Test analysis & fault simulation of microfluidic systems

This work presents a design, simulation and test methodology for microfluidic systems, with particular focus on simulation for test. A Microfluidic Fault Simulator (MFS) has been created based around COMSOL which allows a fault-free system model to undergo fault injection and provide test measurements. A post MFS test analysis procedure is also described.A range of fault-free system simulations have been cross-validated to experimental work to gauge the accuracy of the fundamental simulation approach prior to further investigation and development of the simulation and test procedure.A generic mechanism, termed a fault block, has been developed to provide fault injection and a method of describing a low abstraction behavioural fault model within the system. This technique has allowed the creation of a fault library containing a range of different microfluidic fault conditions. Each of the fault models has been cross-validated to experimental conditions or published results to determine their accuracy.Two test methods, namely, impedance spectroscopy and Levich electro-chemical sensors have been investigated as general methods of microfluidic test, each of which has been shown to be sensitive to a multitude of fault. Each method has successfully been implemented within the simulation environment and each cross-validated by first-hand experimentation or published work.A test analysis procedure based around the Neyman-Pearson criterion has been developed to allow a probabilistic metric for each test applied for a given fault condition, providing a quantitive assessment of each test. These metrics are used to analyse the sensitivity of each test method, useful when determining which tests to employ in the final system. Furthermore, these probabilistic metrics may be combined to provide a fault coverage metric for the complete system.The complete MFS method has been applied to two system cases studies; a hydrodynamic “Y” channel and a flow cytometry system for prognosing head and neck cancer.Decision trees are trained based on the test measurement data and fault conditions as a means of classifying the systems fault condition state. The classification rules created by the decision trees may be displayed graphically or as a set of rules which can be loaded into test instrumentation. During the course of this research a high voltage power supply instrument has been developed to aid electro-osmotic experimentation and an impedance spectrometer to provide embedded test

Design and Optimization Methods for Pin-Limited and Cyberphysical Digital Microfluidic Biochips

<p>Microfluidic biochips have now come of age, with applications to biomolecular recognition for high-throughput DNA sequencing, immunoassays, and point-of-care clinical diagnostics. In particular, digital microfluidic biochips, which use electrowetting-on-dielectric to manipulate discrete droplets (or "packets of biochemical payload") of picoliter volumes under clock control, are especially promising. The potential applications of biochips include real-time analysis for biochemical reagents, clinical diagnostics, flash chemistry, and on-chip DNA sequencing. The ease of reconfigurability and software-based control in digital microfluidics has motivated research on various aspects of automated chip design and optimization.</p><p>This thesis research is focused on facilitating advances in on-chip bioassays, enhancing the automated use of digital microfluidic biochips, and developing an "intelligent" microfluidic system that has the capability of making on-line re-synthesis while a bioassay is being executed. This thesis includes the concept of a "cyberphysical microfluidic biochip" based on the digital microfluidics hardware platform and on-chip sensing technique. In such a biochip, the control software, on-chip sensing, and the microfluidic operations are tightly coupled. The status of the droplets is dynamically monitored by on-chip sensors. If an error is detected, the control software performs dynamic re-synthesis procedure and error recovery.</p><p>In order to minimize the size and cost of the system, a hardware-assisted error-recovery method, which relies on an error dictionary for rapid error recovery, is also presented. The error-recovery procedure is controlled by a finite-state-machine implemented on a field-programmable gate array (FPGA) instead of a software running on a separate computer. Each state of the FSM represents a possible error that may occur on the biochip; for each of these errors, the corresponding sequence of error-recovery signals is stored inside the memory of the FPGA before the bioassay is conducted. When an error occurs, the FSM transitions from one state to another, and the corresponding control signals are updated. Therefore, by using inexpensive FPGA, a portable cyberphysical system can be implemented.</p><p>In addition to errors in fluid-handling operations, bioassay outcomes can also be erroneous due the uncertainty in the completion time for fluidic operations. Due to the inherent randomness of biochemical reactions, the time required to complete each step of the bioassay is a random variable. To address this issue, a new "operation-interdependence-aware" synthesis algorithm is proposed in this thesis. The start and stop time of each operation are dynamically determined based on feedback from the on-chip sensors. Unlike previous synthesis algorithms that execute bioassays based on pre-determined start and end times of each operation, the proposed method facilitates "self-adaptive" bioassays on cyberphysical microfluidic biochips.</p><p>Another design problem addressed in this thesis is the development of a layout-design algorithm that can minimize the interference between devices on a biochip. A probabilistic model for the polymerase chain reaction (PCR) has been developed; based on the model, the control software can make on-line decisions regarding the number of thermal cycles that must be performed during PCR. Therefore, PCR can be controlled more precisely using cyberphysical integration.</p><p>To reduce the fabrication cost of biochips, yet maintain application flexibility, the concept of a "general-purpose pin-limited biochip" is proposed. Using a graph model for pin-assignment, we develop the theoretical basis and a heuristic algorithm to generate optimized pin-assignment configurations. The associated scheduling algorithm for on-chip biochemistry synthesis has also been developed. Based on the theoretical framework, a complete design flow for pin-limited cyberphysical microfluidic biochips is presented.</p><p>In summary, this thesis research has led to an algorithmic infrastructure and optimization tools for cyberphysical system design and technology demonstrations. The results of this thesis research are expected to enable the hardware/software co-design of a new class of digital microfluidic biochips with tight coupling between microfluidics, sensors, and control software.</p>Dissertatio

Multi-scale cellular engineering: From molecules to organ-on-a-chip.

Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches

Biodétection de Legionella pneumophila par biocapteur à photocorrosion digitale à base de peptide antimicrobien

La détection de bactéries pathogènes par culture microbienne est lente, nécessite un milieu de culture spécifique pour garantir la croissance de certaines souches bactériennes fastidieuses telle que Legionella pneumophila (L. pneumophila) et en plus pourrait ne pas déceler les bactéries viables mais non cultivables mais restant dangereuse en termes de pathogénicité. Par conséquent, l’usage de biocapteurs pour la détection de L. pneumophila serait, potentiellement, une approche attrayante permettant une détection précise et rapide. Cependant, la sensibilité et la spécificité des biocapteurs dépendent fortement des molécules de bioreconnaissance utilisées. Jusqu'à présent, différents ligands tels que les anticorps, les enzymes, les acides nucléiques fonctionnels (aptamères) et les bactériophages ont été utilisés comme éléments de bioreconnaissance. En raison de leur haute spécificité, Les anticorps de mammifères ont été largement employés pour le développement de divers biocapteurs. Cependant, les anticorps sont connus pour souffrir de la variabilité des lots produits et d'une stabilité limitée, ce qui réduit l'usage et la constance des performances des biocapteurs à base d'anticorps. Au cours des dernières années, les peptides antimicrobiens (PAM) ont été de plus en plus investigués pour des applications thérapeutiques en plus d’être considérés comme des ligands de bioreconnaissance prometteurs en raison de leur grande stabilité et leurs fortes réactivités aux bactéries. Dans le but d’améliorer les performances du biocapteur à DIP, notre hypothèse reposait sur l’usage de bioarchitectures à base de PAM à courte séquence pour une capture efficace des bactéries et une détection considérablement améliorée en raison du transfert de charge plus facilitée vers dans la biopuce à base de semiconducteur III-V. Dans la première phase du projet, nous avons évalué un biocapteur à DIP consistant en une puce d’arséniure de gallium/arséniure de gallium aluminium (GaAs/AlGaAs) fonctionnalisée par le warnericine RK pour la détection directe in situ de L. pneumophila dans l’eau. Nous avons démontré une détection linéaire de L. pneumophila pour des concentrations allant de 103 à 106 CFU/mL. De plus, le nombre relativement important d'interfaces constituant la bioarchitecture d’un tel biocapteur pourrait affecter sa reproductibilité et sa sensibilité. Dans ce cas, la couche de bioreconnaissance est plus mince (~ 2 nm) permettant une distance plus courte entre les bactéries et la surface du biocapteur, ce qui pourrait jouer un rôle important dans la promotion du transfert de charge entre les bactéries et la biopuce, et ainsi nous avons pu démontrer une détection efficace de L. pneumophila à une concentration de 2 x 102 CFU/mL. Cette configuration a permis d’atteindre des LODs de 50 et 100 UFC/mL, respectivement pour de légionnelle dans du PBS et collectées d’échantillons d’eau de tour de refroidissement. Nous avons observé une détection sélective de L. pneumophila sérogroupe 1 (SG1) comparé au sérogroupe 5 (SG 5). Les biocapteurs à photocorrosion digitale (DIP) en configuration sandwich PAM et Ab pourraient être une approche prometteuse pour développer un biocapteur à faible coût, hautement sensible et spécifique pour la détection rapide de L. pneumophila dans l’eau.Abstract: Culture based detection of pathogenic bacteria is time consuming, and needs specific culture medium to identify bacterial strains such as Legionella pneumophila (L. pneumophila) which does not flourish in typical growth medium. Culture based methods cannot detect viable but unculturable bacteria. Therefore, the detection of L. pneumophila with biosensors potentially could be an attractive approach enabling accurate and rapid detection. The sensitivity and specificity of biosensors depend critically on the biorecognition probes employed for the detection. Until now, different elements such as antibodies, enzymes, functional nucleic acids (aptamers) and bacteriophages have been utilized as biorecognition elements. Due to high specificity of antibodies, and the advanced technology of their production, mammalian antibodies have been widely investigated for the development of various biosensors. However, mammalian antibodies are known to suffer from batch-to-batch variation, as well as limited stability, which could reduce the consistent utility of the proposed biosensors. In recent years, antimicrobial peptides (AMPs) have been increasingly investigated for their therapeutic applications. At the same time, AMPs are considered as promising biorecognition ligands due to their high stability and multiple niches for capturing bacteria. The hypothesis was that AMP-based bioarchitectures allows for highly efficient capturing of bacteria, and the short length of the AMP would significantly enhance detection due to limited obstructive charge transfer in the charge sensing biosensor. In the first phase of the project, we investigated a warnericin RK AMP functionalized gallium arsenide/aluminum gallium arsenide (GaAs/AlGaAs) photonic biosensor for direct detection of L. pneumophila in water environments. This approach allowed for detecting a low to high concentration of L. pneumophila (103 to 106 CFU/mL) with a 103 CFU/mL limit of detection (LOD). In addition, a relatively large number of interfaces constituting the architecture of such biosensors could affect their reproducibility and sensitivity. A thinner biorecognition layer (~2 nm) resulted in a shorter distance between bacteria and the biosensor surface, which played important role in promoting charge transfer between bacteria and biochip. L. pneumophila was detected at concentrations as low of 2 x 102 CFU/mL. This configuration allowed the detection sensitivity of L. pneumophila as low as 50 CFU/mL and 100 CFU/mL in clean water and water originated from cooling tower, respectively, along with the selective detection of whole cell L. pneumophila serogroup 1 (SG1) and serogroup 5 (SG5). The proposed AMP and Ab conjugated sandwich architecture with digital photocorrosion (DIP) biosensors is a promising approach for developing low cost, highly sensitive and specific biosensors for rapid detection of L. pneumophila in water environments

An Outlook on Design Technologies for Future Integrated Systems

The economic and social demand for ubiquitous and multifaceted electronic systems-in combination with the unprecedented opportunities provided by the integration of various manufacturing technologies-is paving the way to a new class of heterogeneous integrated systems, with increased performance and connectedness and providing us with gateways to the living world. This paper surveys design requirements and solutions for heterogeneous systems and addresses design technologies for realizing them

Nanostructured Metal Oxide-Based Microfluidic Biosensors for Point-of-Care Diagnostics

The potential research on microfluidic devices for detection of biomolecules has recently intensified due to its application in point-of-care (POC) diagnostics for global health care. Early detection plays an imperative role to determine predisposition to disease (prevention) or the outcome of disease (monitoring and prognosis). There is a significant need for POC diagnostics devices as perceived from biohazard threats, the spread of infectious disease, home testing and monitoring. The POC diagnostics can provide a convenient and immediate response to a patient test sample. The POC diagnostics can be attained via use of transportable, portable, and handheld instruments such as blood glucometer, cholesterol meter etc. and test kits. It includes testing of blood or urine for pathogens, glucose, cholesterol, blood gas, coagulation, biomarkers, hemoglobin, pregnancy etc. Cheaper, smaller, faster, and smarter devices are the main merits of POC diagnostics for detection of various target analytes. A number of clinical biochemical studies such as blood gas, glucose/lactate/cholesterol, nucleic acid sequence analysis, proteins/peptides, combinatorial synthesis, toxicity monitoring, immunoassays, and forensic analysis are also focused areas for developing microfluidic biochips

Surveillance photonique des activités biologiques de bactéries immobilisées sur des surfaces des semiconducteurs quantiques biofunctionnalisées

Le suivi de la viabilitié, la croissance et le métabolisme cellulaire des bactéries peut contribuer de manière significative au diagnostic précoce de la maladie, mais peut aussi aider à améliorer le rendement des produits bactériens dans des expériences industrielle ou à petite echelle. Les méthodes conventionnelles utilisées pour l'étude de la sensibilité des bactéries aux antibiotiques sont basées principalement sur la culture, une technique qui prend au moins 12 heures pour rendre un résultat. Ce retard conduit au surtraitement d'un large éventail d'infections par des antibiotiques à large spectre, ce qui est coûteux et peut conduire à l'apparition de résistance à ces antibiotiques précieux, tandis que la détection rapide d'une infection virale ou l'absence de bactéries pourrait prévenir de tels traitements et, dans le cas d'une infection bactérienne, l'identification de la sensibilité aux antibiotiques pourrait permettre l'utilisation d'antibiotiques à spectre étroit. Le projet décrit dans le présent document vise à surveiller les activités biologiques des bactéries vivantes immobilisées sur les surfaces biofonctionnalisées de microstructures composées de semi-conducteurs quantiques (QS). Le procédé dépend de la sensibilité de la photoluminescence (PL) émise par des semi-conducteurs à la perturbation du champ électrique induit par la charge électrique des bactéries immobilisées sur la surface de ces structures. Dans la première phase du projet, nous avons étudié une méthode innovante impliquant la surveillance par PL de l'effet de photocorrosion dans des hétérostructures GaAs/AlGaAs. Le maintien d'un équilibre entre la sensibilité et la stabilité du biocapteur dans l'environnement aqueux nous a permis de détecter Escherichia coli K12 dans des solutions salines tamponnées au phosphate (PBS) avec une limite de détection attrayante de 103 UFC/ml en moins de 2 heures. Suite à cette recherche, nous avons émis l'hypothèse que ces hétérostructures pourraient être utilisés pour développer une méthode à faible coût et quasiment en temps reel de la croissance et de la sensibilité des bactéries aux antibiotiques. L'un des éléments clés dans le développement de cette plate-forme de biocapteurs était de démontrer que le GaAs (001), normalement utilisé pour recouvrir les hétérostructures de GaAs/AlGaAs, ne nuira pas à la croissance des bactéries. Dans la deuxième phase du projet, nous avons exploré la capture et la croissance de E. coli K12 sur des surfaces nues et biofonctionnalisées de GaAs (001). Il a été déterminé que la couverture initiale et les taux de croissance de bactéries dépendent de l'architecture de biofonctionnalisation utilisée pour capturer les bactéries: les surfaces biofonctionnalisées avec d'anticorps présentaient une efficacité de capture significativement plus élevée. En outre, on a trouvé que pour des suspensions contenant des bactéries à moins de 105 UFC/ml, la surface des plaquettes de GaAs ne supportait pas la croissance des bactéries, quel que soit le type d'architecture de biofonctionnalisation. Dans la troisième phase du projet, nous avons suivi la croissance et la sensibilité aux antibiotiques de E. coli K12 et E. coli HB101. Tandis que la présence de bactéries retardaient d’apparition du maximum de PL, la croissance des bactéries retardaient encore plus ce maximum. Par contre, en presence d’antibiotiques efficaces, la croissance des bactéries était arrêtée et le maximum de PL est arrivé plus tôt. Ainsi, nous avons pu distinguer entre des E. coli sensibles ou résistantes à la pénicilline ou à la ciprofloxacine en moins de 3h. En raison de la petite taille, du faible coût et de la réponse rapide du biocapteur, l'approche proposée a le potentiel d'être appliquée dans les laboratoires de diagnostic clinique pour le suivi rapide de la sensibilité des bactéries aux antibiotiques.Abstract : Monitoring the viability, growth and cellular metabolism of bacteria can contribute significantly to the early diagnosis of disease, but can also help improve yield of bacterial products in industrial- or small-scale experiments. Conventional methods applied for investigation of antibiotic sensitivity of bacteria are mostly culture-based techniques that are time-consuming and take at least 12 h to reveal results. This delay leads to overtreatment of a wide range of infections with broad spectrum antibiotics which is costly and may lead to the development of resistance to these precious antibiotics, whereas rapid detection of a viral infection or absence of bacteria could prevent such treatments and, in the case of bacterial infection, identification of antibiotic susceptibility could allow use of narrow spectrum antibiotics. The project outlined in this document aims at monitoring biological activities of live bacteria immobilized on biofunctionalized surfaces of quantum semiconductor (QS) microstructures. The method takes advantage of the sensitivity of photoluminescence (PL) emitting semiconductors to the perturbation of the electric field induced by the electric charge of bacteria immobilized on the surface of these structures. Our hypothesis was that bacteria growing on the surface of biofunctionalized QS biochips would modify their PL in a different, and measurable way in comparison with inactivated bacteria. In the first phase of the project, we investigated an innovative method involving PL monitoring of the photocorrosion effect in GaAs/AlGaAs heterostructures. Maintaining the balance between device sensitivity and stability in the biosensing (aqueous) environment allowed us to detect Escherichia coli K12 in phosphate buffered saline solutions (PBS) at an attractive limit of detection of 103 CFU/mL in less than 2 hours. Following this research, we hypothesised that these heterostructures could be employed to develop a method for inexpensive and quasi-real time monitoring of the growth and antibiotic susceptibility of bacteria. One of the key elements in the development of this biosensing platform was to demonstrate that GaAs (001), normally used for capping PL emitting GaAs/AlGaAs heterostructures, would not inhibit the growth of bacteria. In the second phase of the project, we explored the capture and growth of E. coli K12 on bare and biofunctionalized surfaces of GaAs (001). It has been determined that the initial coverage, and the subsequent bacterial growth rates are dependent on the biofunctionalization architecture used to capture bacteria, with antibody biofunctionalized surfaces exhibiting significantly higher capture efficiencies. Moreover, for suspensions containing bacteria at less than 105 CFU/mL, it has been found that the surface of GaAs wafers could not support the growth of bacteria, regardless of the type of biofunctionalization architecture. In the third phase of the project, we used PL to monitor the growth and antibiotic susceptibility of E. coli K12 and E. coli HB101 bacteria. While immobilization of bacteria on the surface of GaAs/AlGaAs heterostructures retards the PL monitored photocorrosion, growth of these bacteria further amplifies this effect. By comparing the photocorrosion rate of QS wafers exposed to bacterial solutions with and without antibiotics, the sensitivity of bacteria to the specific antibiotic could be determined in less than 3 hours. Due to the small size, low cost and rapid response of the biosensor, the proposed approach has the potential of being applied in clinical diagnostic laboratories for quick monitoring of antibiotic susceptibility of different bacteria

Microassembly technology for modular, polymer microfluidic devices

Assembly of modular, polymer microfluidic devices with different functions to obtain more capable instruments may significantly expand the options available for detection and diagnosis of disease through DNA analysis and proteomics. For connecting modular devices, precise, passive alignment structures can be used to prevent infinitesimal motions between the devices and minimize misalignment. The motion and constraint of passive alignment structures were analyzed using screw theory. A combination of three v-groove and hemisphere-tipped post joints constrained all degrees of freedom of the two mating modules without overconstraint. Simulations and experiments were performed to assess the predictability of dimensional and location variations of injection molded components. A center-gated disk with micro scale assembly features was replicated. Simulations using a commercial package (Moldflow) overestimated replication fidelity. Mold surface temperatures and injection speeds significantly affected the experimental replication fidelity. The location of features for better replication, at each mold surface temperature, moved from the edge of the mold cavity to the injection point as the mold surface temperature increased from 100˚C to 150˚C. Prototype modular devices were replicated using double-sided injection molding for the experimental demonstration. Dimensional and location variations of the assembly features and alignment standards were quantified for an assembly tolerance analysis. Monte Carlo methods were applied to the assembly tolerance analysis to simulate propagation and accumulation of variation in the assembly. In simulations, mean mismatches with standard deviations ranged from 115±29 to 118±30 µm and from 17±11 to 19±13 µm along the X- and Y-axes, respectively. Vertical gaps with standard deviations at the X- and Y-axes were 312±37~319±37 µm, compared to the designed value of 287µm. The measured lateral mismatches were 103±7~116±11 µm and 15±9~20±6 µm along the X- and Y-axes, respectively. The vertical gaps ranged from 277±4 µm to 321±7 µm at the X- and Y-axes, respectively. The present study combined an investigation of microassembly technology with a better understanding of the micro injection molding process, to assist in realizing cost-effective mass production of modular, polymer microfluidic devices enabling biochemical analysis