Laccase biosensors based on different enzyme immobilization strategies for phenolic compounds determination.
Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level. The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at -0.10 V versus Ag/AgCl. However, the performance of biosensors - evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability - depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity.
Departamento de Química Analítica y Análisis Instrumental, Facultad de Ciencias, Universidad Autónoma de Madrid (UAM), Campus Internacional de Excelencia UAM+CSIC, c/Francisco Tomás y Valiente No. 7, 28049 Madrid, Spain.
E
E
Casero
M D
MD
Petit-Domínguez
L
L
Vázquez
I
I
Ramírez-Asperilla
A M
AM
Parra-Alfambra
F
F
Pariente
E
E
Lorenzo
D000327:Adsorption; D015374:Biosensing Techniques; D002021:Buffers; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006873:Hydroquinones / Q000032:analysis*; D042845:Laccase / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry; D018625:Microscopy, Atomic Force; D017646:Organosilicon Compounds; D012821:Silanes / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D055454:Trametes / Q000737:chemistry* / Q000201:enzymology; D014867:Water
D002021:Buffers; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D006873:Hydroquinones; D017646:Organosilicon Compounds; D012821:Silanes; D013388:Succinimides; D014867:Water; D006046:Gold; C102833:(3-mercaptopropyl)trimethoxysilane; D042845:Laccase; C011240:dithiobis(succinimidylpropionate); C031927:hydroquinone
Amperometric biosensors; Atomic force microscopy; Enzyme immobilization; Phenolic compounds; Trametes versicolor laccase