Towards Single Bacterium Detection: A Microelectronic/Microfluidic Hybrid System Based on a CMOS Technology

RÉSUMÉ Cette thèse porte sur le développement d'un biocapteur hybride CMOS microfluidique capable de détecter des bactéries pathogènes une à une en temps réel basé sur un principe de spectroscope impédimétrique. Le biocapteur proposé se compose d'une matrice de capteurs qui comportent une matrice de microélectrodes, desmultiplexeurs à commande numérique, et des circuits de détection intégrés sur une puce de silicium CMOS. Cette recherche propose une nouvelle structure de microélectrodes qui permet à une structure de microélectrodes face à face à haute densité intégrable par post-traitement d’une puce CMOS. Au lieu d’être créée par le dépôt et la gravure de couches métalliques supplémentaires, la structure de microélectrodes face à face est construite en exploitant un empilement de couches métalliques disponible avec la technologie CMOS adoptée. Les détecteurs sont obtenus en construisant des microcanaux qui traversent le substrat. Ces microcanaux passent entre les microélectrodes face à face. Lorsque les fluides où se trouvent les échantillons traversent le microcanal, le système détecte de façon continue les changements d'impédance entre les microélectrodes induits par le passage de chaque bactérie . Cette thèse étudie le processus de microfabrication qui permet de libérer la matrice de microélectrodes et de fabriquer les microcanaux traversant le substrat. Les techniques dites de FIB (pours Focused Ion Beam) et de DRIE (pour Deep Reactive Ion Etching) sont utilisées. Les forces et faiblesses de chaque technologie sont analysées et des recettes de processus optimisés sont étudiées. La matrice de microélectrodes a été réalisée avec succès par les deux technologies. Comme preuve de concept, plusieurs microcanaux traversant le substrat sont également formés en utilisant la technologie FIB. Cette thèse propose également un nouveau circuit de détection. Réalisé grâce à la micro-électronique, ce circuit est capable de détecter les changements d'impédance causés par le passage d’une seule bactérie dans un milieu conducteur. Sans conditionnement de signaux et de circuit de traitement complexes, tels que des amplificateurs de haute précision, des filtres ou des convertisseurs analogue à numérique ou numérique à analogique, les circuits de détection sont conçus pour offrir une bonne sensibilité et une configurabilité qui permet de l'adapter aux différentes conditions de détection. Une technique de mise en boîtier biocompatible est également mise en oeuvre pour encapsuler le capteur intégré tout en fournissant des interfaces fluidiques et électriques pour l'injection d'échantillons et de signaux électriques. Une nouvelle approche pour améliorer la sélectivité de détection basée sur l’utilisation de bactéries magnétotactiques est également proposée dans cette thèse. Sous le contrôle d’un champ magnétique extérieur, les bactéries magnétotactiques sont utilisées comme bio-transporteurs, qui peuvent chercher activement et capturer les bactéries pathogènes cibles afin de les amener à la zone de détection. Une puce microfluidique est fabriquée grâce à des techniques de prototypage rapide afin de valider les idées proposées et de fournir des guides de conception d'une puce plus avancés. Les résultats de microfabrication et les résultats des tests préliminaires montrent que l'intégration monolithique des technologies CMOS et microfluidique est possible et qu’elle permet la réalisation de microélectrodes face à face dans une plate-forme capable de détecter le passage d’une seule bactérie en isolation.----------ABSTRACT This thesis reports on the development of a CMOS Microfluidic hybrid biosensor technology that is proposed to detect single pathogenic bacterium in real time based on impedimetric spectroscopy. The proposed biosensor consists of a CMOS silicon die that incorporates a microelectrode array, digitally controlled multiplexers, and sensing circuits. This research proposes a novel microelectrode structure, which is obtained by first manufacturing high density face to face microelectrodes on a CMOS die, possible by a relatively simple CMOS post-processing. Instead of deposition and patterning of additional metal layers, the face to face microelectrode array is constructed by stacking metal and via layers of the adopted CMOS technology. By constructing through substrate microchannels in between pairs of face to face microelectrodes, when a fluid sample flows through the microchannel, the microelectrodes on the wall detect the impedance change induced by bacterium in the fluid in a continuous way. This thesis investigates the microfabrication process of releasing microelectrode arrays and constructing through substrate microchannels. FIB (Focused Ion Beam) and DRIE (Deep Reactive Ion Etching) technologies are utilized. The strength and weakness of each technology are analyzed and optimized process recipes are investigated. Microelectrode array were successfully released using both process technologies. As a proof of concept, several through substrate microchannels were also formed by using the FIB technology. This thesis also proposes a novel sensing microelectronic circuit, which is able to sense the impedance change caused by a single bacterium in a conductive medium. The system does not require complex signal conditioning and processing circuits, such as high precision amplifiers, filters or ADC/DAC. The proposed simple sensing structure offer high sensitivity, reliability and configurability. A dedicated biocompatible packaging is also implemented to encapsulate the CMOS die and provide a microchamber, fluidic and electrical interfaces for sample injection and signal interfaces. A new approach to achieve detection selectivity or specificity assisted by magnetotactic bacterium is also proposed in this thesis. Under the control of an external magnetic field, the viii magnetotactic bacteria are used as bio-carriers, which can actively search and capture some target pathogenic bacteria and bring them to the sensing area. A microfluidic chip is fabricated by rapid prototyping techniques to validate the proposed idea and to provide design guides for a more advanced and highly integrated CMOS chip. The achieved microfabrication results and preliminary testing results show that the monolithic integration of CMOS and microfluidic technology, especially the face to face microelectrode structure is a suitable platform for single bacterium detection and analysis

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 /

An Optofluidic Lens Biochip and an x-ray Readable Blood Pressure Microsensor: Versatile Tools for in vitro and in vivo Diagnostics.

Three different microfabricated devices were presented for use in vivo and in vitro diagnostic biomedical applications: an optofluidic-lens biochip, a hand held digital imaging system and an x-ray readable blood pressure sensor for monitoring restenosis. An optofluidic biochip–termed the ‘Microfluidic-based Oil-Immersion Lens’ (mOIL) biochip were designed, fabricated and test for high-resolution imaging of various biological samples. The biochip consists of an array of high refractive index (n = 1.77) sapphire ball lenses sitting on top of an oil-filled microfluidic network of microchambers. The combination of the high optical quality lenses with the immersion oil results in a numerical aperture (NA) of 1.2 which is comparable to the high NA of oil immersion microscope objectives. The biochip can be used as an add-on-module to a stereoscope to improve the resolution from 10 microns down to 0.7 microns. It also has a scalable field of view (FOV) as the total FOV increases linearly with the number of lenses in the biochip (each lens has ~200 microns FOV). By combining the mOIL biochip with a CMOS sensor, a LED light source in 3D printed housing, a compact (40 grams, 4cmx4cmx4cm) high resolution (~0.4 microns) hand held imaging system was developed. The applicability of this system was demonstrated by counting red and white blood cells and imaging fluorescently labelled cells. In blood smear samples, blood cells, sickle cells, and malaria-infected cells were easily identified. To monitor restenosis, an x-ray readable implantable blood pressure sensor was developed. The sensor is based on the use of an x-ray absorbing liquid contained in a microchamber. The microchamber has a flexible membrane that is exposed to blood pressure. When the membrane deflects, the liquid moves into the microfluidic-gauge. The length of the microfluidic-gauge can be measured and consequently the applied pressure exerted on the diaphragm can be calculated. The prototype sensor has dimensions of 1x0.6x10mm and adequate resolution (19mmHg) to detect restenosis in coronary artery stents from a standard chest x-ray. Further improvements of our prototype will open up the possibility of measuring pressure drop in a coronary artery stent in a non-invasively manner.PhDMacromolecular Science and EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111384/1/toning_1.pd

Optical sensor system for monitoring the pH of cellular media: application to an organ-on-a-chip platform

Dissertação de mestrado integrado em Engenharia FísicapH is a physiological parameter that changes its value according to cellular state of a human organ. When a tumour is being developed, it is known that they have a more acid interstitial pH than normal tissues. This is mainly due to the high metabolic activity of the abnormal cells with increase of acidic sub-products and the absence of organised vasculature of tumours, that leads to poor tissue oxygenation. Indeed, in in vitro animal systems, such as static cell culture experiments or advanced microfluidic devices, the cell’s metabolic activity during incubation causes the alteration of the cell culture pH, which drops the pH from close to the physiological (7.4) to acidic ones (lower than 7.0). Because low environmental pH inhibits cell survival, proliferation and activity, cell culture media has to be consecutively replaced for fresh one. For the pH monitoring, cell culture media is, in general, complemented with a pH colorimetric indicator (e.g. phenol red). However, the colour change of the cell culture media does not quantify the pH value. For this reason, the monitoring and quantification of cell culture medium pH, especially in advanced cell culture devices, such as organ-on-a-chip (OoC), which contain healthy and/or tumour organ models, is still a challenge and a parameter of utmost importance for the maintenance of homeostasis (auto-regulation). The pH of tissues can be measured by a variety of techniques, being pH electrodes the most used. Nevertheless, other methods can be used for pH measurement, such as optical sensors. In general, this technique eliminates the tissue injury effects, but results in an integrated measurement over a long period and demanding a relatively large volume of sample. Particularly, in OoC platforms, the small size of each OoC constituent part, has triggered the development of micro(bio)sensors to be integrated in the microchambers that feed the perfusion chambers containing the organ models, which are used for monitoring the pH of the cell culture media in circulation. In this study, a literature review of pH sensors that can be miniaturised and integrated in OoC was investigated. Based on this previews literature research, and the presence of a colorimetric pH indicator (phenol red) that is commercially added to cell culture media, a miniaturization of an optical pH sensor, for real-time sensing of the cell culture medium feeding advanced microfluidic devices was investigated. This strategy can have several advantages, such as low-cost implementation and improvement of the pH reading based on the beam-splitter phenomenon. For this purpose, a microchamber, processed by micromilling in PMMA, was developed and optimised to support the pH optical sensing system, creating a prototype device that can be directly incorporated into an OoC platform. For the pH sensing experiments, buffered solutions with stablished pH and phenol red were used to test and optimize the optical sensor, by analysing their transmittance signal. In this study, the colorimetric pH indicator (phenol red) was added in the same concentration than the one used in commercial cell culture media. The results shown that the pH reading was successful achieved in intervals of 0.2 pH units, in a range between 6.0 to 8.0.O pH é um parâmetro fisiológico, cujo valor se altera de acordo com o estado celular de um órgão humano. Quando um tumor está em poliferação, este tem um pH intersticial mais ácido do que os tecidos normais. Isto deve-se principalmente à elevada atividade metabólica das células anormais com o aumento de subprodutos ácidos e a ausência de vasculatura organizada dos tumores, que leva à pobre oxigenação dos tecidos. De facto, em sistemas animais in vitro, tais como experiências de cultura celular estática ou dispositivos microfluídicos avançados, a atividade metabólica da célula durante a incubação provoca a alteração do pH da cultura celular, que baixa o pH de perto do fisiológico (7.4) para um mais ácido (inferior a 7.0). O baixo pH fisiológico inibe a sobrevivência, proliferação e atividade celular, e, em consequência, os meios de cultura celular têm de ser substituídos consecutivamente. Para a monitorização do pH, os meios de cultura celular são, em geral, complementados com um indicador colorimétrico de pH (por exemplo, vermelho de fenol). No entanto, a mudança de cor dos meios de cultura celular não quantifica o valor do pH. Por esta razão, a monitorização e quantificação do pH dos meios de cultura celular, especialmente em dispositivos avançados de cultura celular, tais como Organ-on-a-chip (OoC), que contêm modelos de órgãos saudáveis e/ou tumorais, é ainda um desafio e um parâmetro da maior importância para a manutenção da homeostase (autorregulação). O pH dos tecidos pode ser medido por uma variedade de técnicas, sendo os elétrodos de pH os mais utilizados. Contudo, outros métodos podem ser utilizados para a medição do pH, tais como os sensores óticos. Esta técnica elimina os efeitos da lesão tecidual, mas resulta numa medição integrada durante um longo período e a partir de um volume relativamente grande de amostra. Particularmente, nas plataformas de OoC, o pequeno tamanho de cada parte constituinte de OoC, desencadeou o desenvolvimento de micro(bio)sensores para serem integrados nas microcâmaras que alimentam as câmaras de perfusão, as quais contêm os modelos de órgãos, e que podem ser utilizados para monitorizar o pH dos meios de cultura celular em circulação. Neste estudo, foi investigada uma revisão bibliográfica de sensores de pH que podem ser miniaturizados e integrados na OoC. Com base nesta pesquisa bibliográfica prévia, e a presença de um indicador colorimétrico de pH (vermelho fenol) que é comercialmente adicionado aos meios de cultura celular, foi investigada uma miniaturização de um sensor ótico de pH para a deteção em tempo real do meio de cultura celular que alimenta dispositivos microfluídicos avançados. Esta estratégia pode ter várias vantagens, tais como a implementação a baixo custo e a melhoria da leitura do pH com base no fenómeno do separador de feixe. Para este efeito, foi desenvolvida e otimizada uma microcâmara, processada por micromilling em PMMA, para apoiar o sistema de deteção ótica de pH, criando um dispositivo protótipo que pode ser diretamente incorporado numa plataforma OoC. Para as experiências de deteção de pH, foram utilizadas soluções tampão com pH estabilizado e vermelho fenol para testar e otimizar o sensor ótico, através da análise do seu sinal de transmitância. Assim, o indicador colorimétrico de pH vermelho de fenol foi adicionado na mesma concentração encontrada nos meios de cultura celular comerciais. Os resultados mostram que a leitura do pH foi bem-sucedida em intervalos de 0.2 unidades de pH, num intervalo entre 6.0 a 8.0.This work results partially of the project NORTE-01-0145-FEDER-029394, RTChip4Theranostics, supported by Programa Operacional Regional do Norte - Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by Fundação para a Ciência e Tecnologia (FCT), IP, project reference PTDC/EMD-EMD/29394/2017

CMOS IMAGE SENSORS FOR LAB-ON-A-CHIP MICROSYSTEM DESIGN

The work described herein serves as a foundation for the development of CMOS imaging in lab-on-a-chip microsystems. Lab-on-a-chip (LOC) systems attempt to emulate the functionality of a cell biology lab by incorporating multiple sensing modalidites into a single microscale system. LOC are applicable to drug development, implantable sensors, cell-based bio-chemical detectors and radiation detectors. The common theme across these systems is achieving performance under severe resource constraints including noise, bandwidth, power and size. The contributions of this work are in the areas of two core lab-on-a-chip imaging functions: object detection and optical measurements

A Customer Programmable Microfluidic System

Microfluidics is both a science and a technology offering great and perhaps even revolutionary capabilities to impact the society in the future. However, due to the scaling effects there are unknown phenomena and technology barriers about fluidics in microchannel, material properties in microscale and interactions with fluids are still missing. A systematic investigation has been performed aiming to develop A Customer Programmable Microfluidic System . This innovative Polydimethylsiloxane (PDMS)-based microfluidic system provides a bio-compatible platform for bio-analysis systems such as Lab-on-a-chip, micro-total-analysis system and biosensors as well as the applications such as micromirrors. The system consists of an array of microfluidic devices and each device containing a multilayer microvalve. The microvalve uses a thermal pneumatic actuation method to switch and/or control the fluid flow in the integrated microchannels. It provides a means to isolate samples of interest and channel them from one location of the system to another based on needs of realizing the customers\u27 desired functions. Along with the fluid flow control properties, the system was developed and tested as an array of micromirrors. An aluminum layer is embedded into the PDMS membrane. The metal was patterned as a network to increase the reflectivity of the membrane, which inherits the deformation of the membrane as a mirror. The deformable mirror is a key element in the adaptive optics. The proposed system utilizes the extraordinary flexibility of PDMS and the addressable control to manipulate the phase of a propagating optical wave front, which in turn can increase the performance of the adaptive optics. Polydimethylsiloxane (PDMS) has been widely used in microfabrication for microfluidic systems. However, few attentions were paid in the past to mechanical properties of PDMS. Importantly there is no report on influences of microfabrication processes which normally involve chemical reactors and biologically reaction processes. A comprehensive study was made in this work to study fundamental issues such as scaling law effects on PDMS properties, chemical emersion and temperature effects on mechanical properties of PDMS, PDMS compositions and resultant properties, as well as bonding strength, etc. Results achieved from this work will provide foundation of future developments of microfluidics utilizing PDMS

Hybrid microfluidic CMOS capacitive sensors for lab-on-chip applications

Methods and applications of CMOS-based Locs -- Hybrid microfluidic/cmos platform -- Cmos based capacitive sensors for locs -- Direct-write microfluidic packaging procedure -- Core-cbcm capacitive sensor array for locs

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Magnetic bead-based DNA extraction and purification microfluidic chip

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.A magnetic bead-based DNA extraction and purification device has been designed to be used for extraction of the target DNA molecules from whole blood sample. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in the cell lysis buffer in a circular chamber that is sandwiched between two electromagnets. Non-uniform nature of the magnetic field causes temporal and spatial distribution of the beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom electrode. DNA molecules are extracted from the magnetic beads by washing and re-suspension processes. The numerical simulation approach has been adopted in order to design the magnetic field source. The performance of the magnetic field source has been investigated against different physical and geometrical parameters and optimised dimensions are obtained with two different magnetic field sources; integrated internal source and external source. A new magnetic field pattern has been introduced in order to efficiently control the bulk of magnetic beads inside the mixing chamber by dynamic shifting of magnetic field regions from the centre of the coils to the outer edge of the coils and vice versa. A Matlab code has been developed to simulate beads trajectories inside the designed extraction chip in order to investigate the efficiency of the magnetic mixing. A preliminary target molecule capturing simulation has also been performed using the simulated bead trajectories to evaluate the DNA-capturing efficiency of the designed extraction chip. The performance of the designed extraction chip has been tested by conducting a series of biological experiments. Different magnetic bead-based extraction kits have been used in a series of preliminary experiments in order to extract a more automation friendly extraction protocol. The efficiency of the designed device has been evaluated using the spiked bacterial DNA and non-pathogenic bacterial cell cultures (B. subtilis, Gram positive bacteria and E. coli, Gram negative bacteria) into the blood sample. Excellent DNA yields and recovery rates are obtained with the designed extraction chip through a simple and fast extraction protocol

MICROFLUIDIC APPROACHES FOR QUANTITATIVE ANALYSIS AND SCREENING OF SYNTHETICALLY ENGINEERED MICROBES

Department of Biomedical EngineeringRecently, our understanding of complex genomes, proteomes, bio-molecules, and even many metabolic pathways has been developed significantly as growing a fundamental knowledge of the biochemistry of life. These newly revealed findings have incredibly influenced in recent bio-technologies, such as synthetic biology having massive potential to solve the missing connected dots. To have better understanding of microbe, microfluidic approaches were innovatively introduced to the field with the potential to revolutionize high-throughput biological assays. In this study, suggested approach to address limitations of conventional microbiology is microfluidics integrated with synthetic biology. At first, the microbial biosensors will be introduced into microfluidic ratchet platform for a quantitative analysis of microbial bio-signal. The microfluidic device using microfabricated arrowhead-shaped ratchet structures has an intrinsic function that concentrates motile microbes in a microchamber array. Additionally, the ratchet structure provides the concentrated microbes to grow better in a continuous-feed mode. A continuous exposure of detection analytes leads the amplification of fluorescence signal from microbes in a microchamber. Therefore, it was noted that the substantial amplification of bio-signal was achieved from the microfluidic device and measured signals were analyzed in quantitative manner. As a second practical application of the microfluidic approach, for high-throughput screening (HTS) application, a fluid array will be developed by using immiscible character between water and oil for microbial incubation, analysis, selective extraction, recovery process and the demonstration of practical applications. From the characterization of the fluid array platform, HTS will be demonstrated based on two different categories: reporter-gene basis and growth complementation basis. The fluid array device showed not only demonstrations of high-throughput screening, but also advanced screening applications were also demonstrated with higher mutant library screening with 106 and C2C communication screening system. Outstanding mutants were sophisticatedly screened among 106 of a mutant library based on the hybrid type screening method. Also, the proposed C2C screening approach has enabled high-throughput compartmentalization and resulted in 10 possible mutants showing higher extracellular biomolecule secretion performance. The proposed microfluidic approaches can be practically useful combinations showing many advantages: 1) economical and reduced time requirement for real application without complex instruments, 2) facile potentials to enable a multiplex quantitative analysis in a high-throughput manner, and 3) selective, direct and convenient measurement without pre- or post-treatment of sample solutions in near future the entire processes could be fully automated. In this dissertation, different type of microfluidic devices was developed for various collaborative purposes for the bottleneck of conventional microbiology. Therefore, the microfluidic devices have knocked a new door for high-throughput screening application for synthetically engineered microorganisms and quantitative approaches for microbial biosensors. Thus, the research contributions in this doctoral dissertation are the microfluidic approaches to popularize and overcome conventional constraints from biological experimental tools by integration of total analysis system for synthetically engineered microbe cases.ope