Creatinine biosensing by immobilizing creatininase, creatinase and sarcosine oxidase on nanohybrid interface

Commercial enzymes creatininase (CA), creatinase (CI) and sarcosine oxidase (SO) were co-immobilized on the gold nanoparticles/chitosan-multi walled carbon nanotubes (AuNPs/CHIT/c-MWCNT) composite film electrodeposited on the surface of glassy carbon (GC) electrode. A creatinine biosensor was fabricated using enzymes/AuNPs/CHIT/c-MWCNT modified GC electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was further characterized by various methods including chronoamperometric study, cyclic voltammetry study, scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). After polarization of working electrode at 0.2 Vs Ag/AgCl, an optimum response of 4 s at 25 °C in pH 7.5 was achieved. The electrocatalytic response was obtained by a linear dependence on creatinine concentration ranging from 0.5 to 1000 ?M with a detection limit of 0.5 ?M (S/N = 3). In this study, a good correlation (r = 0.98) with a standard colorimetric method was established. The biosensor was used 100 times over the period of 180 days and only the 50% loss of its initial activity at 4 °C was observed.