Whole-Smejkal-thesis-2013.pdf (6.97 MB)
Microfluidics in bioanalytical instrumentation
thesisposted on 2023-05-26, 00:47 authored by Smejkal, P
Portable and field deployable analytical instruments are attractive in many fields, including medical diagnostics where point-of-care and on-site diagnostics systems capable of providing rapid quantitative results have the potential to improve the productivity and quality of medical care. A major limitation and impediment to the usage of portable and field deployable microfluidic chip based analytical instruments in solving real world analytical problems has been the scarcity of commercially available portable or field deployable platforms, which are fully flexible for research. The bench-top analytical instrument , the Agilent Bioanalyzer 2100 used in this research is a microfluidic chip-based platform with fluorescence detection system, available on the market since 1999. Originally, this instrument was capable of electrophoretic analysis of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), with user-tailored application solutions including chips, reagents and pre-developed methods. More applications, specifically electrophoretic analysis of proteins and flow cytometry, were introduced later. The commercial success of this instrument was achieved thanks to an on-a-chip platform which enables fast analysis (separation time around a minute or even less) with minimal sample consumption (microlitres). In this research we built on our long-standing research collaboration with Agilent Technologies, who provided us with access to the Bioanalyzer 'script editor' so that we could develop and implement our own methods, applicable in principle for any chip and analysis. Chapter 1 offers an introductory overview of miniaturised analysis followed by a comprehensive overview focused on ITP on a microfluidic chip techniques. Chapter 2 gives a brief overview of commercially available microfluidic chip based analytical platforms. This is followed by the first experiments aimed at developing a new application for the commercially available microfluidic chip based electrophoretic system, the Agilent Bioanalyzer 2100. The Bioanalyzer was used in this work as fully flexible research tool, which was made possible by using an open software platform in assay developer mode. The new application was investigated for the CE separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) fluorescently labelled oligosaccharides. Their separation was successfully implemented with the analysis completed using the commercially available Agilent DNA chip in under a minute, representing an improvement in the speed of analysis compared to CE by more than an order of magnitude. This result demonstrates that the commercial chips, specifically DNA chips, and the Bioanalyzer, have the capacity for a wider spectrum of applications. To compare with classical CE, the resulting electropherograms were obtained faster but the final resolution was poorer, a result that initiated further investigations aimed at exploring different electrophoretic methods. In Chapter 3, isotachophoresis of carboxylic acids on a DNA chip with electrokinetic injection of a sample and indirect fluorescence detection was investigated. The indirect fluorescence detection was carried out by using fluorescent dye rhodamine 6G (R6G) as a counter ion present in leading electrolyte. Limits of detection (LOD) in millimolar range were obtained for different model analyte acids (oxalate, pyruvate, fumarate, malate, mandelate, 2-hydroxyisobutyrate, succinate, acetate). The method was later used for quantification of benzoate in a variety of soft drinks, which was the first documented use of chip ITP with indirect fluorescence detection for the separation of real samples. In this chapter we fully appreciated the properties of ITP, as no sample preparation, except degassing and sample dilution was necessary prior to the analysis. In Chapter 4, the method developed in the previous chapter, using a hydrodynamic injection of sample, was tested for the separation of lactate in human serum but failed due to the design of the DNA chip, which showed insufficient compatibility with ITP. A twofold improvement of the method sensitivity for lactate in human serum was achieved with in-house designed and microfabricated ITP glass chips. The obtained results still showed significant room for improvement for higher sensitivity and shorter analysis time. In Chapter 5, a twentyfold improvement of sensitivity, if compared with ITP separation of lactate from human serum on a DNA chip, was achieved when specifically designed ITP chips, microfabricated by dry film resist technology were used. Another advantage of the chips introduced in this chapter was smaller resistivity of the separation channel that was used to implement higher separation current and speed up the total analysis time. An under-a-minute separation and quantification of lactate (calculated LOD of lactate was 42 ˜í¬¿M) was achieved in human serum sample. Except for serum dilution, no sample pretreatment was required prior to the analysis. The research results presented here illustrate that the general objective of demonstrating the feasibility of combining the positives of a commercial, field-deployable, chip analytical platform with full research flexibility in designing new chips and methods, has been achieved. It has been shown that from a practical viewpoint, the Bioanalyzer 2100 is convenient to use, not just for different analytes but also for different electrophoretic methods than those for which it was originally designed. The CE separations and analysis of APTS labelled oligosaccharides from human plasma, ITP separations, and analysis of benzoate in soft drinks and of lactate in human serum shown in this work are indicative of the broader application capability and potential of the Bioanalyzer as a field deployable research instrument. Aims The general aim of this research addressed the research question of the possibility of implementing of full research flexibility to a commercially available microfluidic chip based electrophoretic platform. From a practical analytical point of view, this research aimed at a significant broadening of the applicability of the commercially available desktop analytical instrument, the Agilent Bioanalyzer 2100, while implementing a number of strategies to maximally exploit research flexibility. The specific aims of this research were to investigate and implement the above approach, step by step, as follows: ‚Äö Starting from generic chip-CE applicable to, in principle, any chip-CE separation and analysis using the commercial DNA chips, ‚Äö Progressing to ITP separations, using the commercial DNA chips, ‚Äö Following with adding further research flexibility by following onto investigations with in-house designed chips, first fabricated in glass, then using a less cost polymer chips fabricated from dry film photoresist.
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