemical sensor for determining hydroperoxides in nonpolar organic liquids as archetype for sensors consisting of amphiphilic conetworks as immobilisation matrices
- 189 Downloads
This paper reports the successful design of a prototype of an optical biochemical sensor for the determination of hydroperoxides in nonpolar organic liquids. The sensor consists of a matrix of an amphiphilic polymer conetwork (APCN), a novel class of very promising polymeric materials for easy preparation of biochemical sensor matrices. APCNs are characterised by nanoscopic phase separation between the hydrophilic and the hydrophobic phases. For medium ratios of conetwork composition, the domains of both phases are interconnected both on the surface of the conetworks and throughout the bulk. The APCNs have peculiar swelling properties—the hydrophilic phase swells in hydrophilic media and the hydrophobic phase swells in hydrophobic media. In both types of media dissolved reagents can diffuse from the solution into the swollen phase of the polymeric conetwork. This enables loading of the hydrophilic phase of the APCNs with enzymes and indicator reagents by simple impregnation. Hydrophobic analytes can diffuse into the polymeric conetwork via its hydrophobic phase and react with indicator reagents immobilised in the hydrophilic phase at the huge internal interface between the two opposite phases.
To prepare the described hydroperoxide-sensitive biosensors, we used APCN films consisting of 58% (w/w) poly(2-hydroxyethyl acrylate) (PHEA) as hydrophilic chains and 42% (w/w) polydimethylsiloxane (PDMS) as hydrophobic linkers. Horseradish peroxidase (HRP) and diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as indicator reagent were co-immobilised in this optically clear and transparent matrix. In this feasibility study the conditions investigated were principally those relevant to characterisation of the innovative matrix material and the disposable biosensor produced from it; the biosensor was not optimised. Sensitivity toward tert-butylhydroperoxide (tBuOOH) dissolved in n-heptane was acceptable, between approximately 1 and at least 50 mmol L−1, even in the dry state. The response time was 1.7 to 5.0 min. No leaching of immobilised reagents was observed during a period of at least one hour. Pre-swelling the sensors with water increased the reaction rate and the total turnover number of the enzyme. In a dry atmosphere at 4 °C the sensors were found to be stable for at least two weeks.
KeywordsNanophase-separated amphiphilic conetwork Enzyme immobilisation Optical biosensor Nonpolar organic media Horseradish peroxidase Peroxide determination
This work was supported by the European Union (MASTER project), by the Deutsche Forschungsgemeinschaft (SFB 428 and Emmy-Noether-Programm), and by the Fonds der Chemischen Industrie.
- 1.Stöcklein WFM, Scheller FW (1997) In: Scheller FW, Fedrowitz J, Schubert F (eds) Frontiers in biosensorics I. Fundamental aspects. Birkhäuser, Basel, pp 83–96Google Scholar
- 4.Hayes DG (2002) In: Howard GC, Brown WE (eds) Modern protein chemistry: practical aspects. CRC Press, Boca Raton, Fl, pp 179–225Google Scholar
- 5.Castro GR, Knubovets T (2003) Crit Rev Biotechnol 23:195–231Google Scholar
- 13.Bickerstaff GF (ed) (1997) Immobilization of enzymes and cells. Humana Press, Totowa, NJGoogle Scholar
- 14.Tiller J, Berlin P, Klemm D (1999) Biotechnol Appl Biochem 30:155–162Google Scholar
- 32.Friends GD, Künzler JF, Ozark RM (1995) Macromol Symp 98:619–631Google Scholar
- 39.Bruns N, Tiller JC (2005) Ger Offen, DE 2003-10343794, 8 ppGoogle Scholar
- 47.Bourbonnais R, Leech D, Paice M (1998) Biochim Biophys Acta 1379:381–390Google Scholar