Enzymatic method of urea determination in LTCC microfluidic system based on absorption photometry

Abstract

In this paper a novel microanalytical system based on absorption photometry for urea determination in small-volume biological samples is described. The developed microsystem with built-in microreactor with urease immobilised on its surface was fabricated using low temperature co-fired ceramics (LTCC) technology. For detection of the ammonium ions – product of urea hydrolysis, the modified Berthelot’s reaction was applied and conditions of this reaction were optimised in a way to be suitable for analysis in microsystems and for particular application, with special regards to very small alternation of urea concentration in culture medium. The best results were obtained for reagent R1 composed of: 180 mM sodium salicylate, 15 mM sodium nitroprusside, 2 mM EDTA in phosphate buffer solution of pH 7 and reagent R2 containing 16.9 mM sodium hypochlorite in phosphate buffer solution of pH 12.Linear concentration range of urea detection with the use of the developed LTCC microfluidic system for the selected flow rate of 4 μl/min, was in the range up to 1 mM, and the calculated lower limit of detection – 0.002 mM. The developed microsystem enables urea determination in samples of post-culture medium and of cells lysates of minimised volume c.a. 1 μl. Preliminary studies related to determination of urea in real samples, performed using hepatic cells lysates and post-culture medium were successful. As reference methods, commercially available tests were applied. The determined urea concentration measured by means of the QuantiChrom™ and BioMaxima test and by the developed method based on the LTCC microanalytical system differs by c.a. 9% and 2% respectively. To the best of our knowledge, this is the first urea analysis microsystem for use in cells culture studies characterised by: the smallest volume of sample – 1 μl, long lifetime with stable response - reduced by 14%, through experimental time span of 30 days as well as suitability for quasi-monitoring of cells in vitro with sampling rate - 6 samples per hour.