Integrated microchip methods for biological and environmental sample analysis

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.