RESUMO
Amperometric biosensors based on gold planar or nanocomposite electrode containing multiwalled carbon nanotubes for determination of glycerol were developed. The biosensors were constructed by immobilization of a novel multienzyme cascade consisting of glycerol kinase/creatine kinase/creatinase/sarcosine oxidase/peroxidase between a chitosan "sandwich." A measuring buffer contained adenosine 5'-triphosphate (ATP), creatine phosphate, and an artificial electrochemical mediator ferrocyanide. The currents proportional to glycerol concentration were measured at working potential of -50 mV against Ag/AgCl reference electrode. The biosensors showed linearity over the ranges of 5-640 µM and 5-566 µM with detection limits of 1.96 and 2.24 µM and sensitivities of 0.80 and 0.81 nA µM(-1), respectively. Both types of biosensors had a response time of 70s. The biosensors demonstrated satisfactory operational stability (no loss of sensitivity after 90 consecutive measurements) and excellent storage stability (90% of the initial sensitivity after 15 months of storage at room temperature). The results obtained from measurements of wines correlated well with those obtained with an enzymatic-spectrophotometric assay. The presented multienzyme cascade can be used also for determination of triglycerides or various kinase substrates when glycerol kinase is replaced by other kinases.
