Langmuir 2007, 23, 6807-6811
6807
Enzyme Activity Control by Responsive Redoxpolymers Birgit Nagel,† Axel Warsinke,‡ and Martin Katterle*,† Fraunhofer Institute for Biomedical Engineering, Am Mu¨hlenberg 13, 14476 Potsdam, Germany, and Analytical Biochemistry, UniVersity of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany ReceiVed February 6, 2007. In Final Form: March 26, 2007 A new thermoresponsive poly-N-isopropylacrylamide (PNIPAM)-ferrocene polymer was synthesized and characterized. PNIPAMFoxy bears additional oxirane groups which were used for attachment by a self-assembly process on a cysteamine-modified gold electrode to create a thin hydrophilic film. The new redox polymer enabled electrical communication between the cofactor pyrrolinoquinoline quinone (PQQ) of soluble glucose dehydrogenase (sGDH) and the electrode for sensitive detection of this enzyme as a prospective protein label. The temperature influence on the redox polymer/enzyme complex was investigated. An inverse temperature response behavior of surface bound PNIPAMFoxy compared to the soluble polymer was found and is discussed in detail. The highest efficiency of mediated electron transfer for the immobilized PNIPAMFoxy with sGDH was observed at 24 °C, which was twice as high as that of its soluble counterpart. A steady-state electrooxidation current densitiy of 4.5 µA‚cm-2 was observed in the presence of 10 nM sGDH and 5 mM glucose. A detection limit of 0.5 nM of soluble PQQ-sGDH was obtained.
Introduction Polymers undergoing thermally and electrochemically controllable phase transition can be used as gel actuators,1,2 drug release membranes,3-5 permeability-controlled films,6-8 thermal switches, or as immobilizing matrices for enzymes.9-11 N-isopropylacrylamide based polymers (PNIPAM) are known to be thermoresponsive in aqueous solution. This behavior is based on a widespread hydrogen bond network between the amide groups and water molecules at lower temperatures, whereas at higher temperatures the stabilizing H-bonds break up and the hydrophobic interactions become predominant, which results in a reversible volume phase transition. Tatsuma et al. described an electrochemically controllable phase transition and a thermally controllable electrochemistry of a PNIPAM-ferrocene polymer (PAF).10 The phase transition is influenced by the change of hydrophobicity of the copolymerized ferrocene by electrochemical oxidation. A similar PAF polymer is used to “wire” glucose oxidase (GOD) to an electrode surface by placing a cold enzyme-polymer mixture on the electrode followed by subsequent drying and heating steps (>35 °C). This procedure caused a shrunken gel film with the entrapped enzyme.10-14 To unload the enzyme from the PAF gel-coated * To whom correspondence should be addressed. E-mail: Martin.
[email protected]. † Fraunhofer Institute for Biomedical Engineering. ‡ University of Potsdam. (1) Tsutsui, H.; Moriyama, M.; Nakayama, D.; Ishii, R.; Akashi, R. Macromolecules 2006, 39, 2291-2297. (2) Tsutsui, H.; Akashi, R. J. Appl. Polym. Sci. 2007, 103, 2295-2303. (3) Lee, W. F.; Lin, Y. H. J. Mater. Sci. 2006, 41, 7333-7340. (4) Ito, T.; Yamaguchi, T. Langmuir 2006, 22, 3945-3949. (5) Malmsten, M. Soft Matter 2006, 2, 760-769. (6) Sumaru, K.; Ohi, K.; Takagi, T.; Kanamori, T.; Shinbo, T. Langmuir 2006, 22, 4353-4356. (7) Matsukuma, D.; Yamamoto, K.; Aoyagi, T. Langmuir 2006, 22, 59115915. (8) Baldi, A.; Lei, M.; Gu, Y. D.; Siegel, R. A.; Ziaie, B. Sens. Actuators, B 2006, 114, 9-18. (9) Suzuki, D.; Kawaguchi, H. Colloid Polym. Sci. 2006, 284, 14431451. (10) Tatsuma, T.; Takada, K.; Matsui, H.; Oyama, N. Macromolecules 1994, 27, 6687-6689. (11) Tatsuma, T.; Saito, K.; Oyama, N. J. Chem. Soc., Chem. Commun. 1994, 1853-1854.
electrode, the temperature was decreased to
