Lab-on-a-chip platforms for pathogen analysis

Infectious diseases caused by pathogenic microorganisms are a big burden in developed and developing countries. The emergence and rapid global spread of virus and antimicrobial resistant bacteria is a significant threat to patients, healthcare systems and the economy of countries. Early pathogen detection is often hampered by low concentrations present in complex matrices such as food and body fluids.Microfluidic technologies offer new and improved approaches for detection of pathogens on the microscale. Here, two microfluidic platforms for pathogen sorting and molecular identification were investigated: (1) inertial focusing and (2) microscale immiscible filtration. Inertial focusing in two serpentine channel designs etched in glass at different depths was evaluated with different microparticles, bacteria and blood. The shallow design allowed 2.2-fold concentration of Escherichia coli O157 cells, whereas the deep design accomplished recovery of 54% E. coli O157 depleted from 97% red blood cells in 0.81% haematocrit at flowrates of 0.7 mL min-1.A lab-on-a-chip platform based on microscale immiscible filtration was investigated for capture and detection of nucleic acids and bacteria. For nucleic acids, oligo (dT) functionalised magnetic beads or silica paramagnetic particles in GuHCl were used to capture genomic RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and genomic DNA from Neisseria gonorrhoeae, respectively. On-chip amplification and detection were performed via colorimetric loop-mediated isothermal amplification (LAMP). Results showed sensitive and specific detection of targeted nucleic acids (470 RNA copies mL-1 and 5 × 104 DNA copies mL-1) with no cross-reactivity to other RNAs and DNAs tested. The whole workflow was integrated in a single device and time from sample-in to answer-out was within 1h. The platform only required power for a heat source and showed potential for point of care diagnostics in resource-limited settings. For bacteria detection, anti-E. coli O157 functionalised magnetic beads were used to capture cells with > 90% efficiency and on-chip fluorescence in situ hybridisation and a staining assay were explored for bacteria identification.A wide variety of microfluidic approaches for pathogen analysis have been devised in the literature with different advantages and drawbacks. Careful evaluation based on their purpose, integrated steps and end user is critical. Input from stakeholders right from the start of a project and throughout is vital to success. The platforms investigated herein have potential for applications such as sample preparation, pathogen concentration and specific molecular detection of E. coli O157, N. gonorrhoeae DNA, and SARS-CoV-2 RNA. With further development and clinical validation, the widespread use of these systems could facilitate early diagnosis of infectious diseases, allowing timely management of outbreaks and treatment and slowing the incidence of antimicrobial resistance

Computational inertial microfluidics:a review

Since the discovery of inertial focusing in 1961, numerous theories have been put forward to explain the migration of particles in inertial flows, but a complete understanding is still lacking. Recently, computational approaches have been utilized to obtain better insights into the underlying physics. In particular, fundamental aspects of particle focusing inside straight and curved microchannels have been explored in detail to determine the dependence of focusing behavior on particle size, channel shape, and flow Reynolds number. In this review, we differentiate between the models developed for inertial particle motion on the basis of whether they are semi-analytical, Navier-Stokes-based, or built on the lattice Boltzmann method. This review provides a blueprint for the consideration of numerical solutions for modeling of inertial particle motion, whether deformable or rigid, spherical or non-spherical, and whether suspended in Newtonian or non-Newtonian fluids. In each section, we provide the general equations used to solve particle motion, followed by a tutorial appendix and specified sections to engage the reader with details of the numerical studies. Finally, we address the challenges ahead in the modeling of inertial particle microfluidics for future investigators

Harvinaisten merkkaamattomien solujen kokoon perustuva erottelu mikrofluidistiikalla

Separating and isolating rare cells with cheap methods and rapid prototyping is attractive in analysing DNA in foetal cells circulating in maternal blood with non-invasive methods. Inertial microfluidics as a separation method is based on equilibrium points or trajectories that the particles migrate during flow. In this work, three different separation devices and one with a main purpose is concentration of diluted solutions after separation were investigated. The purpose of the thesis is to gain inertial focusing and particle separation. The chips were fabricated by using soft-lithography process with SU-8 masters for transferring patterns on PDMS. After curing, the chips were cut, punched and treated with oxygen plasma to bond on microscope slides. Flow thru the devices was produced with syringe pumps. Devices were optimized first using de-ionized water, then investigated with different concentrations of polystyrene microparticles and finally with cells to gain preliminary results. Devices were tested within 0.006 ml/min to 10 ml/min range. Non-equilibrium inertial array chip (NISA chip) is based on wall induced inertial lift force and siphoning by geometry induced pressure difference. NISA chip was investigated to separate 8 µm from 10 µm particles. The device had optimization issues with back-flow to feed, particle attachment to islands and low concentration of output samples. Spiral chip is based on net inertial lift forces and Dean secondary flow induced by curvature of the device. The device was investigated with large throughput (above 4.9 ml/min flow rate) to separate 10 µm particles from 15 µm particles. Spiral chip had suboptimal outlet design, which let to large deviation in data. However, the results showed particle focusing during videoing of the flow although with low separation efficiency. Labyrinth chip is likewise based on net inertial forces and Dean secondary flow as well as alternating corner design that induces additional mixing of particles. The chip was investigated with reasonably high throughput (above 1.75 ml/min flow rate) to separate 10 µm particles from 15 µm particles. The device showed inertial focusing both in video results and the particle analysis. Separation was seen in particle analysis. Concentrator chip is based on inertial focusing and siphoning. Chip was investigated to concentrate solution of 10 µm 105 particles/ml tenfold. The device showed effective concentration using optimal flow rate with particles and using slightly slower flow rate with preliminarily cell experiments.Harvinaisten solujen erottelu ja eristäminen edullisilla menetelmillä ja nopealla prototypoinnilla on houkuttelevaa, kun analysoidaan verenkierrossa olevia kasvainsoluja tai DNA:ta sisältäviä sikiönsoluja äidin verestä ei-invasiivisesti. Inertiaalinen mikrofluidistiikka erottelumenetelmänä perustuu erikokoisten solujen taipumukseen asettua eri tasapainopisteisiin tai lentoradoille virtauksessa. Tässä työssä kolme erottelulaitetta sekä yksi solujen konsentrointiin erottelun jälkeen tähtäävä laite tutkittiin, jotta selvitettäisiin laitteiden kyky fokusoida ja erotella partikeleita. Sirut valmistettiin pehmyt-litografialla, jossa SU-8 mastereilla siirrettiin mikrofluidistiset kanavat PDMS:ään. Kovettumisen jälkeen sirut leikattiin, rei’itettiin, käytettiin happiplasmassa ja bondattiin mikroskooppilaseihin. Virtauskokeissa käytettiin ruiskupumppua. Laitteet tutkittiin ja optimoitiin alustavasti ensin deionisoidulla vedellä ja sitten erilaisilla polystyreeni mikropartikkeli liuoksilla ja lopuksi vielä alustavilla solukokeilla. Laitteet tutkittiin virtausnopeusvälillä 0.006 ml/min - 10 ml/min. Inertiaaliarray siru (NISA siru) perustuu kanavan saarien seinien aiheuttamaan inertiaalivoimaan ja lappoon, joka syntyy geometriasta johtuvista paine-eroista. Laite tutkittiin tarkoituksena erottaa 8 µm partikkelit 10 µm partikkeleista. Laitteen optimointi osoittautui haasteelliseksi johtuen nesteen takaisinvirtauksesta syötteeseen, partikkelien tarttumisesta saariin ja ulostulojen matalasta konsentraatiosta. Spiraali siru perustuu inertiaalivoimiin ja Deanin sekundaari virtauksiin, jotka johtuvat kanavan kaareutuvuudesta, sekä kanavan pylväiden virtausta supistavaan vaikutukseen. Laite tutkittiin korkeilla virtausnopeuksilla tarkoituksena erottaa 10 µm partikkelit 15 µm partikkeleista. Spiraali sirun ulostulon design aiheutti korkeaa hajontaa partikkelianalyysissa. Toisaalta, laitteen virtauksen videotuloksissa ja partikkelianalyysissa on nähtävissä selkeä inertiaalifokusointi. Labyrintti siru perustuu myös inertiaalivoimiin ja Deanin sekundaari virtoihin sekä laitteen designissa olevien kulmien partikkeleita sekoittavaan vaikutukseen. Laite tutkittiin korkeahkoilla virtausnopeuksilla keskittyen 10 µm ja 15 µm partikkelien erotteluun. Laitteen virtauksen videotuloksissa ja partikkelianalyysissa on selkeä fokusointi. Lisäksi erottelua oli havaittavissa partikkelianalyysissa. Konsentraattori siru perustuu inertiaalifokusointiin ja lappoon. Laite tutkittiin tarkoituksena konsentroida 10 µm 105 partikkelia/ml kymmenkertaisesti. Laitteessa oli havaittavissa selkeää konsentroitumista partikkeleilla optimaalisella virtausnopeudella ja alustavissa solukokeissa hieman matalammalla virtausnopeudella

Microscale methods to investigate and manipulate multispecies biological systems

The continuing threats from viral infectious diseases highlight the need for new tools to study viral interactions with host cells. Understanding how these viruses interact and respond to their environment can help predict outbreaks, shed insight on the most likely strains to emerge, and determine which viruses have the potential to cause significant human illness. Animal studies provide a wealth of information, but the interpretation of results is confounded by the large number of uncontrolled or unknown variables in complex living systems. In contrast, traditional tissue culture approaches have provided investigators a valuable platform with a high degree of experimental control and flexibility, but the static nature of flask-based cell culture makes it difficult to study viral evolution. Serial passaging introduces un-physiological perturbations to cell and virus populations by drastically reducing the number of species with each passage. Low copy, high fitness viral variants maybe eliminated, while in vivo these variants would be essential in determining the virus’ evolutionary fate. Bridging technologies are urgently needed to mitigate the unrealistic dynamics in static flask-based cultures, and the complexity and expense of in vivo experiments. This thesis details the development of a continuous perfusion platform capable of more closely mimicking in vivo cell-virus dynamics, while surpassing the experimental control and flexibility of standard cell culture. First, a microfluidic flow through acoustic device is optimized to enable efficient and controllable separation of cells and viruses. Repeatable isolation of cell and virus species is demonstrated with both a well-characterized virus, Dengue Virus (DENV), and the novel Golden Gate Virus. Next, a platform is built around this device to enable controllable, automated, continuous cell culture. Beads are used to assess system performance and optimize operation. Subsequently, the platform is used to culture both murine hybridoma (4G2) and human monocyte (THP-1) cell lines for over one month, and demonstrate the ability to manipulate population dynamics. Finally, we use the platform to establish a multispecies culture with THP-1 cells and Sindbis Virus (SINV). This work integrates distinct engineering feats to create a platform capable of enhancing existing cell virus studies and opening the door to a variety of high-impact investigations

Space Shuttle flying qualities and flight control system assessment study, phase 2

A program of flying qualities experiments as part of the Orbiter Experiments Program (OEX) is defined. Phase 1, published as CR-170391, reviewed flying qualities criteria and shuttle data. The review of applicable experimental and shuttle data to further define the OEX plan is continued. An unconventional feature of this approach is the use of pilot strategy model identification to relate flight and simulator results. Instrumentation, software, and data analysis techniques for pilot model measurements are examined. The relationship between shuttle characteristics and superaugmented aircraft is established. STS flights 1 through 4 are reviewed from the point of view of flying qualities. A preliminary plan for a coordinated program of inflight and simulator research is presented

Modeling and simulation of a Stewart platform type parallel structure robot

The kinematics and dynamics of a Stewart Platform type parallel structure robot (NASA's Dynamic Docking Test System) were modeled using the method of kinematic influence coefficients (KIC) and isomorphic transformations of system dependence from one set of generalized coordinates to another. By specifying the end-effector (platform) time trajectory, the required generalized input forces which would theoretically yield the desired motion were determined. It was found that the relationship between the platform motion and the actuators motion was nonlinear. In addition, the contribution to the total generalized forces, required at the actuators, from the acceleration related terms were found to be more significant than the velocity related terms. Hence, the curve representing the total required actuator force generally resembled the curve for the acceleration related force. Another observation revealed that the acceleration related effective inertia matrix I sub dd had the tendency to decouple, with the elements on the main diagonal of I sub dd being larger than the off-diagonal elements, while the velocity related inertia power array P sub ddd did not show such tendency. This tendency results in the acceleration related force curve of a given actuator resembling the acceleration profile of that particular actuator. Furthermore, it was indicated that the effective inertia matrix for the legs is more decoupled than that for the platform. These observations provide essential information for further research to develop an effective control strategy for real-time control of the Dynamic Docking Test System

Multinode Acoustic Systems for High Throughput Cellular Analysis

For decades, flow cytometry is used as the gold standard for cellular analyses as it measures multiple properties of single cells. Traditional flow cytometry uses the hydrodynamic focusing technique where the sheath fluid focuses cells in the sample into a narrow stream. Although such precise focusing provides accurate optical measurements, high sheath fluid pressure and high linear velocities limit analysis rate to 50,000 particles/s. Such rates are too low for detecting rare events where one cell may have to be detected in a population of about a billion cells. Therefore, it is necessary to eliminate the sheath fluid and improve throughput by several orders of magnitude by using a suitable alternate focusing mechanism that allows focusing cells in high volume samples into multiple focused streams. Toward this aim, this dissertation presents the multinode acoustic technique that uses high frequency ultrasonic waves to focus particles and cells into highly parallel focused streams. One challenge, however, is that acoustic attenuation is significant at high frequencies. Therefore, to optimize acoustic energy density within multinode acoustic flow cells, a simple model is derived based on exhaustive literature review, suggesting that attenuation may be minimized by proper choice of fabrication material. Using such acoustically transparent materials, multinode acoustic flow cells were fabricated by three approaches, which include using machined aluminum frame and glass slides, disposable rectangular glass capillaries and etched silicon flow cells fabricated using standard photolithography and deep reactive ion etching techniques. Among these, flexibility in design using microfabrication approach has allowed fabricating etched flow cells having multiple parallel channels. Such parallelization improves acoustic energy density within each channel and precisely focuses particles at volumetric throughput of few tens of mL/min. Finally, this dissertation presents a proof-of-concept flow cytometry instrumentation using laser line generation optics and microscopy image sensors for imaging parallel focused streams in multinode acoustic flow cells. High throughput and precise focusing suggest that multinode focusing is a suitable alternative to conventional hydrodynamics, and multinode acoustic flow cells integrated with such optical imaging systems incorporating real-time signal processing circuitry will provide throughput matching that required for the detection of circulating tumor cells

Modeling and Control of a Multibody Hinge-BargeWave Energy Converter

Wave Energy Converters (WECs) are devices used to extract energy from the waves. The particular WEC considered in this thesis is a three-body hinge-barge WEC, which is an articulated floating structure composed of 3 rectangular bodies interconnected by hinges, and it operates longitudinally to the direction to the incoming wave. The relative motion between each pair of bodies drives a Power Take-Off (PTO) system, which extracts the energy from the waves. The objective of this thesis is to increase the energy that can be extracted by a three-body hinge-barge WEC using an optimal control strategy, which computes the optimal loads applied by the PTOs driven by the relative motion between the bodies. The optimal control is formulated in the time domain, and computes the PTO loads in a coordinated way, so that the total energy extracted by the device is maximized. The optimal control strategy is formulated for a three-body hinge-barge WEC that is equipped with either passive or active PTOs. In this thesis, an optimal control strategy, for the maximization of the energy extracted by a three-body hinge-barge WEC, is derived with Pseudo-Spectral (PS) methods, which are a subset of the class of techniques used for the discretisation of integral and partial differential equations known as mean weighted residuals. In particular, PS methods based on Fourier basis functions, are used to derive an optimal control strategy, for a finite time horizon. Therefore, an optimal control strategy, with PS methods based on Fourier basis functions, cannot be applied for realtime control of the WEC, as Fourier basis functions can only represent the steady-state response of the WEC. However, PS methods based on Fourier basis functions provide a useful framework for the evaluation of the achievable power absorption performance of the WEC, with both active and passive PTOs. The Receding Horizon (RH) real-time optimal control of a three-body hingebarge WEC is derived with PS methods based on Half-Range Chebyshev-Fourier (HRCF) basis functions. The RH optimal real-time controller, with PS methods based on HRCF basis functions, maximizes the energy extracted by the WEC at each time step over a moving control horizon. In contrast to Fourier basis functions, HRCF basis functions are well suited for the approximation of non-periodic signals, allowing the representation of both the transient and steady-state response of the WEC. The optimal control strategy, with PS methods based on either Fourier or HRCF basis functions, is based on a dynamic model of the device, which is derived with two different modeling methodologies, that can be also applied to other types of multiple body WECs. The modeling methodologies are validated against wave-tank tests carried out on a 1/7th scale two-body hingebarge device, and a 1/25th and 1/20th scale three-body hinge-barge device