A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode

Abstract

A hydrogen gas (H₂) biosensor was developed in which hydrogenase (H₂ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H₂ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay–PBV–H₂ase modified electrode. The clay–PBV–H₂ase electrode catalyzed the oxidation of H₂ to protons (H⁺) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38±2 kJ/mol at pH 7. The catalytic current of the clay–PBV–H₂ase electrode increased linearly when exposed to increasing concentrations of H₂ gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H₂ by the clay–PBV–H₂ase electrode was from 7 to 10. Electron transfer process in the clay–PBV–H₂ase electrode is discussed.

Introduction

The reversible interconversion of hydrogen (H₂) to protons and electrons catalyzed by hydrogenases (H₂ases) is a central metabolic feature of some microorganisms, including most prokaryotic genera and some lower eucaryotes (Adams, 1990, Kleihues et al., 2000). Electrochemical studies have revealed that H₂ases catalyze the reversible reaction 2H⁺+2e⁻↔H₂ for a potential of about −390 mV at pH 7.0 30 °C and 0.1 bar H₂ with appropriate electron carriers, such as ferredoxin and cytochrome c₃ (Albracht, 1994). The activation energy E_a of the H₂ase from Azotobacter vinelandii is 10 kcal/mol for H₂ oxidation and 22 kcal/mol for H₂ evolution, indicating that the enzyme catalyzes H₂ oxidation at much greater rate than the H₂ evolution (McTavish et al., 1996). Pershad et al. (1999) concluded that H₂ oxidation is a very rapid process even at potentials much lower than the formal potential of the [3Fe-4S]^1+/0 cluster, and that the catalytic rate is controlled by transport of H₂ to the H₂ase layer. Thus, the electrocatalytic H₂ oxidation by the H₂ases closely relates to the structure of H₂ase electrode and H₂ concentration, the properties of which might be utilized for H₂ biosensors. Biosensors can be designed to transduce highly specific biomolecular interactions into amplifiable signals and to assure significant anti-interference from the environment. The purpose of our present study was the development of a highly efficient, stable H₂ase electrode for the H₂ biosensor.

The H₂ase used was a thermal stable [NiFe] H₂ase from the phototrophic bacterium Thiocapsa roseopersicina (Zorin et al., 1995). It is a hydrophobic protein and contains one nickel atom and a single iron–sulfur cluster per αβ-heterodimer (Sherman et al., 1991). Immobilization of the H₂ase on an electrode is the first step for preparing the H₂ biosensor. Although the simplest way to do this is to directly immobilize the H₂ase onto the electrode surface, large biological molecules capable of electron transfer often do not respond or they show weak signals at conventional electrodes because it is difficult for the redox center to get close enough to the electrode for reaction (Lewis and Wrighton, 1981, Li et al., 1997). To improve the electron transfer efficiency, some electron transfer mediators are commonly used to modify the electrode before immobilization of the enzyme (Lewis and Wrighton, 1981, Zakeeruddin et al., 1996). Ferredoxin and cytochrome c₃ are examples of natural electron carriers for H₂ase (Meuer et al., 1999), while viologens often act as artificial electron carriers (De Lacey et al., 2000, Nedoluzhko et al., 2001). When viologens are used, they are usually dissolved in solutions or absorbed on the electrode surface, and H₂ases are in solutions or covered on or immersed in the viologen layer (De Lacey et al., 2000, Nedoluzhko et al., 2001, Qian et al., 2000b). In previous studies, we prepared several kinds of viologen-modified electrodes by using self-assemble and Langmuir–Blodgett methods (Qian et al., 2000a), and found that although the signal is not so strong, these electrodes can act as electrode carriers for the H₂ase (Qian et al., 2000b, Noda et al., 1998). Eng et al. (1994) constructed a poly(viologen)–H₂ase modified electrode, which showed a distinct and reproducible response current to H₂. Based on hydrophobic interactions of an H₂ase with a bilayer assembly consisting of octadecyltrichlorosilane and octadecylviologen, Parpaleix et al. (1992) immobilized Desulfovlbrio gigas H₂ase at the electrode surface, which could catalyze H₂ oxidation mediated by the octadecylviologen molecules.

In our design of a H₂ biosensor, we used a clay-sandwich method to immobilize the electron carrier, poly(butylviologen) (PBV), between the electrode and H₂ase. The advantages of this method are that colloidal clays have appreciable surface area, intercalation properties, low cost, high stability and high cation exchange capacity (Zen et al., 1997). The composite enzyme–clay films have been confirmed to improve the analytic performances including adhesion, mechanical strength, and enzymatic activity (Besombes et al., 1995, Besombes et al., 1997). A glucose biosensor was prepared by the clay-sandwich method (Zen and Lo 1996, Zen et al., 1997). In this current study, we used a mixture of clay and PBV instead of pure clay as the sandwich layer and found that, compared with a pure clay layer, the mixture layer showed a significantly higher catalytic current. Based on the electrochemical results, we developed a scheme of the electron transfer process in the clay–PBV–H₂ase electrode. Because the stability, temperature, pH, and anti-inference are important for practical applications of the clay–PBV–H₂ase electrode, we further evaluated the long-term stability and the effects of the solution temperature, combustible material (i.e. carbon monoxide, CO; methane, CH₄; and methanol, CH₃OH), and pH of the buffer solution on the activity of the clay–PBV–H₂ase electrode.