Portable microfluidic system for determination of urinary creatinine
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.
Section snippets
Chemicals, reagents and samples
All chemicals were of analytical reagent grade. Creatinine hydrochloride and picric acid were obtained from Sigma. Sodium hydroxide and chemicals for buffer preparation were supplied by Merck (Darmstadt, Germany). Poly(dimethylsiloxane) (PDMS, Sylgard 184) kits were purchased from Dow Corning (USA). Photoresist (SU-8 2100) and developer were supplied by MicroChem (USA). All solutions were prepared in Milli-Q water.
A working Jaffé reagent was prepared daily from a stock solution of 0.05 mol L−1
Reaction time optimisation
With the Jaffé reaction, a non-specific reaction derived from non-creatinine chromogen can occur late in the reaction and lead to a plateau graph. In addition, the slower reacting substances such as protein, glucose and ascorbic acid can substantially interfere by reducing alkaline picrate to picramate [30]. For this reason, the initial rate of colour development was investigated with real urine samples. Urine was diluted 50-fold before analysis. Results shown in Fig. 3 imply that the optimal
Conclusions
In this work, a microfluidic system for determination of creatinine has been developed. A universal absorbance detection was performed directly on-chip by utilisation of a portable miniature fibre optic spectrometer. The sufficient sensitivity is required for the proposed system because several folds dilutions of urine sample were achieved before assay, and the small injected sample was unavoidably diluted by the large volume of flow cell. By utilising a custom-made flow cell, these together
Acknowledgements
This research was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and the Thailand Graduate Institute of Science and Technology scholarship under the contracts no. TGIST 01-50-086, from the National Science and Technology Development Agency (NSTDA).
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