Development of oxalate sensor using paper-based analytical device

Abstract

Herein, oxalate sensors were developed using paper-based analytical devices (PADs) coupled with electrochemical and colorimetric detection. For electrochemical paper-based analytical device (ePAD), the amount of oxalate in the solution was determined through the measurement of hydrogen peroxide (H2O2) produced from the enzymatic reaction between oxalate and oxalate oxidase (OxOx) obtained from an oxalate test kit. A carbon paste electrode (CPE) as a working electrode (WE) was modified with silver nanoparticles (AgNPs) and multi-walled carbon nanotubes (MWCNTs) to enhance electrochemical signal of H2O2. A CPE was composed of 50% w/w oil phase (mineral oil and PDMS) and 50% w/w graphite powder. The CPE was modified with 400 µL of 10,000 ppm AgNPs and 1% w/w MWCNTs. The AgNPs-MWCNTs-CPE was used to measure H2O2 using amperometric detection with an applied potential of -0.6 V. The linear ranges of the method for determination of H2O2 were in the ranges of 0.05-10 and 10-1,000 mM. From the first linear range, limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.02 and 0.08 mM, respectively. For detection of oxalate using the AgNPs-MWCNTs-CPE, oxalate solution was reacted with OxOx in a reagent B consisting of OxOx and horseradish peroxidase (HRP). However, electrochemical signal was not observed, which could be because the produced H2O2 was reacted with HRP from the reagent B. Therefore, the electrochemical determination of oxalate using ePADs was not successful.

In addition, a PAD was developed with colorimetric detection as an oxalate sensor using the same enzymatic reaction. The produced H2O2 was reacted with 3-(Dimethylamino) benzoic acid (DMAB) and 3-Methyl-2-benzothiazolinine (MBTH) to produce an indamine dye which is a blue color. The color intensity corresponded to the amount of oxalate in the solution. Interfering effect of AA contained in urine was minimized by adding a masking reagent (CuSO4, H3BO3 and NaOH) to react with AA for 30 min. The optimized amounts of OxOx, HRP, DMAB and MBTH were 3 x 10-3 u L-1, 1 x 10-4 u L-1, 2.4 mM and 0.0176 mM, respectively, which were used to react with oxalate for 10 min before measuring color intensity. The linearity of the method was in the range of 5-50 ppm. LOD and LOQ were found to be 3.38 and 11.27 ppm, respectively. The developed oxalate sensor based on PADs with colorimetric detection was successful to determine oxalate in urine with high accuracy and precision, in which %recovery of spiked oxalate in urine was found in the range of 80.7-110.0% and %RSD values of the amounts of oxalate from intra and inter-day measurements were lower than 5%. Therefore, the developed oxalate sensor based on PADs will hold a great promise to be a simple, low-sample and reagent volume, reliable and portable tool for determination of oxalate, especially for on-site measurements.