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Magnetic bead-based DNA extraction and purification microfluidic chipAzimi, Sayyed Mohamad January 2010 (has links)
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.
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Immunoaffinity Monoliths for Multiplexed Extraction of Preterm Birth Biomarkers from Human Blood Serum in 3D Printed Microfluidic DevicesAlmughamsi, Haifa Mohammad 06 August 2021 (has links)
Preterm birth (PTB) results in over 15 million early births annually and is the leading cause of neonatal deaths. There are no clinical methods currently available to evaluate risk of PTB at early stages in pregnancy; thus, a rapid diagnostic to analyze PTB risk would be beneficial. Microfluidic immunoaffinity extraction is a promising platform for preparing complex samples, such as maternal serum with PTB risk biomarkers. 3D printed microfluidic devices have advantages over conventional microfluidic systems including simple fabrication and potential for iterative optimization to improve designs. In this work, I developed immunoaffinity monoliths in 3D printed microfluidic devices modified with antibodies to enrich PTB biomarkers from human blood serum. I retained and eluted a peptide PTB biomarker in both buffer and blood serum using an immunoaffinity column. An additional three PTB biomarkers were also successfully extracted either from buffer or blood serum on single-antibody columns. Both polyclonal and monoclonal antibodies to PTB biomarkers were characterized by dot blots, biolayer interferometry, and surface plasmon resonance to determine their specificity and dissociation constants. I created multiplexed immunoaffinity columns to simultaneously enrich three PTB biomarkers from depleted human blood serum in a single extraction. This is the first demonstration of multiplexed immunoaffinity columns for PTB biomarkers in a 3D printed microfluidic device. My work is a key step towards the future development of 3D printed microfluidic devices for rapid PTB testing.
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Absorption Flow-Cytometry for Point-of-Care DiagnosticsBanoth, Earu January 2017 (has links) (PDF)
Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology.
Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated.
In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs.
In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells.
In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well.
As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.
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Quantification des immunoglobulines A par résonance des plasmons de surface pour identifier les individus IgA-déficientsDubois, Caroline 12 1900 (has links)
Le but de ce projet, en collaboration avec Héma-Québec, était de développer une méthode
point de service (POC) pour la quantification des immunoglobulines A (IgA) dans un petit volume
de plasma humain. Les IgA jouent un rôle important au niveau de notre système immunitaire,
surtout au niveau des bio fluides sur les muqueuses comme la salive. On peut les retrouver
normalement dans le plasma à des concentrations plus grandes que 2000 ng/mL pour la majorité
des gens. Dans le cas d’une personne immunosupprimée, une concentration plus basse que 500
ng/mL est observée. La déficience en IgA est souvent associée à des maladies auto-immunes
comme la maladie de Crohn. Ces individus peuvent éprouver un choc anaphylactique à la suite
d’une transfusion de sang, ce qui nous motive à utiliser la résonance des plasmons de surface (SPR)
sur place à des collectes de sang et dans les milieux hospitaliers.
Pour réaliser ce capteur d’immunodéficience en IgA, un anticorps primaire spécifique aux
IgA fut lié à un peptide formant une monocouche auto-assemblée sur une surface d’or du capteur
SPR. Un deuxième anticorps a été utilisé pour quantifier spécifiquement cet analyte dans une
matrice humaine complexe. Plusieurs étapes ont été optimisées pour obtenir une limite de détection
basse, soit l’étape d’immobilisation de l’anticorps de capture, le type d’anticorps, le pH pour
l’immobilisation, la détection secondaire, la validation dans différentes matrices biologiques, parmi
d’autres. Le capteur a été validé avec quelques échantillons cliniques et la réponse SPR fut
comparée avec ELISA. Ainsi, nous anticipons que cette méthode pourrait être utile à des collectes
de sang pour assurer les réserves de sang déficient en IgA. / The goal of this project, in collaboration with Héma-Québec, was to develop a point-ofcare (POC) method to quantify immunoglobulin A (IgA) in a small volume of human plasma.
Immunoglobulin A play an important role in immunity, specifically in biofluids on mucosaes like
saliva. It can generally be found in plasma at concentrations higher than 2000 ng/mL for most
people. In the case of an immunosuppressed individual, a concentration under 500 ng/mL is
observed. IgA-deficiency is often associated with autoimmune diseases, such as Crohn’s disease.
These individuals may experience an anaphylactic shock following a blood transfusion, which
motivated us to use surface plasmon resonance (SPR) on site at blood drives and in a hospital
setting.
SPR quantifies the interactions between biomolecules. It is a complementary technique to
ELISA that can test more rapidly individuals susceptible of being IgA-deficient in a hospital setting
or at blood drives. To develop an IgA SPR sensor, a specific capture antibody to IgA was bound
to a peptide monolayer on the gold surface. A second antibody was used to specifically quantify
IgA in a complex matrix. Multiple steps were optimized to achieve the low detection limits required
for IgA detection, such as the capture antibody immobilisation step, type of antibody, pH of
immobilisation buffer, secondary detection, validation in different biological matrices, among
others. The SPR sensor was validated with a few clinical samples, and compared to ELISA, to
demonstrate the potential. As such, we envision that this rapid method may be used at blood drives
to ensure sufficient reserves of IgA-deficient blood.
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Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food controlCalero Alcarria, María del Señor 02 May 2022 (has links)
Tesis por compendio / [ES] La sociedad actual demanda un mayor control en la seguridad y calidad de los alimentos que se consumen. Esta preocupación se ve reflejada en los diferentes planes estatales y europeos de investigación científica, los cuales, plantean la necesidad de innovar y desarrollar nuevas técnicas analíticas que cubran los requerimientos actuales. En el presente documento se aborda el problema de la presencia de residuos químicos en la miel. El origen de los mismos se debe, fundamentalmente, a los tramientos veterinarios con los que se tratan enfermedades y parásitos en las abejas, y a los tratamientos agrícolas con los que las abejas se ponen en contacto cuando recolectan el néctar en cultivos próximos a las colmenas. La Agencia Europea de Seguridad Alimentaria (EFSA) confirma esta realidad al notificar numerosas alertas sanitarias en la miel.
En los últimos años, los métodos de análisis basados en inmunosensores piezoeléctricos se han posicionado como la base de una técnica de cribado muy prometedora, la cual puede ser empleada como técnica complementaria a las clásicas de cromatografía, gracias a su sencillez, rapidez y bajo coste. La tecnología de resonadores High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detección directa en tiempo real, alta sensibilidad y selectividad con un fácil manejo y coste reducido en comparación con otras técnicas. Además, está tecnología permite aumentar el rendimiento del análisis mediante el diseño de arrays de resonadores en un mismo sustrato (Monolithic HFF-QCMD). En este documento se presenta el diseño de un array de 24 sensores HFF-QCMD, junto con un cartucho de micro-fluídica que traza diversos microcanales sobre los diferentes elementos sensores, a los que hace llegar la muestra de miel diluida a analizar. El cartucho actúa también como interfaz para realizar la conexión entre el array de resonadores y el instrumento de caracterización de los mismos. Para obtener el máximo partido del array diseñado, se desarrolla un método de medida robusto y fiable que permite elevar la tasa de adquisición de datos para facilitar la toma de registros eléctricos de un elevado número de resonadores de forma simultánea, e incluso en varios armónicos del modo fundamental de resonancia. La gran sensibilidad de la tecnología HFF-QCMD a los eventos bioquímicos a caracterizar se extiende también a otro tipo eventos externos, como son los cambios de temperatura o presión, lo que es necesario minimizar con el fin de reducir el impacto que estas perturbaciones no deseadas provocan en la estabilidad y fiabilidad de la medida. Con este fin, se desarrolla un algoritmo de procesado de señal basado en la Discrete Transform Wavelet (DTW). Finalmente, todos los desarrollos tecnológicos realizados se validan mediante la implementación de un inmunoensayo para la detección simultánea, en muestras de mieles reales, de residuos químicos de naturaleza química muy diferente, a saber, el fungicida tiabendazol y el antibiótico sulfatiazol. / [CA] La societat actual demanda un major control en la seguretat i qualitat dels aliments que es consumeixen. Aquesta preocupació es veu reflectida en els diferents plans estatals i europeus d'investigació científica, els quals, plantegen la necessitat d'innovar i desenvolupar noves tècniques analítiques que cobrisquen els requeriments actuals. En el present document s'aborda el problema de la presència de residus químics en la mel. L'origen dels mateixos es deu, fonamentalment, als tractaments veterinaris amb els quals es tracten malalties i paràsits en les abelles, i als tractaments agrícoles amb els quals les abelles es posen en contacte quan recol·lecten el nèctar en cultius pròxims als ruscos. L'Agència Europea de Seguretat Alimentària (EFSA) confirma aquesta realitat notificant nombroses alertes sanitàries en la mel.
En els últims anys, els mètodes d'anàlisis basades en immunosensors piezoelèctrics s'han posicionat com la base d'una tècnica de garbellat molt prometedora, la qual pot ser emprada com a tècnica complementària a les clàssiques de cromatografia, gràcies a la seua senzillesa, rapidesa i baix cost. La tecnologia de ressonadors High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detecció directa en temps real, alta sensibilitat i selectivitat amb un fàcil maneig i cost reduït en comparació amb altres tècniques. A més, està tecnologia permet augmentar el rendiment del anàlisi mitjançant el disseny d'arrays de ressonadors en un mateix substrat (Monolithic HFF-QCMD). En aquest document es presenta el disseny d'un array de 24 sensors HFF-QCMD, juntament amb un cartutx de microfluídica que estableix diversos microcanals sobre els diferents elements sensors, als quals fa arribar la mostra de mel diluïda a analitzar. El cartutx actua també com a interfície per a realitzar la connexió entre l'array de ressonadors i l'instrument de caracterització d'aquests. Per a traure el màxim partit a l'array dissenyat, es desenvolupa un mètode de mesura robust i fiable que permet elevar la taxa d'adquisició de dades per a facilitar la presa de registres elèctrics d'un elevat nombre de ressonadors de manera simultània, i fins i tot en diversos harmònics del mode fonamental de ressonància. La gran sensibilitat de la tecnologia HFF-QCMD als esdeveniments bioquímics a caracteritzar s'estén també a un altre tipus esdeveniments externs, com són els canvis de temperatura o pressió, la qual cosa és necessari minimitzar amb la finalitat de reduir l'impacte que aquestes pertorbacions no desitjades provoquen en l'estabilitat i fiabilitat de la mesura. A aquest efecte, es desenvolupa un algorisme de processament de senyal basat en la Discrete Transform Wavelet (DTW). Finalment, tots els desenvolupaments tecnològics realitzats es validen mitjançant la implementació d'un immunoassaig per a la detecció simultània, en mostres de mel reals, de residus químics de naturalesa química molt diferent, a saber, el fungicida tiabendazol i l'antibiòtic sulfatiazol. / [EN] Currently, society demands greater control over the safety and quality of the food consumed. This concern is reflected in the different states and European plans for scientific research, which establish the necessity to innovate and develop new analytical techniques that meet current requirements. This document addresses the problem of the presence of chemical residues in honey. Its origin is fundamentally due to the veterinary treatments against diseases and parasites in bees, and also to the agricultural treatments with which the bees come into contact when they collect the nectar in crops close to the hives. The European Food Safety Agency (EFSA) confirms this reality by notifying numerous health alerts in honey.
In recent years, analysis methods based on piezoelectric immunosensors have been positioned as the basis of a very promising screening technique, which can be used as a complementary technique to the classic chromatography, thanks to its simplicity, speed and low cost. High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) resonator technology combines direct real-time detection, high sensitivity and selectivity with easy handling and low cost compared to other techniques. In addition, this technology allows increasing the performance of the analysis through the design of resonator arrays on the same substrate (Monolithic HFF-QCMD). This document presents the design of an array of 24 HFF-QCMD sensors, together with a microfluidic cartridge that establish various microchannels on the different sensor elements, to provide them the diluted honey sample to be analyzed. The cartridge also acts as an interface to make the connection between the array of resonators and the characterization instrument. To get the most out of the designed array, a robust and reliable measurement method has been developed that allows increasing the data acquisition rate to facilitate electrical parameters readout from a high number of resonators simultaneously, and even in several harmonics of the fundamental resonance mode. The great sensitivity of the HFF-QCMD technology to the biochemical events to be characterized also is extended to other types of external events, such as changes in temperature or pressure, which must be minimized in order to reduce the impact that these unwanted disturbances cause in the stability and reliability of the measurement. To this end, a signal processing algorithm based on the Discrete Transform Wavelet (DTW) is developed. Finally, all the technological developments carried out are validated through the implementation of an immunoassay for the simultaneous detection, in real honey samples, of chemical residues of very different chemical nature, namely, the fungicide thiabendazole and the antibiotic sulfathiazole. / The authors would also like to thank Jorge Martínez from the Laboratory of High Frequency Circuits (LCAF) of the Universitat Politècnica de València (UPV) for assistance with profilometry, and Manuel Planes, José Luis Moya, Mercedes Tabernero, Alicia Nuez and Joaquin Fayos from the Electron Microscopy Services of the UPV for helping with the AFM, and SEM measurements. M.Calero is the recipient of the doctoral fellowship BES-2017-080246 from the Spanish Ministry of Economy, Industry and Competitiveness (Madrid, Spain). This research was funded by Spanish Ministry of Economy and Competitiveness with FEDER funds (AGL 2016-77702-R) and European Commission Horizon 2020 Programme (Grant Agreement number H2020-FETOPEN-2016-2017/737212-CATCH-U-DNA - Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics) for which the authors are grateful. Román Fernández is with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain and with Advanced Wave Sensors S.L., Paterna, València, Spain. (e-mail: rfernandez@awsensors.com); Yolanda Jiménez, Antonio Arnau and María Calero are with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain; Ilya Reiviakine is with Advanced Wave Sensors S.L., Paterna, Valencia, Spain and with the Department of Bioengineering, University of Washington, Seattle, WA, 98150 USA; María Isabel Rocha-Gaso and José Vicente García are with Advanced Wave Sensors S.L., Paterna, València, Spain. / Calero Alcarria, MDS. (2022). Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food control [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182652 / TESIS / Compendio
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