Chemiluminescent choline biosensor using histidine-modified peroxidase immobilised on metal-chelate substituted beads and choline oxidase immobilised on anion-exchanger beads co-entrapped in a photocrosslinkable polymer.
A novel sensing layer design is presented based on the non-covalent immobilisation of enzymes on derivatized Sepharose beads subsequently entrapped in PVA-SbQ photopolymer. Two different modified Sepharose beads were used, IDA- and DEAE-Sepharose, for the immobilisation, respectively, of horseradish peroxidase (HRP) modified with histidine, and choline oxidase (Chx). The HRP-IDA-Sepharose-based sensing layer was used in a flow injection analysis chemiluminescent system as the basis of an H2O2 biosensor. It was shown that the pre-immobilisation on IDA-Sepharose beads enhanced the sensing layer stability and enabled the immobilisation of a larger amount of enzyme. A 1.8 mg charge of HRP-IDA-Sepharose beads in the sensing layer produced the most sensitive H2O2 biosensor. Such an analytical system exhibited very good performances, with a cycle time of 2 min and a detection limit of 15 pmol (detection ranging over four decades at least), and an unusual long operational stability of 200 measurements (CV, 3.5%). The HRP-IDA-Sepharose beads were then combined with Chx-DEAE-Sepharose. With this modified Sepharose-based biosensor the limit of detection for choline (S/N, 3) was equal to 0.5 pmol and the working range was 0.35 pmol-10 nmol. Moreover, the cycle time was only 2.5 min with the new sensing layer, and a long operational stability of 150 successive assays was found, with a variation coefficient of 2.6%.
Tsafack
V C
VC
Laboratoire de Génie Enzymatique, UPRESA CNRS 5013, Université Claude Bernard Lyon 1, Villeurbanne, France.
Marquette
C A
CA
Pizzolato
F
F
Blum
L J
LJ
D000429:Alcohol Oxidoreductases; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002794:Choline / Q000032:analysis*; D004800:Enzymes, Immobilized; D006639:Histidine; D006735:Horseradish Peroxidase; D006861:Hydrogen Peroxide / Q000032:analysis; D008163:Luminescent Measurements; D012685:Sepharose
D004800:Enzymes, Immobilized; D006639:Histidine; D012685:Sepharose; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D006735:Horseradish Peroxidase; D002794:Choline