Simultaneous determination of renal function biomarkers in urine using microfluidic paper-based analytical device

Author(s)

Eduardo Luiz Rossini

• Document type: Master's Dissertation
• Press: Araraquara. 2016-02-24.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
• Defense date: 2016-02-05
• Advisor: Helena Redigolo Pezza

Abstract

Nitrogen is present in human organism mainly as a nucleic acid and proteins constituent. The catabolism of these substances forms nitrogen compounds known as nonproteinaceous. Among the many nonproteinaceous nitrogen compounds, uric acid and creatinine can be used as biomarkers of renal function in humans. Creatinine is derived from a non-enzymatic reaction of creatine dehydretion or dephosphorylation of phosphocreatine in the muscle contraction process; creatinine concentration is used as clinic marker to evaluate the renal function. Uric acid is formed from metabolism of purine bases and, by being a slightly soluble compound, its deposition in the joints of the extremities or other soft tissues creates a case known as gout. In this way, creatinine and uric acid are constantly dosed in urine laboratory exams. The methodologies used to determine these two compounds range from classic methods, to the used of HPLC, electrophoresis and in flow analysis, in other words, slightly portable techniques and, some of them, very expensive. Thus, this work proposes the development of microfluidic paper-based analytical devices (μPAD) to determinate creatinine and uric acid. Chromogenic reagents were selected for both analytes in study. Picric acid in alkaline medium was chose for creatinine determination and Fe3+ and 1,10-phenantroline were chose for uric acid. Experimental conditions were optimized for both reactions using 23 factorial design and a central composite design. The factores optimized were reagents concentration and time of heating. The analytical curve to uric acid were repetitive, with a line equation equal to A = 0,01651 + 0,00038764 CAU, with determination coefficient (R²) equal to 0,997, and limit of detection equal to 16,5 mg L-1 and limit of quantification equal to 54,9 mg L-1. Of the interferents tested, only ascorbic acid was shown to be interfering the reaction and may be removed by bubbling O2 in the sample. Calibration curve of creatinine shows itself repetitive with a linear equation equal to A = -0,02229 + 0,0004010 CCRN, with linear coefficient (R²) of 0,998; limits of detection and quantification were, respectively, 15,7 mg L-1 and 52,4 mg L-1; and none of the interferentes tested present interference during the determination. The proposed method was applied to three artificial urine samples and four natural urine samples. The obtained results were compared with a chromatographic methodology already described in literature. Statistic Student t-test shows that there is no significant difference between the results obtained by the proposed method and the comparative method. (AU)

• FAPESP's process: 14/17749-7 - Simultaneous determination of biomarkers of renal function in urine using microfluidic paper: based analytical device
• Grantee: Eduardo Luiz Rossini
• Support Opportunities: Scholarships in Brazil - Master