A bioanalytical protocol based on the enzymatic-spectrophotometric micro-method for determination of citrate with immobilized citrate lyase (CL) is described. In the design of this spectrophotometric bioanalytical system, L-malate dehydrogenase (L-MDH), oxaloacetate decarboxylase (OACD) and L-lactate dehydrogenase (L-LDH) were applied as a biological recognition system. Immobilized CL catalysed the conversion of citrate to oxaloacetate and acetate. In the presence of the enzymes L-MDH and L-LDH, oxaloacetate and its decarboxylation product pyruvate were reduced to L-malate and L-lactate, respectively. The concentration of citrate is stoichiometric to the concentration of nicotinamide adenine dinucleotide (NADH) formed in enzymatic reaction. NADH was determined by means of its light absorbance at 340 nm. The maximal difference in light absorbance was detected when CL had been immobilized on porous carbon. If CL had been immobilized on wool fibres or carbon rod the absorbance difference after 360 min was almost 2 times and 3.2 times lower, respectively, as compared with the results observed when CL had been immobilized on porous carbon. The method proposed here has several advantages if compared with the technique based on dissolved CL. Moreover, this method allows achieving a higher rate of CL-catalysed reaction and to reduce the duration of analysis versus the previously suggested methods. We expect that this method can be employed for determination of other analytes by applying the corresponding enzymes in the design of the bioanalytical system.

Keywords: bioanalytical chemistry, enzymatic detection, biosensors, citrate lyase, citric acid, citrate