title: Immobilization of enzymes into nanocavities for the improvement of biosensor stability.
abstract: Nanoporous materials with different pore sizes are evaluated as immobilization and stabilization matrices of proteins for the development of highly stable biosensors. It has been proven experimentally that confinement of proteins in cages with a diameter that is 2-6 times larger than their size increases considerably the stability of the biomolecules, as has been shown earlier by theoretical calculations. Porous silica beads with pore sizes of 10nm were utilized for the immobilization of the enzymes HRP and GOx with diameters in the order of 5 and 7 nm, respectively. The sensitivity of the corresponding biosensor systems was monitored for 70 h under continuous operation conditions (+600 mV) and it was found that the stabilization factor of GOx is 1.7 times higher compared to HRP. Also the stabilization efficiency of enzymes against leaching and inactivation in porous polymer beads with pore diameters of 10 and 30 nm was examined. The leaching rate of the enzyme AChE from the 30 nm polymer beads was found to be 1.1 times higher than that from the 10nm beads. At the same time the remaining activity of GOx biosensors after 5 days of continuous operation conditions (+600 mV) was found to be 2.1 times higher when the enzyme had been immobilized in the 10nm beads compared to the 30 nm beads. It is thus evident that the matching between the pore size of nanoporous materials and the molecular size of enzymes is essential for the development of biosensors with extended shelf and operational lifetimes.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, University of Crete, Voutes, P.O. Box 2208, 71003 Iraklion, Crete, Greece.
authors.forename: Vicky
authors.initials: V
authors.lastname: Vamvakaki
authors.forename: Nikos A
authors.initials: NA
authors.lastname: Chaniotakis
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005947:Glucose / Q000032:analysis*; D005949:Glucose Oxidase / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000032:analysis*; D008839:Microelectrodes; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004800:Enzymes, Immobilized; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D005947:Glucose