Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals

Microfluidics for Biosensing and Diagnostics

Efforts to miniaturize sensing and diagnostic devices and to integrate multiple functions into one device have caused massive growth in the field of microfluidics and this integration is now recognized as an important feature of most new diagnostic approaches. These approaches have and continue to change the field of biosensing and diagnostics. In this Special Issue, we present a small collection of works describing microfluidics with applications in biosensing and diagnostics

The development of microfluidic based processes

Doctor of Science (DSc) thesis.Full version unavailable due to 3rd party copyright restrictions

Biosensing for the analysis of raw milk

Appropriate methods for monitoring raw milk in food sectors are required in order to prevent health-related issues from consuming milk or fermented dairy products such as cheese and yogurt. Conventional systems used for this purpose require sophisticated instruments, highly technical staff and several days to yield an estimated contaminant concentration profile. Currently, there is no technology available for fast and sensitive identification of unwanted substances that evidences several concentration levels in raw milk. As a contribution to the progress of innovative biochemical sensing systems, this thesis presents the design, development and construction of a prototype, which combines the expertise of sensitive immunoassay process with micro total analysis system (µTAS) to identify specific compounds encountered in raw milk. Since the concept of µTAS appeared, the development of microfluidic devices and their application have increased enormously. Microfluidic devices offer a highly efficient platform for analysis of biomolecules due to the capacity for manipulating small amounts of liquid quickly and with high precision. Particle size estimation, particle separation, cell collection, manipulation and cell detection are some of many functions which could be performed through microfluidic systems. Based on these advantages, microfluidic devices in combination with an associated immunoassay system were used in the prototype to concentrate contamination patterns or specific compounds encountered in raw milk. The NANODETECT prototype developed in this thesis should provide an economic, efficient and sensitive platform for multicomponent detection of specific substances in raw milk without requiring sophisticated instruments and trained staff. This thesis was performed for the European project NANODETECT (Development of nanosensors for the detection of quality parameters along the food chain) funded by European Commission within the 7th framework program

Development and evaluation of a calibration free exhaustive coulometric detection system for remote sensing.

Most quantitative analytical measurement techniques require calibration to correlate signal with the quantity of analyte. The purpose of this study was to employ exhaustive coulometry, an implementation of coulometric analysis in a stopped-flow, fixed-volume, thin-layer cell, to attain calibration-free measurements that would directly benefit intervention-free analysis systems designed for remote deployment. This technique capitalizes on the short diffusion lengths (\u3c 100 µm) to dramatically reduce the time for analysis (\u3c 30 sec). For this work, a thin-layer fluidic cell was designed in software, fabricated via CNC machining, and evaluated using Ferri/Ferrocyanide {Fe(CN)63-/4-} as a model analyte. The 2 µL fixed volume incorporated an oval, 8mm by 4 mm, thin-film gold electrode sensor with an integrated Ag|AgCl pseudo-reference electrode. The flow cell area matched the shape of the sensor, with a volume set by the thickness of a laser-cut silicone rubber gasket (~80 µm). A semi-permeable membrane isolated the working electrode and counter electrode chambers to prevent analyte diffusion. A miniaturized custom potentiostat was designed and built to measure reaction currents ranging from 10 mA to 0.1 nA. Software was developed to perform step voltammetry as well as cyclic voltammetry analysis for verifying electrode condition and optimal redox potential levels. Experimentally determined oxidation/reduction potentials of -100 mV and 400 mV, respectively, were applied to the working electrode for sample concentrations ranging from 50 µM to 10,000 µM. The current generated during the reactions was recorded and the total charge captured at each concentration was obtained by integrating the amperograms. When compared to the expected charge for a 2 µL sample, the total charge vs. concentration plots displayed a near perfect linearity over the full concentration range, and the expected charge (100 % converted) was reached within 20 seconds. The reaction currents ideally should have decayed to background levels, but exhibited constant offset values slightly larger than the background signal, a phenomenon assumed to be lingering residual flow from sample injection. After adding rigid tubing and external valves, the thin-layer cell was shown to remain within 6% of the theoretical charge after 200 seconds. Continued development of this system will offer the possibility of remote, calibration-free determinations of real-world analytes such mercury and lead