Flow amperometric uric acid biosensors based on different enzymatic mini-reactors: A comparative study of uricase immobilization

This is the first report of a novel strategy for uric acid (UA) determination via newly developed highly stable flow biosensors based on different enzymatic mini-reactors in combination with a four-electron reduction of the consumed oxygen. The design of the biosensors provides a spatial separation of the detection (working electrode) and biorecognition (uricase-based mini-reactor) part. To prepare the most efficient enzymatic mini-reactor, a comparative study focused on three different techniques of the covalent uricase immobilization at two kinds of mesoporous silica powders (SBA-15 and MCM-41) and the physical enzyme adsorption at four types of carbonaceous powders (glassy carbon, graphite, Starbon (R) 300, and Starbon (R) 800) has been performed. All investigations were carried out for two kinds of uricase: from Candida sp. (UOx(c)) and from Bacillus fastidiosus (UOx(b)) to estimate the influence of uricase source. The flow-through cell with tubular detector of silver solid amalgam was used for amperometric monitoring of four-electron oxygen reduction at the highly negative po-tential (-1100 mV vs. SCE). It was found that the developed UA biosensors responses are strongly dependent on the enzyme immobilization strategy. UOx(c) bounded to glassy carbon by physical adsorption has provided good results in terms of sensitivity, stability, and repeatability for UA determination. The biosensor with the mini-reactor based on the covalent immobilization of UOx(b) at glutaraldehyde activated -NH2 functionalized MCM-41 has shown extremely high stability (more than 1 year) and reusability (at least 600 measurements) The biosensor's practical applicability has been tested by successful determination of UA in human urine.