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

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline ba cterial cell surface layers (S-layers) as immobilization matrix for th e biological components.Isoporous, crystalline surface layers (S-layer s) have been identified as outermost cell envelope layer in many bacte ria. Since they are composed of identical protein or glycoprotein subu nits with functional groups in well defined positions and orientations , they represent ideal matrices for the controlled and reproducible im mobilization of functional macromolecules, as required for the develop ment of biosensors. Apart from single enzyme sensors, which were descr ibed earlier, a strikingly simple method for the assembly and optimiza tion of multistep systems was developed. For the fabrication of an amp erometric sucrose sensor invertase, mutarotase and glucose oxidase wer e individually immobilized on S-layer fragments isolated from Clostrid ium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecule s. Subsequently, appropriate mixtures of enzyme loaded S-layer fragmen ts were deposited on a microfiltration membrane and finally, the compo site multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements bas ed on H2O2 oxidation revealed a high signal level (1 muA1/cm2*mmol su crose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.