Evaluation of coloured materials in microfluidic flow-cells for chemiluminescence detection☆
Graphical abstract
Introduction
Chemiluminescence can provide highly sensitive detection of analytes of importance in clinical, agricultural and industrial applications, including some compounds that can be difficult to detect by other approaches [1], [2], [3], [4], [5], [6]. The instrumentation required to measure the emission of light is relatively simple, and amenable to portable analytical devices, but precise chemiluminescence measurements require reproducible mixing of two or more reactant solutions [7], [8]. Moreover, chemiluminescence reactions with fast kinetics place even greater demands on the efficiency of solution mixing [9]. For these reasons, chemiluminescence has been widely applied in flow-based analytical systems, such as flow injection analysis [10], [11], [12], high performance liquid chromatography [13], [14], [15], [16], and microfluidic devices [17], [18], [19], where the eluate/carrier stream containing the analyte(s) can be merged with the reagent(s) under highly controlled conditions within a simple flow manifold.
Chemiluminescence detection flow-cells have traditionally comprised a coil of glass or translucent polymer tubing that can be mounted flush against the circular window of a photomultiplier tube [7], [8], [15], [20]. Conventionally, a T- or Y-piece is used to merge the solutions prior to the central entrance of the coil. For fast chemiluminescence reactions, the distance between the points of confluence and detection must be very short, to ensure that the transient emission reaches its maximum intensity as the reacting mixture moves through the coil in front of the photodetector [9].
This simple and effective coiled-tubing design has remained the most popular approach for several decades [15], but in recent years, researchers have exploited engraving/machining [9], [21], [22], [23], [24], [25], [26], 3D printing [27], [28] and other microfabrication techniques [29], [30], [31], [32], [33] to not only create flow-cells with more reproducible construction, greater mixing efficiency, and superior transfer of light to the photodetector, but also integrate chemiluminescence detection zones into microfluidic chips. These approaches enable a wide range of materials to be utilised, but most flow-cells and microfluidic chips for chemiluminescence detection are constructed from transparent polymers [22], [23], [30], [31], [32], [34], [35], [36], sometimes with a reflective backing to direct more light towards the photodetector [25], [31], [32], [34]. Previous experiments from our research group have shown that flow-cells constructed by machining channels into an opaque white polymer (and sealed with a thin transparent film) transferred light of approximately 4-fold greater intensity to the photodetector than a transparent polymer flow-cell of the same design that was placed on a mirror, due to the light lost through the sides of the clear polymer [25]. Similarly, Stieg and Nieman reported that the chemiluminescence intensity from a flow-cell constructed by stacking white polymer plates decreased by up to two-fold when the back plate was replaced by a dark-brown material, and that the change was most prominent with lower channel depths [37]. These studies indicate that reflective materials for all surfaces other than that facing the photodetector may be ideal [25], [37], but little is known of the extent that channel walls fabricated from coloured materials will influence the transfer of light to the photodetector. We anticipate that this will become increasingly important as chemiluminescence detection zones are integrated with other analytical operations within complex microfluidic devices [17], [18], [19]. Moreover, the smaller volumes of sample and reagent within typical microfluidic devices compared to conventional flow-analysis systems reduce the chemiluminescence intensity considerably [25], placing even greater demands on detector efficiency.
Herein, we describe a comparison of flow-cells fabricated from five different coloured polymer sheets (clear, white, black, red, blue), using two flow-cell designs, four different chemiluminescence reactions to provide a range of different emission colours, and two modes of photodetection. Considering the anticipated areas of application, we selected channel widths and depths typical of microfluidic devices, and thus smaller than those considered optimal for conventional flow injection analysis or high performance liquid chromatography [15].
Section snippets
Chemicals
Reagents were prepared daily using Milli-Q deionised water unless otherwise stated. Morphine was supplied by SunPharma (Port Fairy, VIC, Australia) and prepared as a 1 mM stock solution and diluted as required. Potassium permanganate was purchased from ChemSupply (Gillman, SA, Australia). Sulfuric acid was obtained from Merck (Bayswater, VIC, Australia). Ofloxacin, cerium(IV) sulfate, luminol, sodium thiosulfate and sodium polyphosphate were purchased from Sigma-Aldrich (Castle Hill, NSW,
Selection of materials and chemiluminescence reactions
In addition to visibly transparent and white flow-cell materials similar to those of previous investigations [24], [27], in this study we included polymers that were black, red or blue in appearance (Fig. 1f–h) to examine the extent that these materials would affect the intensity of light from the chemiluminescence reactions that reached the photodetector.
The light emitted from any particular chemiluminescence system normally emanates from only one luminophore and therefore no additional
Conclusions
The direct transfer of light from the chemiluminescence reaction within the flow-cell channel to the photodetector, through the thin transparent film used to seal the channel (i.e., pathway 1), is independent of the material into which the channel is machined. Therefore, the large variation in chemiluminescence intensity measured using the different flow-cells shows the importance of pathways 2 and 3. The relatively low responses from the clear and black flow-cells indicate that pathway 1 makes
Acknowledgements
The authors thank Deakin University and the Australian Research Council (DP140100439) for funding this research.
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Cited by (0)
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Selected paper from the 23rd Annual RACI R&D Topics Conference in Analytical and Environmental Chemistry (R&D Topics 2015), 6th-9th December, at the University of Melbourne, Parkville Campus, Australia.