Development of a Composite ion-exchange membrane for PDMS-based microfluidic devices

The purpose of this research is to investigate potential methods to produce an ion-exchange membrane that can be integrated directly into a polydimethylsiloxane Lab-on-a-Chip or Micro-Total-Analysis-System. The majority of microfluidic membranes are based on creating microporous structures, because it allows flexibility in the choice of material such that it can match the material of the microfluidic chip. This cohesion between the material of the microfluidic chip and membrane is an important feature to prevent bonding difficulties which can lead to leaking and other practical problems. However, of the materials commonly used to manufacture microfluidic chips, there are none that provide the ion-exchange capability. The DuPont product Nafion{TM} is chosen as the ion-exchange membrane, a copolymer with high conductivity and selectivity to cations and suitable for many applications such as electrolysis of water and the chlor-alkali process. The use of such an ion-exchange membrane in microfluidics could have multiple advantages, but there is no reversible/irreversible bonding that occurs between PDMS and Nafion{TM}. In this project multiple methods of physical entrapment of the ion-exchange material inside a film of PDMS are attempted. Through the use of the inherent properties of PDMS, very inexpensive sugar granulate can be used to make an inexpensive membrane mould which does not interfere with the PDMS crosslinking process. After dissolving away this sacrificial mould material, Nafion{TM} is solidified in the irregular granulate holes. Nafion{TM} in this membrane is confined in the irregular shape of the PDMS openings. The outer structure of the membrane is all PDMS and can be attached easily and securely to any PDMS-based microfluidic device through reversible or irreversible PDMS/PDMS bonding. Through impedance measurement, the effectiveness of these integrated membranes are compared against plain Nafion{TM} films in simple sodium chloride solutions.

Capillary electrophoresis characterisation of a rapid prototyped PMMA chip for particle analysis

Màster en Nanociència i Nanotecnologia

Digital microfluidic devices: the role of the dielectric layer

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.

Natural-rubber-based flexible microfluidic device

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microfabrication processes for microfluidic devices on a single laser Workstation: direct writing lithography on SU-8, laser ablation on polymers and mask manufacturing

We demonstrate the capability of a laser micromachining workstation for cost-effective manufacturing of a variety of microfluidic devices, including SU-8 microchannels on silicon wafers and 3D complex structures made on polyimide Kapton® or poly carbonate (PC). The workstation combines a KrF excimer laser at 248 nm and a Nd3+:YVO4 DPSS with a frequency tripled at 355 nm with a lens magnification 10X, both lasers working at a pulsed regime with nanoseconds (ns) pulse duration. Workstation also includes a high-resolution motorized XYZ-tilt axis (~ 1 um / axis) and a Through The Lens (TTL) imaging system for a high accurate positioning over a 120 x 120 mm working area. We have surveyed different fabrication techniques: direct writing lithography,mask manufacturing for contact lithography and polymer laser ablation for complex 3D devices, achieving width channels down to 13μ m on 50μ m SU-8 thickness using direct writing lithography, and width channels of 40 μm for polyimide on SiO2 plate. Finally, we have tested the use of some devices for capillary chips measuring the flow speed for liquids with different viscosities. As a result, we have characterized the presence of liquid in the channel by interferometric microscopy.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Microfluidic Paper-based Devices For Bioanalytical Applications.

Paper has become increasingly recognized as a very interesting substrate for the construction of microfluidic devices, with potential application in a variety of areas, including health diagnosis, environmental monitoring, immunoassays and food safety. The aim of this review is to present a short history of analytical systems constructed from paper, summarize the main advantages and disadvantages of fabrication techniques, exploit alternative methods of detection such as colorimetric, electrochemical, photoelectrochemical, chemiluminescence and electrochemiluminescence, as well as to take a closer look at the novel achievements in the field of bioanalysis published during the last 2 years. Finally, the future trends for production of such devices are discussed.

In vitro Microfluidic Model For The Study Of Vaso-occlusive Processes.

Vaso-occlusion, responsible for much of the morbidity of sickle-cell disease, is a complex multicellular process, apparently triggered by leukocyte adhesion to the vessel wall. The microcirculation represents a major site of leukocyte-endothelial interactions and vaso-occlusive processes. We have developed a biochip with subdividing interconnecting microchannels that decrease in size (40 μm to 10 μm in width), for use in conjunction with a precise microfluidic device, to mimic cell flow and adhesion through channels of sizes that approach those of the microcirculation. The biochips were utilized to observe the dynamics of the passage of neutrophils and red blood cells, isolated from healthy and sickle-cell anemia (SCA) individuals, through laminin or endothelial adhesion molecule-coated microchannels at physiologically relevant rates of flow and shear stress. Obstruction of E-selectin/intercellular adhesion molecule 1-coated biochip microchannels by SCA neutrophils was significantly greater than that observed for healthy neutrophils, particularly in the microchannels of 40-15 μm in width. Whereas SCA red blood cells alone did not significantly adhere to, or obstruct, microchannels, mixed suspensions of SCA neutrophils and red blood cells significantly adhered to and obstructed laminin-coated channels. Results from this in vitro microfluidic model support a primary role for leukocytes in the initiation of SCA occlusive processes in the microcirculation. This assay represents an easy-to-use and reproducible in vitro technique for understanding molecular mechanisms and cellular interactions occurring in subdividing microchannels of widths approaching those observed in the microvasculature. The assay could hold potential for testing drugs developed to inhibit occlusive mechanisms such as those observed in SCA and thrombotic diseases.

Dual contactless conductivity and amperometric detection on hybrid PDMS/glass electrophoresis microchips

A new approach for the integration of dual contactless conductivity and amperometric detection with an electrophoresis microchip system is presented. The PDMS layer with the embedded channels was reversibly sealed to a thin glass substrate (400 mu m), on top of which a palladium electrode had been previously fabricated enabling end-channel amperometric detection. The thin glass substrate served also as a physical wall between the separation channel and the sensing copper electrodes for contactless conductivity detection. The latter were not integrated in the microfluidic device, but fabricated on an independent plastic substrate allowing a simpler and more cost-effective fabrication of the chip. PDMS/glass chips with merely contactless conductivity detection were first characterized in terms of sensitivity, efficiency and reproducibility. The separation efficiency of this system was found to be similar or slightly superior to other systems reported in the literature. The simultaneous determination of ionic and electroactive species was illustrated by the separation of peroxynitrite degradation products, i.e. NO(3)(-) (non-electroactive) and NO(2)(-) (electroactive), using hybrid PDMS/glass chips with dual contactless conductivity and amperometric detection. While both ions were detected by contactless conductivity detection with good efficiency, NO(2)(-) was also simultaneously detected amperometrically with a significant enhancement in sensitivity compared to contactless conductivity detection.

Rapid prototyping of polymeric electrophoresis microchips with integrated copper electrodes for contactless conductivity detection

A simple and easy approach to produce polymeric microchips with integrated copper electrodes for capacitively coupled contactless conductivity detection (CD) is described. Copper electrodes were fabricated using a printed circuit board (PCB) as an inexpensive thin-layer of metal. The electrode layout was first drawn and laser printed on a wax paper sheet. The toner layer deposited on the paper sheet was thermally transferred to the PCB surface working as a mask for wet chemical etching of the copper layer. After the etching step, the toner was removed with an acetonitrile-dampened cotton. A poly(ethylene terephthalate) (PET) film coated with a thin thermo-sensitive adhesive layer was used to laminate the PCB plate providing an insulator layer of the electrodes to perform CID measurements. Electrophoresis microchannels were fabricated in poly(dimethylsiloxane) (PDMS) by soft lithography and reversibly sealed against the PET film. These hybrid PDMS/PET chips exhibited a stable electroosmotic mobility of 4.25 +/- 0.04 x 10(-4) V cm(-2) s(-1), at pH 6.1, over fifty runs. Efficiencies ranging from 1127 to 1690 theoretical plates were obtained for inorganic cations.

Chips de pneus – substituto de brita calcária em aterros sanitários

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção de grau de Mestre em Engenharia Sanitária

Análise de chips de pneus usados para aplicações em sistemas de drenagem de cobertura final de aterros sanitários

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente, Perfil gestão e sistemas ambientais

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.

Evaluating biomaterial-and microfluidic-based 3D tumor models

Cancer is a major cause of morbidity and mortality worldwide, with a disease burden estimated to increase in the coming decades. Disease heterogeneity and limited information on cancer biology and disease mechanisms are aspects that 2D cell cultures fail to address. We review the current "state-of-the-art" in 3D Tissue Engineering (TE) models developed for and used in cancer research. Scaffold-based TE models and microfluidics, are assessed for their potential to fill the gap between 2D models and clinical application. Recent advances in combining the principles of 3D TE models and microfluidics are discussed, with a special focus on biomaterials and the most promising chip-based 3D models.