A MULTISTEP ENZYME SENSOR FOR SUCROSE BASED ON S-LAYER MICROPARTICLES AS IMMOBILIZATION MATRIX

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline bacterial cell surface layers (S-layers) as immobilization matrix for the biological components. Isoporous, crystalline surface layers (S-layers) have been identified as outermost cell envelope layer in many bacteria. Since they are composed of identical protein or glycoprotein subunits with functional groups in well defined positions and orientations, they represent ideal matrices for the controlled and reproducible immobilization of functional macromolecules, as required for the development of biosensors. Apart from single enzyme sensors, which were described earlier, a strikingly simple method for the assembly and optimization of multistep systems was developed. For the fabrication of an amperometric sucrose sensor invertase, mutarotase and glucose oxidase were individually immobilized on S-layer fragments isolated from Clostridium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecules. Subsequently, appropriate mixtures of enzyme loaded S-layer fragments were deposited on a microfiltration membrane and finally, the composite multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements based on H2O2 oxidation revealed a high signal level (1 muA*1/cm2*mmol sucrose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.