Improving enzyme–electrode contacts by redox modification of cofactors

Abstract

Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox groups. Generally these methods involve contacting the enzymes at random with electron relay units. Here we report an approach that allows site-specific positioning of electron-mediating units in redox proteins. We strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way.

Emprego de monocamadas auto-organizadas no desenvolvimento de sensores eletroquímicos

“…Uma outra maneira de se estabelecer melhor contato elétrico entre o centro redox da enzima e a superfície do eletrodo é por meio do método de reconstituição da enzima. Neste processo, o mediador é ligado diretamente ao centro redox da enzima, sendo a apoenzima (enzima sem o centro redox) posteriormente reconstituída sobre a superfície do eletrodo, de acordo com o esquema mostrado na Figura 8 118,120 . Empregando-se este método foi construído um sensor para glicose 121 , onde o mediador pirroloquinolina quinona (PQQ) foi covalentemente ligado a uma monocamada de cisteamina e, posteriormente, reagido com um derivado amina do FAD, o N(6)-(2-aminoethyl)-FAD.…”

Glucose Amperometric Biosensor Based on the Co-immobilization of Glucose Oxidase (GOx) and Ferrocene in Poly(pyrrole) Generated from Ethanol / Water Mixtures

“…Schuhmann 13 used ferrocene modified glucose oxidase, both of them attached to each other via long and flexible spacer chains within conducting polymer films. Katz et al 15,16 reported the preparation of a glucose biosensor by removing FAD (flavine adenine dinucleotide) from GOx and reintroducing the ferrocene-modified FAD again into the GOx. In this way, authors improved the electrical communication between the electrode and the reconstituted enzyme.…”

Nanostructured Inorganic Materials at Work in Electrochemical Sensing and Biofuel Cells

“…To enhance the sensitivity of the multiplexed bioelectrodes, either a GOx solution is mixed thoroughly with a chitosan/carbon nanotubes one or a LOx layer is sandwiched between two layers of chitosan/carbon nanotubes at pH 7.2. The mixture stands on a thinner Prussian blue layer mediator that has been preliminary electrodeposited on a bare Au electrode (promotes better immobilization [19,[44][45][46]). In such a configuration, the input will be the substrate (glucose, lactate) and the output current (between the working electrode and the counter-electrode) of the sensor can be converted to a voltage through a transimpedance amplifier.…”