Integrated microchip methods for biological and environmental sample analysis

摘要

The introduction of the “microscale total analysis system (μTAS)” concept in theudlate 80’s triggered the evolution of microfluidic devices that cover a vast range ofudapplications. Automation, integration of multiple processes, and near zero deadudvolume for separation techniques are some benefits. Closing the gap between researchudand commercialization in a resource-limited environment is the main aim of thisudresearch.udThis project feeds into two main streams. The first is to integrate on-chip sampleudpreparation for biological applications, like therapeutic drug monitoring (TDM) anduddiagnostics, using nanojunctions created by controlled dielectric breakdown (ChaptersudOne - Five). The second part focuses on fast prototyping of microfluidic devices withudmultiple integrated functionalities using a consumer-based 3D-printer (Chapters Sixud& Seven). These two approaches were tailored to solve specific problems inherent toudeach sample type and application.udChapter One starts with a general introduction to the unique ion transportudphenomena associated with nanojunctions. Many factors act together to determineudwhether a certain ion will be blocked or preferentially transported through theudnanojunction. I developed controlled dielectric breakdown as a cost-effectiveudalternative to conventional nanolithography methods. Pore size control was achievedudby tuning the breakdown voltage in response to the feedback current measuredudthrough the formed nanojunction. Higher pre-set current limits result in larger poreudsize and hence the nanojunction will be permeable to larger molecules. I demonstrateudthe use of single nanojunction for simple extraction and the use of two nanojunctionsudacting together to form a size/mobility trap (SMT) for the simultaneous extraction,udconcentration, and desalting. In the SMT format, the second nanojunction wasudintroduced on the other side of the separation channel and offset by a 500 μm. Whileudthe role of the first junction remains the same, extraction, the second junction madeudwith smaller pore size blocks the analyte but permits smaller ions. The twoudnanojunctions work together as a trap that concentrates the injected plug andudsimultaneously desalt it. This approach is very flexible and can be tuned for differentudapplications as demonstrated in the following chapters.udChapter Two is an introduction to microfluidic systems used for analysis of smalludmolecules, especially pharmaceuticals, in biological samples. The methods wereudreviewed regarding the hardware and fluid handling processes. The chapter concludesudby discussing the requirements for point-of-care devices and decision making basedudon the results obtained. There are still many challenges and issues that need to beudaddressed before the wide spread use of these devices becomes a reality.udIn Chapter Three, the pore size of the nanojunctions was optimized for theudanalysis of small molecules in blood. First, a single nanojunction was integratedudbetween the sample compartment and the separation channel of the microfluidicuddevice. The nanojunction will permit the analyte of interest and small ions but blockudblood cells and other macromolecules. Isotachophoresis (ITP) and blue light emittinguddiodes (LEDs) were employed for the determination of small organic acids in bloodudwith indirect fluorescence detection. The acids chosen in this study were pyruvate,udlactate, and 3-hydroxy butyrate due to their significance as biomarkers for diabetesudand ketoacidosis. The single nanojunction allowed for the extraction of acids directlyudfrom whole blood within 60 s without interference from other macromolecules. Theudlimit of detection (LOD) was 12.5 mM and can be further improved by changing theudmicrochannel geometry near the detection point.udThe need for point-of-care devices for TDM was addressed through twoudexamples: quinine (an example for positively charged drug) and ampicillin (anudexample for negatively charged drug). Quinine is a counter-ion at the experimentaludconditions employed, which is also the case for many pharmaceuticals likeudantidepressants, and hence its transport is favoured through the negatively chargedudnanojunction. A single nanojunction was integrated between the sample compartmentudand the separation channel of the microfluidic device for extraction. Peak mode ITPudwas employed to concentrate the injected plug and achieve a linear response thatudcovers the therapeutically relevant range. Direct fluorescence detection was feasibleuddue to the native fluorescence of quinine.udFinally, SMTs were employed for TDM of ampicillin. This eliminated the needudto use other preconcentrating techniques like ITP. The electroosmotic flow (EOF) canudbe tuned in relation to the electrophoretic mobility by carefully selecting the buffers inudthe separation channel and the waste/desalting channel. This enables trapping of ionsudwithin a certain size/mobility range. Ampicillin is one of the front line antibioticsudused for managing sepsis, a critical condition with 30-50% mortality rate. The deviceudmay facilitate accurate dose adjustment and improve the survival of septic patients.udChapter Four is a general introduction to different electrokinetic methods forudbiological sample pretreatment with an interest in biopolymers like proteins andudDNA. A special attention was given to devices that incorporate nanojunctions as theyudexhibit unique behaviour and have already being demonstrated for DNAudmanipulation, protein concentration, and single molecule detection. Their use wasudhighlighted for sample pretreatment processes like purification, extraction, andudconcentration.udChapter Five demonstrates the use of the developed nanojunction methods forudbiopolymer applications. The single nanojunction format was employed toudconcentrate sodium dodecyl sulphate (SDS)-protein complexes from high ionicudstrength buffers. Enhancement factors up to 80-fold were achieved within 200 s. Theudabove mentioned SMTs were employed for the direct extraction of short single strandudDNA (ssDNA), 20 bases, from blood. As examined with small molecules, DNAudmolecules were extracted into the separation channel while cells and proteins wereudblocked. The second nanojunction trapped the DNA in the separation channel leadingudto simultaneous concentration and desalting. The LOD achieved for fluoresceinudlabelled DNA was 12.5 nM.udChapter Six is an introduction to 3D-printing. Different modes were discussedudand compared regarding their capabilities and suitability for microfluidic applications.udThis was followed by brief discussion of the recent portable systems reported forudenvironmental analysis and design requirements in comparison to biological samples.udChapter Seven explores the microfabrication capabilities of a desktop 3D-printerudbased on stereolithography (SL). The printer employed for this work is audcommercially available low-cost printer that photocures a clear resin that resemblesudpolymers commonly used for large-scale manufacturing. A wide range of microfluidicudprocesses was demonstrated like mixing, gradient generation, droplet extraction andudITP. Multiple functionalities were integrated into one device for nitrate analysis inudwater. The final design features standard addition at five levels to correct for theudmatrix effect, passive mixers to shorten reaction time, and detection at different pathudlengths to extend the linear response range and accommodate samples regardless ofudtheir initial concentration. Development and refining of the design was accelerated byudthe short turn-around times as 3D objects were printed at 2 cm/h speed, in heightudregardless of xy dimensions. The low price of the printer makes it a very accessibleudtool for small research laboratories.udIn Chapter Eight, I summarise the findings of this project and suggest futureuddirections. The outcomes of this research provide valuable solutions for multipleudprocess integration for on-site analysis. Whether it is dielectric breakdown forudcontrolled integration of nanojunctions or fast prototyping of complex devices, bothudapproaches are simple and low-cost. They are suitable for disposable devices and onsiteudanalysis and there is still a great opportunity for improvement in this area.