Development of a biparametric bioanalyser for creatinine and urea. Validation of the determination of biochemical parameters associated with hemodialysis

Abstract

The construction and evaluation of an automated urea and creatinine biparametric biosystem using flow injection analysis (FIA) are described. The biosystem uses enzyme reactions that hydrolyse urea and creatinine producing ammonium ions. The enzymes used were creatinine deiminase and urease, which are immobilized covalently in flow reactors. The reactor with creatinine deiminase has the enzyme immobilized on controlled-pore glass beads, whereas urease is immobilized on a nylon open tubular reactor. Detection is realised with a flow-through ammonium ion-selective electrode with an inner solid-state contact (graphite-epoxy composite). Ammonium ions are separated from alkali ion interferents through a gas-diffusion cell. The bioanalyser is fully automated using software and electronics developed ex profeso in our laboratories. The analyser was validated off-line by measuring urea and creatinine from discrete effluent samples from hemodialysis equipment. Results agreed with concurrent analyses realised using hospital laboratory methods. There were no significant differences between the two sets of results at the 95% confidence level. Finally, the biparametric bioanalyser was validated on-line by measuring creatinine and urea levels in artificial kidney effluents. These measurements were useful in the determination of key biochemical parameters of clinical interest such as the mass of urea and creatinine extracted from the patient as well as the initial concentration of creatinine and urea in blood plasma. When the results of the bioanalyser were compared with those yielded by the usual methods, they showed no significant differences at the 95% confidence level when determining the mass of the analytes extracted by the hemodialyser or when determining the urea concentration in blood plasma. However, when measuring the creatinine concentration in blood plasma using the developed bioanalyser, significant differences appeared.

Potentiometric Biosensor Based on the Recombinant Urease for the Control of the Urea Contrentation in Real Biological Samples

“…До методів першої групи умов-но можна віднести хімічні методи з колориме-тричною детекцією. До колориметричних ре-агентів відносяться диацетилмонооксим, фта-лальдегід, нафтилетилендіамін, хромотропова кислота [4]. Одним із найбільш популярних реактивів в сучасній клінічній діагностиці се-човини являється диацетилмонооксим [5].…”

Calibrationless Determination of Creatinine and Ammonia by Coulometric Flow Titration

“…[9] The developed coulometric flow titrator is working automatically with respect to the control of all active hardware components, the evaluation of the titration results, and the protocoll of the analytical results. To use the highly selective creatinine iminohydrolase for the creatinine determination in combination with potentiometric (13,29), fluorimetric (30) and photometric (10) procedures, two calibration graphs must be measured either for two different sensors or for determination channels. The difference between the sum signal and the ammonium signal provides a higher imprecision than the proposed method, which does not need to be calibrated or recalibrated.…”

Biosensors and flow-through system for the determination of creatinine in hemodialysate

“…The enzyme creatinine deiminase (creatinine iminohydrolase, EC 3.5.4.21), which was isolated by Szulmajster [19] in 1958, converts creatinine to N-methylhydantoin and ammonia. Both the detection of ammonia by potentiometric, ion-selective electrodes [21,22,23,24,25,26,27,28,29] and, in few cases, by amperometric electrodes [30,31] have been described. Some other approaches used the pH-shift associated with the ammonia production for the determination of creatinine by potentiometric devices [32,33,34] or by AC impedance measurements [35].…”