Portable microfluidic system for determination of urinary creatinine

Abstract

A simple, low cost and portable microfluidic system based on a two-point alkaline picrate kinetic reaction has been developed for the determination of urinary creatinine. The creatinine reacts with picric acid under alkaline conditions, forming an orange-red colour, which is monitored on PDMS microchip using a portable miniature fibre optic spectrometer at 510 nm. A linear range was displayed from 0 to 40 mg L⁻¹ creatinine (r² = 0.997) with a detection limit of 3.3 mg L⁻¹ (S/N = 3). On-chip absorbance signals are reproducible, with relative standard deviations (RSDs) of 7.1%, when evaluated with 20 mg L⁻¹ creatinine (n = 10). The standard curves in which the intra-run CVs (4.7–6.8%) and inter-run CVs (7.9%) obtained were performed on three different days and exhibited good reproducibility. The method was highly correlated with the conventional spectrophotometric method when real urine samples were evaluated (r² = 0.948; n = 15).

Introduction

Creatinine is an end-product of creatine metabolism and well recognised as one of the most common analytes providing an assessment of renal function. Because creatinine is eliminated by the kidneys at a constant excretion rate of about 1.6–1.7% per day [1], increasing levels of creatinine in serum or decreasing levels in urine can be used to evaluate kidney function. In addition, its concentration is also employed as a correction factor for fluctuations in urine volume, which is useful for determination of the microalbumin/creatinine ratio [2], [3]. Due to its clinical importance, a sensitive and accurate assay for creatinine in blood and urine samples is in demand.

Numerous approaches for creatinine assay have been reported in the literature, such as enzymatic methods [4], [5], strip assay [6], HPLC [7], mass spectroscopy [8], capillary zone electrophoresis [9], [10], potentiometric biosensors [11], [12], [13], flow [14], [15] and sequential injection analysis systems [16]. However, the very first and most commonly used method for creatinine analysis is a colourimetric method based on the alkaline picrate of Jaffé reaction [17], [18]. Here, creatinine reacts with picric acid under alkaline conditions and subsequently forms an orange-red colour complex, which is monitored by spectrophotometry. Of several methods presently used in clinical laboratories, the methods based on the classical Jaffé reaction continue to be the method of choice in most laboratories [19], despite continuous attempts to overcome the interferences known to exist with this method. For example, without deproteinisation steps, a common practice for overcoming interference in the Jaffé reaction is the two-point kinetic assay, which possesses several advantages in terms of simplicity, rapidity, and ability to run with an automatic analyser [20], [21].

In recent research, there has been a growing interest in the concept of micro-total analysis systems (μTAS) or lab-on-a-chip systems. Microfluidic systems offer many advantages over conventional ones, including less reagent consumption, shorter analysis time, and portability. Several applications of microfluidic system have been documented in the area of clinical diagnostics for monitoring and diagnosis of various diseases [22] such as cardiovascular disease [23], diabetes [24], cancer [25], [26], and renal disease [27].

Poly(dimethylsiloxane) (PDMS) has been widely adopted as a polymer for microfluidics devices because of its simplicity, ease of fabrication and low cost [28]. The creatinine assay can be adapted to a PDMS flow-through microsystem as described by Grabowska et al. [27]. Absorbance detection is the most universal detection technique, but is classified as an unconventional detection method for microfluidic devices [29]. The general limitations are due to short optical path length and the difficulties in coupling the light into and out of microchannels [29].

This study was initiated to present a simple, low cost and sufficiently sensitive on-chip absorbance detection method for the determination of urinary creatinine based on a classical alkaline picrate Jaffé reaction. The optical path length of the microchip could be increased to a 1-cm path length similar to a standard one by means of custom-made flow cell, where the fibre optic cables were horizontally aligned in the opposite direction. The effects of various parameters affecting the assay sensitivity were studied and optimised. We also present validation results for real sample analysis in comparison with the conventional method.