Towards rapid 3D direct manufacture of biomechanical microstructures

The field of stereolithography has developed rapidly over the last 20 years, and commercially available systems currently have sufficient resolution for use in microengineering applications. However, they have not as yet been fully exploited in this field. This thesis investigates the possible microengineering applications of microstereolithography systems, specifically in the areas of active microfluidic devices and microneedles. The fields of micropumps and microvalves, stereolithography and microneedles are reviewed, and a variety of test builds were fabricated using the EnvisionTEC Perfactory Mini Multi-Lens stereolithography system in order to define its capabilities. A number of microneedle geometries were considered. This number was narrowed down using finite element modelling, before another simulation was used to optimise these structures. 9 × 9 arrays of 400 μm tall, 300 μm base diameter microneedles were subjected to mechanical testing. Per needle failure forces of 0.263 and 0.243 N were recorded for the selected geometries, stepped cone and inverted trumpet. The 90 μm needle tips were subjected to between 30 and 32 MPa of pressure at their failure point - more than 10 times the required pressure to puncture average human skin. A range of monolithic micropumps were produced with integrated 4 mm diameter single-layer 70 μm-thick membranes used as the basis for a reciprocating displacement operating principle. The membranes were tested using an oscillating pneumatic actuation, and were found reliable (>1,000,000 cycles) up to 2.0 PSIG. Pneumatic single-membrane nozzle/diffuser rectified devices produced flow rates of up to 1,000 μl/min with backpressures of up to 375 Pa. Another device rectified using active membrane valves was found to self-prime, and produced backpressures of up to 4.9 kPa. These devices and structures show great promise for inclusion in complex, fully integrated and active microfluidic systems fabricated using microstereolithography alone, with implications for both cost of manufacture and lead time

Towards rapid 3D direct manufacture of biomechanical microstructures

The field of stereolithography has developed rapidly over the last 20 years, and commercially available systems currently have sufficient resolution for use in microengineering applications. However, they have not as yet been fully exploited in this field. This thesis investigates the possible microengineering applications of microstereolithography systems, specifically in the areas of active microfluidic devices and microneedles. The fields of micropumps and microvalves, stereolithography and microneedles are reviewed, and a variety of test builds were fabricated using the EnvisionTEC Perfactory Mini Multi-Lens stereolithography system in order to define its capabilities. A number of microneedle geometries were considered. This number was narrowed down using finite element modelling, before another simulation was used to optimise these structures. 9 × 9 arrays of 400 μm tall, 300 μm base diameter microneedles were subjected to mechanical testing. Per needle failure forces of 0.263 and 0.243 N were recorded for the selected geometries, stepped cone and inverted trumpet. The 90 μm needle tips were subjected to between 30 and 32 MPa of pressure at their failure point - more than 10 times the required pressure to puncture average human skin. A range of monolithic micropumps were produced with integrated 4 mm diameter single-layer 70 μm-thick membranes used as the basis for a reciprocating displacement operating principle. The membranes were tested using an oscillating pneumatic actuation, and were found reliable (>1,000,000 cycles) up to 2.0 PSIG. Pneumatic single-membrane nozzle/diffuser rectified devices produced flow rates of up to 1,000 μl/min with backpressures of up to 375 Pa. Another device rectified using active membrane valves was found to self-prime, and produced backpressures of up to 4.9 kPa. These devices and structures show great promise for inclusion in complex, fully integrated and active microfluidic systems fabricated using microstereolithography alone, with implications for both cost of manufacture and lead time.EThOS - Electronic Theses Online ServiceEngineering and Physical Sciences Research Council (EPSRC)GBUnited Kingdo

Slotted photonic crystal biosensors

Optical biosensors are increasingly being considered for lab-on-a-chip applications due to their benefits such as small size, biocompatibility, passive behaviour and lack of the need for fluorescent labels. The light guiding mechanisms used by many of them result in poor overlap of the optical field with the target molecules, reducing the maximum sensitivity achievable. This thesis presents a new platform for optical biosensors, namely slotted photonic crystals, which engender higher sensitivities due to their ability to confine, spatially and temporally, the peak of optical mode within the analyte itself. Loss measurements showed values comparable to standard photonic crystals, confirming their ability to be used in real devices. A novel resonant coupler was designed, simulated, and experimentally tested, and was found to perform better than other solutions within the literature. Combining with cavities, microfluidics and biological functionalization allowed proof-of-principle demonstrations of protein binding to be carried out. High sensitivities were observed in smaller structures than most competing devices in the literature. Initial tests with cellular material for real applications was also performed, and shown to be of promise. In addition, groundwork to make an integrated device that includes the spectrometer function was also carried out showing that slotted photonic crystals themselves can be used for on-chip wavelength specific filtering and spectroscopy, whilst gas-free microvalves for automation were also developed. This body of work presents slotted photonic crystals as a realistic platform for complete on-chip biosensing; addressing key design, performance and application issues, whilst also opening up exciting new ideas for future study

Thermally driven Knudsen gas pump enhanced with a thermoelectric material.

The thesis focuses on improving the flowrate of the Knudsen gas pump. The Knudsen pump uses thermal transpiration as the driving mechanism to pump gas. It is a motionless gas pump as the pump does not require any moving actuators for pumping. The thermally driven gas flow is accomplished in the molecular or transitional gas flow regime. The advantage of this pump is that without any moving parts it avoids friction losses and stiction problems which devices in micro scale are prone to suffering due to scaling issues. Thus, this pump is highly robust and reliable. Knudsen pumps in the past have suffered from the drawback of low flowrates and inability to operate at atmospheric pressure. In the early days lack of micromachining technologies limited minimum channel size which had to be operated at lower than atmospheric pressure to achieve free molecular flow. Various designs have been implemented with an impetus on increasing the flowrate of the pump. The key to this pump is establishing a temperature difference along the length of the channel. A higher temperature difference over a shorter channel length makes the pump more efficient. Pump channels have been made out of various materials like silicon, glass and polymer. The silicon microfabricated single channel conventional design pump suffered from the high thermal conductivity of silicon, which limited the thermal gradient that could be achieved. Silicon was replaced by glass, which has a lower thermal conductivity. The glass micro fluidic pump could pump water in reservoirs but at a slow rate. Renewable forms of Knudsen pump were also made by using nanoporous silica colloidal crystals which are robust and could use solar energy and body heat to create a temperature difference and achieve pumping. The pump powered by body heat produced a maximum pressure differential of 1.5 kPa. However, the use of these pumps is restricted to certain applications due to slow pumping. The polymer material, made of mixed cellulose ester, has a very low thermal conductivity, which aids in maintaining a higher temperature difference between the ends of a channel to achieve a higher flowrate. The polymer material used is in the form of a nanoporous template which has numerous pores each of which acts as a pump and thus the pump\u27s conductance to gas flow is also increased which makes it faster. The pore sizes range from 25 nm to 1200 nm. It has been proven that a smaller channel diameter pump is more efficient. Efficiency decreases as the channel size approaches viscous flow regime. The initial design used a resistive heater to actively heat one end of the channel and a heat sink was used to passively cool the other end of the channel. This design was ineffective in achieving a significant temperature difference for a decent flowrate with the materials like silicon and glass. The conventional Knudsen pump design using a porous polymer matrix as channel material attained a normalized maximum no load flowrate of 135 µL/min-cm2 at 3.81 Watts of input power. This number is low compared to other micropumps. This led to the use of a thermoelectric material, which could actively heat and cool the pump channel ends and provide a much higher temperature difference over the same channel length as compared to the conventional Knudsen pumps which used only active heating of the channel\u27s hot side. The thermoelectric strategy also eliminates the need for a heat sink in the pump. This transforms the design to bi-directional modes of operation. The first design using thermoelectrics is a lateral design in which the pump channels closer to the thermoelectric element developed a higher temperature difference across them compared to the channels away from the thermoelectric element. Thus, the thermoelectric energy was underutilized. Changing to the radial design made the pump more efficient compared to the lateral design since the thermoelectric energy was uniformly distributed on all the pump channels. The radial design also reduced air gap resistances and minimized energy losses which enhanced the output for the same input power. At an input power of 4.18 Watts it achieved a normalized no load flowrate of 408 µL/min-cm2. It also recorded a maximum normalized flowrate of 1.5 mL/min-cm2 while moving a drop of water which to date is the maximum flowrate reported by any Knudsen pump. A theoretical model has been developed to compute the pump\u27s efficiency based on the flowrate and pressure difference obtained by the pump. The efficiency of the radial design pump with the thermoelectric is higher when compared to a conventional pump using a resistive heater whose channels are also made from the same material as that of the thermoelectric pump

Disposable Lab-on-Chip Systems for Biotechnological Screening

The main goal of this work was to develop different disposable Lab-on-Chip (LoC) systems for the application of biotechnological screening e.g. for bioprocess development through microorganisms or drug testing with human cell lines. Nowadays, microfluidics represents a highly promising field for the fabrication of microtools, as the increasing demand for screening data are difficult to meet with current platforms. This is mainly due to time and cost aspects as well as a limited amount of newly developed drugs. The ideal microfluidic platform for biotechnological screening should include three different groups of elements: (i) microbioreactors (MBR) in which cultivation takes place; (ii) auxiliary microfluidic systems (for transportation, filtration or mixing), and (iii) enzymatic biosensors for onchip analysis of substrate concentrations which are difficult to measure offline due to small available sample volumes. Within the scope of this work, various horizontally and vertically positioned MBR designs (resembling plug flow reactors, micro stir tanks or bubble columns) were developed, fabricated and successfully applied to the screening of different biological expression systems, such as yeast cells (S. cerevisiae), fungal spores (A. ochraceus) and primary human endothelial cells. Different integrated functional structures based on geometrical, optical or electrical elements allowed for online monitoring of various physical, chemical and biological process parameters during cultivation. In terms of the second group, passive and active microvalves, PZTand pneumatically actuated micropumps, passive filtration and mixing elements were produced. The third group comprised different types of enzymatic biosensors based on a hybrid detection principle (electrochemical-optical) and on different types of enzymatic responses. In general, the unique LoC setup (patterned element made of poly(dimethylsiloxane) and bonded to a glass substrate) allows an easy integration of systems into one monolithic LoC platform which are usually better suited for technically mature systems. Modular systems are advantageous for prototyping of new microfluidic applications. Therfore, an LoC construction kit was developed that offers a user friendly, standardized modular platform.Im Rahmen der Dissertation wurden verschiedene Einweg-Lab-on-Chip Systeme entwickelt, die beispielsweise bei biotechnologischen Parameterstudien von Mikroorganismen zur Bioprozesssteigerung oder von humanen Zelllinien zum Wirkstoffscreening Anwendung finden. Die Mikrofluidik ist ein vielversprechendes Forschungsgebiet für die Herstellung von kostengünstigen Mikrochips, womit der steigende Bedarf für Screening-Daten aufgrund von Vorteilen wie Zeit- und Kostenreduzierung erfüllt werden kann. Eine ideale mikrofluidische Plattform zum biotechnologischen Screenen sollte aus folgenden Gruppen bestehen: (i) dem Mikrobioreaktor zur Kultivierung, (ii) mikrofluidische Komponenten zum Transportieren, Filtrieren und Mischen von Suspensionen, und (iii) einem enzymatischen Biosensor für die on-Chip Analyse von Substratkonzentrationen. Innerhalb der Arbeit wurden diverse horizontal und vertikal positionierte Mikrobioreaktoren entwickelt, hergestellt und erfolgreich zum Screenen von unterschiedlichen biologischen Expressionssystemen (wie S. cerevisiae, A. ochraceus und humane Endothelzellen) angewendet. Die Integration von geometrischen, optischen und elektrischen Funktionselementen erlaubte eine online Überwachung von verschiedenen physikalischen, chemischen und biologischen Prozessparametern während der Kultivierung. Im Bereich der Gruppe (ii) wurden passive und aktive Mikoventile, PZT- und pneumatisch aktuierte Mikropumpen, Filtrations- und Mischkomponenten hergestellt und charakterisiert. Gruppe (iii) umfasste die Entwicklung eines enzymatischen Biosensors mit hybridem (elektrochemisch-optisch) Messumformer. Der einheitliche Chipaufbau aller Lab-on-Chip Systeme – bestehend aus einer Kombination von strukturiertem Polydimethylsiloxan und Glas – erlaubt das monolithische und modulare Zusammenschalten der Einzelsysteme zu der gewünschten Plattform. Da für erste Prototypen eine modulare Verschaltung zu bevorzugen ist, wurde ein Baukastensystem entwickelt, welches eine standardisierte und benutzerfreundliche Plattform für flexible Versuchsaufbauten bietet

Implementation of SELEX technique on Lab-on-Chip systems

The thesis presents the design and the experimental development of a compact and high sensitive Lab-on-Chip (LoC) system suitable for the implementation of SELEX technique. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a combinatorial technique used in molecular biology to produce copies of the same nucleotide and to select a strand of DNA (aptamer) specific for a target molecule. The proposed Lab-On-Chip system includes the following functional units: an amplification module based on the PCR technique; a separation module able to obtain a single strand DNA from a double strand DNA and a selection module for the specific selection of the aptamer. These functionalities are implemented combining microfluidic components (micro-channels and micro-valves), electronic devices (amorphous silicon photosensors, thin film heaters, temperature sensors) and bioanalytical procedure

Development and application of microtechnologies in the design and fabrication of cell culture biomimetic systems

“Lab-On-a-chip” systems have proved to be a promising tool in the field of biology. Currently, cell culture is performed massively on Petri dishes, which have traditionally been used in cell culture laboratories and tissue engineering. However, having proved to be a widely used tool until now, the scientific community has largely described the lack of correlation between the results obtained in the laboratory and the clinical results. This lack of connection between what has been studied in the laboratories and what has been observed in the clinic has led to the search for more advanced alternative tools that allow results to be obtained closer to reality. Thus, the use of microtechnologies in the field of biomedical engineering, presents itself as the perfect tool as an alternative to obsolete traditional media. Thanks to the low volumes of liquid it presents for its use, it also makes it an essential technology for the testing of drugs, new compounds and materials. By being able to more accurately reproduce the biomimetic environment of cell cultures and tissues, they make this technique fundamental as an intermediate step between basic in vitro laboratory tests and preclinical animal tests, resulting from this way in the best alternative for the reduction of both the use of animal models, as in times and costs. For a biomimetic system to be as such, it also needs another series of complementary devices for its better functioning. Micro-valves, micro pumps, flow sensors, O2 sensors, pH, CO2 are fundamental for the correct functioning andsophistication of biomimetic systems. This complexity, on the other hand, is often not perceived by the user since the miniaturization of all these components makes “Lab-On-a-Chip” systems smaller every day, despite numerous control components that can be incorporated.This thesis presents some examples of different microfluidic devices designed and manufactured through the use of microtechnologies, with all applications, focused on their use in biomimetic systems.<br /

Autonomous capillary systems for life science research and medical diagnostics

In autonomous capillary systems (CS) minute amounts of liquid are transported owing to capillary forces. Such CSs are appealing due to their portability, flexibility, and the exceptional physical behavior of liquids in micrometer sized microchannels, in particular, capillarity and short diffusion times. CSs have shown to be a promising technology for miniaturized immunoassays in life science research and diagnostics. Building on existing experimental demonstrations of immunoassays in CSs, a theoretical model of such immunoassays is implemented, tools and CSs for performing immunoassays are developed, key functional elements of CSs such as capillary pumps and valves are explored experimentally, and a proof-of-concept of the ultimate goal of one-step immunoassays are given in this work. For the theoretical modeling of immunoassays in CSs a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance. A method using evaporation for controlling the flow rate in CSs was developed for maximum flexibility for developing assays. The method allows to use small CSs that initially are filled by capillary forces and then provide a well defined area of the liquid-air interface from which liquid can evaporate. Temperature and humidity are continuously measured and Peltier-elements are used to adjust the temperatures in multiple areas of the CSs relative to the dew-point. Thereby flow rates in the range from ~1.2 nL s−1 to ~30 pL s−1 could be achieved in the microchannels. This method was then used for screening cells for surface receptors. CSs, that do not need any peripherals for controlling flow rates become even more appealing. We explored the filling behavior of such CSs having microchannels of various length and large capillary pumps. The capillary pumps comprise microstructures of various sizes and shapes, which are spaced to encode certain capillary pressures. The spacing and shape of the microstructures is also used to orient the filling front to obtain a reliable filling behavior and to minimize the risk of entrapping air. We show how two capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in CSs. Liquid filling CSs can hardly be stopped, but in some cases it might be beneficial to do so. In a separate chapter we explore how microstructures need to be designed to use capillary forces to stop, time, or trigger liquids. Besides well-defined flow rates in CSs accurately patterned capture antibodies (cAbs) are key for performing high-sensitive surface immunoassays in CSs. We present a method compatible with mass fabrication for patterning cAbs in dense lines of up to 8 lines per millimeter. These cAbs are used with CSs that are optimized for convenient handling, pipetting of solutions, pumping of liquids such as human serum, and visualization of signals for fluorescence immunoassays to detect c-reactive protein (CRP) with a sensitivity of 0.9 ng mL−1 (7.8 pM) from 1 uL of CRP-spiked human serum, within 11 minutes, with 4 pipetting steps, and a total volume of sample and reagents of <1.5 uL. CSs for diagnostic applications have different requirements than CSs that are used as a research tool in life sciences, where a high flexibility and performance primes over the ease of use and portability of the CSs. We give a proof-of-concept for one-step immunoassays based on CSs which we think can be the base for developing portable diagnostics for point-of-care applications. All reagents are preloaded in the CSs. A sample loaded in the CSs redissolves and reconstitutes the detection antibodies (dAbs), analyte-dAb-complexes are formed and detected downstream in the CSs. A user only needs to load a sample and measure the result using a fluorescence microscope or scanner. C-reactive protein was detected in human serum at clinical concentrations within 10 minutes and using only 2 uL of sample