A mechanized urease activity assay

Abstract

Two fully mechanized flow analysis systems for urease activity assays have been developed, characterized and compared. Both of them are based on almost the same compact system of solenoid micropumps and microvalves controlled and actuated by highly effective, low-power and economic Arduino microcontroller. For photometric detection of ammonia formed in the course of enzymatic hydrolysis of urea, the Berthelot method and the Nessler reaction have been examined. For both these detection schemes very simple dedicated optoelectronic flow-through detectors made of paired light emitting diodes have been developed. In both systems single enzyme assay lasting a few minutes allows determination of urease in activity range 0.02–5.3 U mL−1 with detection limit 0.02 U mL−1 and in 1.3–5.3 U mL−1 range with 0.75 U mL−1 detection limit for Nessler reaction and Berthelot method based systems, respectively. When compared with mechanized Berthelot method, the bioanalytical system based on Nessler reaction offers higher sensitivity, lower detection/determination limits, better selectivity and lower cost of the assay. It has been demonstrated that the developed bioanalytical flow systems could be useful for urease determination in complex biological matrix like plant extracts and media for microbial cultures as well as for inhibitive determination of heavy metals at sub-ppm levels.