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Integrated microchip methods for biological and environmental sample analysis

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Shallan, AI (2015) Integrated microchip methods for biological and environmental sample analysis. PhD thesis, University of Tasmania.

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Abstract

The introduction of the “microscale total analysis system (μTAS)” concept in the
late 80’s triggered the evolution of microfluidic devices that cover a vast range of
applications. Automation, integration of multiple processes, and near zero dead
volume for separation techniques are some benefits. Closing the gap between research
and commercialization in a resource-limited environment is the main aim of this
research.
This project feeds into two main streams. The first is to integrate on-chip sample
preparation for biological applications, like therapeutic drug monitoring (TDM) and
diagnostics, using nanojunctions created by controlled dielectric breakdown (Chapters
One - Five). The second part focuses on fast prototyping of microfluidic devices with
multiple integrated functionalities using a consumer-based 3D-printer (Chapters Six
& Seven). These two approaches were tailored to solve specific problems inherent to
each sample type and application.
Chapter One starts with a general introduction to the unique ion transport
phenomena associated with nanojunctions. Many factors act together to determine
whether a certain ion will be blocked or preferentially transported through the
nanojunction. I developed controlled dielectric breakdown as a cost-effective
alternative to conventional nanolithography methods. Pore size control was achieved
by tuning the breakdown voltage in response to the feedback current measured
through the formed nanojunction. Higher pre-set current limits result in larger pore
size and hence the nanojunction will be permeable to larger molecules. I demonstrate
the use of single nanojunction for simple extraction and the use of two nanojunctions
acting together to form a size/mobility trap (SMT) for the simultaneous extraction,
concentration, and desalting. In the SMT format, the second nanojunction was
introduced on the other side of the separation channel and offset by a 500 μm. While
the role of the first junction remains the same, extraction, the second junction made
with smaller pore size blocks the analyte but permits smaller ions. The two
nanojunctions work together as a trap that concentrates the injected plug and
simultaneously desalt it. This approach is very flexible and can be tuned for different
applications as demonstrated in the following chapters.
Chapter Two is an introduction to microfluidic systems used for analysis of small
molecules, especially pharmaceuticals, in biological samples. The methods were
reviewed regarding the hardware and fluid handling processes. The chapter concludes
by discussing the requirements for point-of-care devices and decision making based
on the results obtained. There are still many challenges and issues that need to be
addressed before the wide spread use of these devices becomes a reality.
In Chapter Three, the pore size of the nanojunctions was optimized for the
analysis of small molecules in blood. First, a single nanojunction was integrated
between the sample compartment and the separation channel of the microfluidic
device. The nanojunction will permit the analyte of interest and small ions but block
blood cells and other macromolecules. Isotachophoresis (ITP) and blue light emitting
diodes (LEDs) were employed for the determination of small organic acids in blood
with indirect fluorescence detection. The acids chosen in this study were pyruvate,
lactate, and 3-hydroxy butyrate due to their significance as biomarkers for diabetes
and ketoacidosis. The single nanojunction allowed for the extraction of acids directly
from whole blood within 60 s without interference from other macromolecules. The
limit of detection (LOD) was 12.5 mM and can be further improved by changing the
microchannel geometry near the detection point.
The need for point-of-care devices for TDM was addressed through two
examples: quinine (an example for positively charged drug) and ampicillin (an
example for negatively charged drug). Quinine is a counter-ion at the experimental
conditions employed, which is also the case for many pharmaceuticals like
antidepressants, and hence its transport is favoured through the negatively charged
nanojunction. A single nanojunction was integrated between the sample compartment
and the separation channel of the microfluidic device for extraction. Peak mode ITP
was employed to concentrate the injected plug and achieve a linear response that
covers the therapeutically relevant range. Direct fluorescence detection was feasible
due to the native fluorescence of quinine.
Finally, SMTs were employed for TDM of ampicillin. This eliminated the need
to use other preconcentrating techniques like ITP. The electroosmotic flow (EOF) can
be tuned in relation to the electrophoretic mobility by carefully selecting the buffers in
the separation channel and the waste/desalting channel. This enables trapping of ions
within a certain size/mobility range. Ampicillin is one of the front line antibiotics
used for managing sepsis, a critical condition with 30-50% mortality rate. The device
may facilitate accurate dose adjustment and improve the survival of septic patients.
Chapter Four is a general introduction to different electrokinetic methods for
biological sample pretreatment with an interest in biopolymers like proteins and
DNA. A special attention was given to devices that incorporate nanojunctions as they
exhibit unique behaviour and have already being demonstrated for DNA
manipulation, protein concentration, and single molecule detection. Their use was
highlighted for sample pretreatment processes like purification, extraction, and
concentration.
Chapter Five demonstrates the use of the developed nanojunction methods for
biopolymer applications. The single nanojunction format was employed to
concentrate sodium dodecyl sulphate (SDS)-protein complexes from high ionic
strength buffers. Enhancement factors up to 80-fold were achieved within 200 s. The
above mentioned SMTs were employed for the direct extraction of short single strand
DNA (ssDNA), 20 bases, from blood. As examined with small molecules, DNA
molecules were extracted into the separation channel while cells and proteins were
blocked. The second nanojunction trapped the DNA in the separation channel leading
to simultaneous concentration and desalting. The LOD achieved for fluorescein
labelled DNA was 12.5 nM.
Chapter Six is an introduction to 3D-printing. Different modes were discussed
and compared regarding their capabilities and suitability for microfluidic applications.
This was followed by brief discussion of the recent portable systems reported for
environmental analysis and design requirements in comparison to biological samples.
Chapter Seven explores the microfabrication capabilities of a desktop 3D-printer
based on stereolithography (SL). The printer employed for this work is a
commercially available low-cost printer that photocures a clear resin that resembles
polymers commonly used for large-scale manufacturing. A wide range of microfluidic
processes was demonstrated like mixing, gradient generation, droplet extraction and
ITP. Multiple functionalities were integrated into one device for nitrate analysis in
water. The final design features standard addition at five levels to correct for the
matrix effect, passive mixers to shorten reaction time, and detection at different path
lengths to extend the linear response range and accommodate samples regardless of
their initial concentration. Development and refining of the design was accelerated by
the short turn-around times as 3D objects were printed at 2 cm/h speed, in height
regardless of xy dimensions. The low price of the printer makes it a very accessible
tool for small research laboratories.
In Chapter Eight, I summarise the findings of this project and suggest future
directions. The outcomes of this research provide valuable solutions for multiple
process integration for on-site analysis. Whether it is dielectric breakdown for
controlled integration of nanojunctions or fast prototyping of complex devices, both
approaches are simple and low-cost. They are suitable for disposable devices and onsite
analysis and there is still a great opportunity for improvement in this area.

Item Type: Thesis (PhD)
Keywords: Microchip electrophoresis, 3D printing, portable point-of~care devices, dielectric breakdown
Copyright Information:

Copyright 2015 the Author

Additional Information:

Chapter 4 appears to be the equivalent of a post-print version of an article published as: Shallan, A., Guijt, R., Breadmore, M., 2014, Electrokinetics for sample preparation of biological molecules in biological samples using microfluidic systems, Bioanalysis, 6 (14), 1961-1974

Chapter 7 appears to be the equivalent of a post-print version of an article published as: Shallan, A., Smejkal, P., Corban, M., Guijt, R., Breadmore, M., 2014, Cost-effective three-dimensional printing of visibly transparent microchips within minutes., Analytical chemistry, 86, 3124-3130

Date Deposited: 16 Nov 2016 23:56
Last Modified: 09 Feb 2017 23:57
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