A ferrocene-labeled high molecular weight coenzyme derivative (PEI-Fc-NAD) and a thermostable NAD-dependent l-lysine 6-dehydrogenase (LysDH) from thermophile Geobacillus stearothermophilus were used to fabricate a reagentless l-lysine sensor. Both LysDH and PEI-Fc-NAD were immobilized on the surface of a gold electrode by consecutive layer-by-layer adsorption (LBL) technique. By the simple LBL method, the reagentless l-lysine sensor, with co-immobilization of the mediator, coenzyme, and enzyme was obtained, which exhibited current response to llysine without the addition of native coenzyme to the analysis system. The amperometric response of the sensor was dependent on the applied potential, bilayer number of PEI-Fc-NAD/LysDH, and substrate concentration. A linear current response, proportional to l-lysine concentration in the range of 1 -120 mM was observed. The response of the sensor to l-lysine was decreased by 30% from the original activity after one month storage.
SummaryA glassy carbon electrode (GC) was modified by multi-walled carbon nanotubes (MWCNTs). The modified electrode showed a pair of redox peaks that resulted from the oxygen-containing functional groups on the nanotube surface. A recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH) from hyperthermophilic archaeon (Thermococcus profundus) was further immobilized by physical adsorption. The modified electrode (GC/MWCNTs/L-proDH) exhibited an electrocatalytic signal for L-proline compared to bare GC, GC/L-proDH and GC/MWCNTs electrodes, which suggested that the presence of MWCNTs efficiently enhances electron transfer between the active site of enzyme and electrode surface. The immobilized L-proDH showed a typical Michaelis–Menten catalytic response with lower apparent constant.
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