Elsevier

Analytica Chimica Acta

Volume 803, 25 November 2013, Pages 135-142
Analytica Chimica Acta

Analytical isotachophoresis of lactate in human serum using dry film photoresist microfluidic chips compatible with a commercially available field-deployable instrument platform

https://doi.org/10.1016/j.aca.2013.01.046Get rights and content

Abstract

A dry film resist (DFR) chip compatible with the Agilent Bioanalyzer 2100 was designed and fabricated for use in the analysis of lactate in serum by chip isotachophoresis (ITP). The Agilent Bioanalyzer 2100 is a commercially available field deployable analytical instrument originally developed for the electrophoretic analysis of DNA, RNA and proteins. The DFR chip was designed for the ITP separation of lactate in human serum within 1 min and was made compatible with the Bioanalyzer after packaging in the plastic caddies normally used for the DNA chips. A 20-fold improvement in sensitivity was obtained for the DFR chips in comparison with the standard chips used in earlier work. The limit of detection and limit of quantification for lactate were 24 μM and 80 μM, respectively. This new approach enables the use of commercial platforms like the Agilent Bioanalyzer for new applications including the analysis of small molecules.

Highlights

► A dry film resist polymer chip designed for analysis of lactate in human serum. ► LIF/LEDIF indirect fluorescence detection used. ► Polymer ITP chip compatible with a field-deployable commercial platform. ► Full research flexibility using a commercial microfluidic separation platform.

Introduction

Miniaturization has been a major focus in the development of modern highly sensitive separation techniques, such as liquid chromatography [1], [2] and electrophoresis [3] in the last two decades. While the transition from classical analytical separation systems to a microfluidic format has been widely adapted in the research, only few microfluidic chip-based platforms appeared on the market as commercial devices [4], [5], [6]. Therefore the scarcity of commercially available portable or field deployable platforms, which are fully flexible for research, has been a major limitation and impediment to the use of portable and field deployable microfluidic chip based analytical instruments in solving real world analytical problems. This has led to the development of simple microfluidic devices that are compatible with existing and common laboratory instrumentation such as centrifuges and PCR thermocyclers [7].

One of the first commercial microfluidic systems on the market was the Agilent Bioanalyzer 2100, a platform for chip electrophoresis with fluorescence detection for analysis of DNA, RNA and proteins, and later for pressure driven flow cytometry. The general advantage of electrophoresis is that it enables high resolution separations to be easily achieved in a small instrument. The Bioanalyzer 2100 is a small footprint desktop instrument, and its dimensions and weight make it suitable as a portable and field deployable device [8].

In our previous research we examined the use of the Bioanalyzer as a general research-flexible microfluidic platform for chip capillary electrophoresis (chip-CE) [9], which opens the Bioanalyzer up for a more diverse range of applications than what it was originally designed for. Later, Lloyd et al. used the Bioanalyzer 2100 for the separation of amphetamines labeled with fluorescein isothiocyanate [10]. While these illustrate the possibility of performing simple zone electrophoretic separations in this platform, the separation performance was inferior to that obtained in capillaries due to the short channels and low field strengths that can be applied. To overcome this, we established the applicability of the Agilent Bioanalyzer 2100 for analytical isotachophoresis (ITP) with indirect fluorescence detection using the commercially available Agilent DNA chips and demonstrated this for the separation of carboxylic acids in soft drinks [11]. ITP is an ideal approach for miniaturization because it retains all of the separation power of zone electrophoresis while the self-sharpening boundaries counter-act diffusion allowing high resolution separations to be obtained at relatively low field strengths. In this work we further develop chip-ITP separations using a range of in-house designed Bioanalyzer-compatible polymer chips.

ITP has been shown to be a powerful separation technique with the ability to deal with high salinity and complex samples (serum) without difficult and time consuming sample pretreatment a further benefit for its use in portable instrumentation [12], [13], [14], [15], [16], [17], [18]. The technique was developed in the 1980s and 1990s and a detailed description of the theory, applications and instrumentation can be found in a monographs such as by Bocek et al. [19]. After the development of a protocol for the determination of benzoate in soft drinks using the DNA chips, we demonstrated the method could also be applied to serum samples by changing the injection conditions using a commercial DNA chip [20]. While this appears attractive from the point of using an existing commercial glass chip, the isotachopherogram from the commercial DNA chip did not display fully developed ITP steps, and therefore a new microfluidic ITP chip was in-house designed and fabricated from soda-lime glass, and demonstrated for separation of lactate in human serum [20]. The results although successful were far from practical with long separation times and poor limits of detection.

Here, we introduce a new design ITP microchip and show that compared to our previous work we can improve the repeatability and significantly reduce the analysis time. The new chip was designed to allow a vacuum injection and improve the repeatability of injection. The channels dimensions were varied to reduce the hydrodynamic flow between reservoirs, improve detection sensitivity [21], [22], [23] and also to allow the use of a higher current, and therefore faster isotachophoretic separation.

Despite superior properties of glass as microfluidic chip material including optical transparency and chemical stability, Lab on a Chip research has moved to the use of polymer devices mainly because of their lower manufacturing costs [24]. The material used for the fabrication of our newly designed microchips compatible with the Agilent Bioanalyzer 2100 was dry film photoresist (DFR). The Ordyl SY300 series DFR was previously introduced for fabrication of microfluidics chips at low cost using budget infrastructure, such as a LED light source and office laminator [25] and was used here to make microchips compatible with the Agilent Bioanalyzer 2100. The DFR ITP chips were used for the determination of lactate in human serum using a Bioanalyzer 2100 with indirect fluorescence detection and the results were confirmed using a CE method [26].

Section snippets

In-house dry-film resist chip fabrication

The ITP chip was designed using CAD freeware Draft Sight (Dassault Systemes, Velizy Villacoublay, France). The DFR chips were fabricated using the same equipment as described previously by Guijt et al. [25] and a slightly modified procedure as follows. A 70 mm × 50 mm × 1 mm piece of polycarbonate (PC) sheet (Polytech Plastics, WA, Australia) was used as a support upon which the DFR microchip was constructed. A layer of 30 μm thick DFR (Ordyl 330, Elga Europe, Italy) was laminated using an office

Design of DRF ITP chips

As explained above the microfabrication method uses DRF technology [25], where the selected thickness of dry film layer used for chip fabrication determines the depth of microchannels – in this work DRF layer of 30 μm was used. Other factors that have to be taken into account when designing the chip compatible with the Bioanalyzer are the positions of the reservoirs to maintain compatibility with array of 16 (4 × 4) platinum electrodes and the position of LIF detection.

The most important aspect of

Conclusions

A new ITP chip designed for determination of lactate in serum could be fabricated easily and at low-cost in Ordyl SY-300 series dry-film photoresist. Importantly, the DFR chips were shown to be fully compatible with the Agilent Bioanalyzer 2100, while combining a full research flexibility based on own in-house chip designs, with the advantage of a commercial compact instrumental platform with a sensitive LIF detection. Using 300 μm wide channels, twenty times more sample could be injected than

Acknowledgements

This work was supported by the Grant Agency of the Czech Republic (P301/11/2055 and P206/12/G014) and the Institutional Research Plan (UIACH 68081715). MCB would like to thank the Australian Research Council for funding and provision of a QEII Fellowship (DP0984745), and MM would like to acknowledge the Australian Research Council for funding and provision of a Future Fellowship (FT120100559).

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