Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detection.
Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.
Rabinovich
Mikhail L
ML
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia. mirabi@mail.ru
Vasil'chenko
Liliya G
LG
Karapetyan
Karen N
KN
Shumakovich
Galina P
GP
Yershevich
Olga P
OP
Ludwig
Roland
R
Haltrich
Dietmar
D
Hadar
Yitzhak
Y
Kozlov
Yuri P
YP
Yaropolov
Alexander I
AI
D001685:Biological Factors / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002395:Catecholamines / Q000032:analysis*; D044602:Cellulases / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007202:Indicators and Reagents / Q000737:chemistry; D009097:Multienzyme Complexes / Q000737:chemistry; D012680:Sensitivity and Specificity
D001685:Biological Factors; D002395:Catecholamines; D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D002482:Cellulose; D044602:Cellulases