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1
Harrison, Olivia Jane. "Integrated platform to assay melanoblast development in vitro." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31164.
Full textAbstract:
Melanoblasts are the embryonic precursors of melanocytes, the pigment producing cells of the skin and hair. Melanoblasts are of key interest to developmental biologists for numerous reasons, including their ability to migrate throughout the body from a single origin in the neural crest (NC). Current methods for the study of the melanocyte lineage are limited by the heavy reliance on animal models. To challenge this, a platform of in vitro tools were designed to replace and complement current studies. A major obstacle is the transition from 2D cultures, which provide only limited behavioural information, to 3D models which are able to recapitulate the environmental conditions. 3D cultures are regularly created using tissue samples and synthetic matrices for attachment, but building a model from cell lines only has not been achieved. A co-culture model using immortalised keratinocyte (COCA) and melanoblast cell lines proved unsuitable for observing developmental processes, due to lack of movement at high cell densities, but may be practical in pigmentation research. Other methods were explored to examine melanoblast behaviour, including the use of cell derived matrices (CDMs) integrated with melanoblast cell lines, and aggregates formed by hanging drop (HD) culture. CDMs were successfully generated from the COCA line, as well as NIH3T3 fibroblasts which has been shown previously. These structures are denuded of cells to leave the deposited extracellular matrix (ECM) components intact, representative of the dermal (fibroblast) and epidermal (keratinocyte) layers of the skin. HDs were prepared from cultured melanoblast cell lines, and form tight aggregates which disseminate when plated, in a manner similar to the dissemination of cells from the NC in explant cultures. The receptor tyrosine kinase KIT and its ligand (KITL), are vital for melanoblast development. Previous study of this signalling complex has often focussed on the haematopoietic lineage and spermatogenesis, where they perform essential roles. KITL is expressed in a membrane localised form found on the surface of keratinocytes thought to promote melanoblast/melanocyte survival, and a soluble isoform found sequestered in the ECM which promotes cell migration. Cell lines expressing fluorescently tagged KIT and KITL were created to visualise their interactions using live-cell confocal imaging. Firstly, cell lines were generated to perform co-culture experiments with KIT and KITL, and we showed that these constructs are able to interact by uptake of KITL into KIT cells. Secondly, tandem fluorescent protein timers of KIT and KITL were generated which were used to observe protein kinetics. We showed that these protein timers can be manipulated using cycloheximide to block protein production, or by increasing ligand availability. These protein timers reveal that soluble KITL (sKITL) has a faster turnover than membrane bound KITL (mKITL), and that in all three proteins, there is distinct change in spatial localisation as the proteins age. Using a novel melanoblast reporter mouse, Pmel-CMN, primary mouse melanoblasts between E12.5 and E14.5 were isolated for RNA sequencing. This time period is the earliest reported for melanoblast isolation for use in gene expression analysis. We show that within this time course, there are significant changes in the RNA expression profiles, including decreasing expression of other NC cell markers, and huge increasing expression of pigmentation genes. To assess the biological relevance of using in vitro assays, cells of the immortalised melanoblast cell line, melb-a, were cultured under different conditions and examined via RNA sequencing. Results reveal differences in several areas between primary cells and those in culture, including loss of melanocyte specificity. The different tools described in this thesis provide a platform on which to study various aspects of cell behaviour, including migration, morphology and cell adhesion at both the individual cell and population levels.
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Sun, Han. "Novel microfluidic platform for bioassays." HKBU Institutional Repository, 2019. https://repository.hkbu.edu.hk/etd_oa/699.
Full textAbstract:
Microfluidics have been created to acquire, operate, and process complex fluids in extremely tiny volumes with high efficiency and high speed, and without the requirement for an experienced operator. In addition, microfluidic systems also enable miniaturization and incorporation of different complex functions, which can help bring intricate diagnostic tools out of the laboratories. Ideally, these systems should be inexpensive, precise, reliable, robust, and well-suited to the medical diagnostic systems. Most of the microfluidic devices reported previously were based on devices made of polydimethylsiloxane (PDMS). PDMS is a material that dissolves in many common organic solvents. Meanwhile, it is also prone to absorb small molecules like the proteins, which is detrimental to a stable and reliable result. Current work focuses on bioassays that are badly needed in our life and these bioassays are addressed based on microfluidic platform with different materials. The translation of microfluidic technology into large scale implementations highly relies on new materials that address the limitations of PDMS. Firstly, we fabricated two different microfluidic platforms for rapid antimicrobial susceptibility testing (AST). One was made of hydrogel, and the bacterial cells were cultured on the top of the device; the other was of polypropylene (PP), and bacterial cells were cultured inside the microchannels. Meanwhile, we developed a novel "barcode" sensor, a microscope-free method for cell accumulation and cell counting, as the downstream of the PP-based chips. As a result, AST can be accomplished simply through an application on a mobile phone rather than using an expensive and sophisticated microscope. Secondly, we presented a self-contained paper-based system for lead(II) ion detection based on G-quadruplex-based luminescence switch-on assay, comprising a novel type of paper-based chip and a matching portable device. Different from the reported paper-based devices, the paper substrate we chose was art paper, which is used for printing magazines. This type of paper could prevent the absorption of liquid into the paper matrix and hold the liquid in place for a period of time; and it could also be used for temporary liquid containing like a plastic substrate (such as polypropylene (PP) and polystyrene (PS)), but the surface of the paper is inherently hydrophilic. In such a design, liquid drops are suspended on the surface of the device in designed reservoirs, rather than absorbed into the paper; when the chip is tilted, the liquid drops will move to other reservoirs according to the guidance of channels defined on the surface. To differentiate it from reported μPAD devices that are fabricated with water-permeable paper, we name this new type of paper-based devices suspending-droplet mode paper-based microfluidic devices (SD-μPAD). Different from the conventional μPADs that use capillary force to drive liquid, our SD-μPADs uses wetting and gravity as driving force. To fabricate the superhydrophobic pattern on the paper device, we developed a new microcontact printing-based method to produce inexpensive and precisely patterned superhydrophobic coating on paper. The coating material is poly(dimethylsiloxane) (PDMS), a hydrophobic and transparent silicone that has long been used for fabricating microfluidic devices. Importantly, the negative-relief stamp we used is made of Teflon, a non-stick polymer, so that the PDMS-coated paper could be peeled from the stamp flawlessly. After such fabrication process, the stamped area of the paper is coated with a textured PDMS layer that is decorated with arrays of micropillars, which could provide superhydrophobic effect and most effectively hold the droplets in place; the remaining area of the paper is still hydrophilic. As a demonstration of this new design, we developed a method using the reaction characteristics of iridium(III) complex for rapid, onsite detection of lead(II) ions in liquid samples. As the reagents have already been loaded onto the paper device during fabrication, the only reagent the users need to add is water. Because of the large Stokes shift of the iridium(III) complex probe, inexpensive optical filters can be employed, and we were able to make an inexpensive, battery-powered compact device for routine portable detection using a smartphone as a detector, allowing the rapid analysis and interpretation of results on site as well as the automatic dissemination of data to professional institutes, including tests even in poor rural areas in developing countries. Thirdly, we upgraded our suspending-droplet mode paper-based microfluidic device (SD-μPAD), which is used for the detection of lead(II) ions in liquid solution. The reason is that our paper-based SD chips are not suitable for long reaction process (> 20 min) detection of biomolecules due to the potential permeation and contaminating problems of art papers. Hence, we chose polypropylene (PP), a hydrophobic, cheap, and thermal stable material (< 110°C), as the material for the fabrication of the SD microfluidic chip. We established a convenient, low-cost, portable and reliable platform for monitoring VEGF165 accurately, which can be applied for point-of-care (POC) testing. In this project, we also employed the label-free oligonucleotide-based luminescence switch-on assay on the microfluidic platform, which possesses the advantages of high sensitivity and high selectivity. Based on the detection of VEGF165 in a three-step reaction process, we adopted a new design for the droplet transfer throughout the channels. This design could migrate the droplet through the chambers via controlling the orientation of the chip, which systematically combined the superhydrophobic force of the coating, the gravity of the droplet and the surface tension between PP and droplet. Therefore, traditional micro pump could be avoided and the total cost for the device could be substantially reduced. In addition, we developed an automatic, matched and portable device for the detection of VEGF165, which assembled by a rotatable chip holder, a UV lamp, a filter, and a camera. Finally, we developed a new whole Teflon membrane-based chip for the aptamer screening. Our article "Whole-Teflon microfluidic chips" introduced the fabrication of a microfluidic device entirely using Teflon materials, one group of the most inert materials in the world. It was a successful and representative introduction of new materials into the fabrication of microfluidic devices, which show dramatically greater anti-fouling performance. However, even such device was inadequate for current purpose, as it is rigid and lacks convenient valve control functions for particle suspensions used in systematic evolution of ligands by exponential enrichment (SELEX). For this project, we propose a SMART screening strategy based on a highly integrated microfluidic chip. This new type of whole-Teflon devices, which are made of flexible Teflon membranes, offering convenient valving control for the whole SELEX process to be performed on chip and fulfilling the anti-fouling requirement in the meantime. The SELEX cycles including positive and negative selections could be automatically performed inside tiny-size microchambers on a microchip, and the enrichment is real-time monitored. The selection cycles would be ended after the resulted signal of the aptamers with high specificity reached a plateau, or no target aptamer is captured after a number of cycles of enrichment. Owning to the antifouling property of the chip materials, the loss of the sample is tremendously reduced. The SMART platform therefore is not only free of complicated manual operations, but also high-yield and well reproducible over conventional methods
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Spetsare, Ebba. "Optimization of a pharmacokinetic assay in a bridging assay format using the Gyrolab immunoassay platform." Thesis, Uppsala universitet, Biokemi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-396493.
Full textAbstract:
Anti-TNF alpha antibodies were among the first approved antibody drugs and now belongs to the best-selling drugs. Today, several companies are developing biosimilars to those drugs which will increase the access of medications and potentially reduce health care costs. There is a great demand for pharmacokinetic assays for anti-TNF-alpha drugs and the bridging assay format is a potential tool, mostly due to its high serum tolerance. This project at Gyros Protein Technologies AB aimed to investigate the properties of the solid phase on the Gyrolab and to utilize this to optimize the bridging assay to be used as a pharmacokinetic assay for a human antibody in the presence of serum. The solid phase was optimized by incorporating three reagents with increasing molecular weight and examining the column profiles generated. Furthermore, the capture reagent was titrated with b-BSA to avoid cross-binding of both arms of the antibody to the capture reagent. Since the background was relatively high, further optimization was done to reduce background and increase the signal to noise ratio. The performance of the optimized bridging assay was compared to alternative PK assay formats. The estimated sensitivity of the bridging assay was 5 ng/ml compared to 250 ng/ml for the indirect antibody assay and 2.5 ng/ml for the bridging assay using an anti-idiotypic antibody as detect. The optimized bridging assay performed well without dilution in buffer and was therefore used for affinity determination of Humira in neat serum. Variable concentrations of TNF-alpha were added to a fix concentration of Humira to compete with the interaction. Calculated KD-values were similar regardless of whether the measurements were performed in neat serum or after dilution in Rexxip buffer.
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Bucco, Olgatina, and olgatina@gmail com. "Preparing, measuring and capturing G-protein coupled receptor (GPCR) signalling complexes for future development of cell-free assay technologies." Flinders University. medicine, 2006. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20060703.114912.
Full textAbstract:
G-protein coupled receptors (GPCRs) are integral membrane proteins which
represent primary cellular targets for intracellular signalling. Many of these receptors
are altered in disease states and hence are the target for over 50% of marketed drugs.
Despite their physiological importance, high-throughput, cell-free assays which
measure functional or signalling activity are only recently being investigated. The
current approach by the pharmaceutical industry to initially screen compounds for
functionality is to use heterologous cell-based assay formats. The aim of this work
was to reconstitute a cell-free GPCR signalling system on an appropriate platform
(surface) as a prototype for future rapid drug screening and other applications. The
proof-of-concept approach involved using the �2A-adrenergic receptor (�2A-AR)
containing cell membrane preparations as the model GPCR, reconstituted with a set
of heterotrimeric G-proteins; G�i1 and �1�2 (the signal transducing complex being
termed a �transductosome�). However, other receptors and G-proteins were also
investigated. Receptors were initially obtained from natural (tissue) sources, however
in the later stages they were expressed in a heterologous system (insect or
mammalian expression system). G-proteins were expressed in Spodoptera
frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor
expression was verified by radioligand binding assays and endogenous G-proteins
were removed from membrane preparations using the chaotropic agent urea to allow
for reconstitution with purified G-proteins. Signal transduction through the
transductosome was measured using the [35S]GTP�S binding assay. Receptor
activated [35S]GTP�S binding was used to determine functional reconstitution and to
validate that the system was working in the normal physiological manner both on and
off a surface (with surface attachment being via histidine attachment on the G�i1
(6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent �2-AR antagonists), Prazosin (potent �1-
AR antagonist) and Propranolol (�-AR antagonist) displayed the appropriate rank
order for this class of receptor. This cell-free, surface-attached signalling complex
prototype may have use in the future development of drug screening and discovery
assay technologies as well as other applications as an alternative to cell-based assays
which are not readily amendable to miniaturisation, long term storage and therefore
stable robust microarray formats.
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Tothill, Alexander M. "Developing a proof of principle 3D-printed lab-on-a-disc assay platform." Thesis, Cranfield University, 2017. http://dspace.lib.cranfield.ac.uk/handle/1826/13496.
Full textAbstract:
A 3D-printed microfluidic lab-on-a-disc (LOAD) device was designed and manufactured using a low cost ( ̃£1600) consumer grade fused deposition modelling (FDM) Ultimaker 2+ 3D printer with imbedded microfluidic channels 1 mm wide, 400 μm depth and with a volumetric capacity of approximate 23 μl. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication; in addition 3D-printed objects can suffer from poor optical transparency. However, in this work, imbedded microfluidic channels were produced and the optical transparency of the device was improved though manufacture optimisation to such a point that optical colourimetric assays can be performed in a microfluidic cuvette device with sample path length of 500 μm and volumetric capacity of 190 μl. When acetone vapour treatment was used, it was possible to improve transparency of plastic samples by up to a further 30%. The LOAD device is capable of being spun using an unmodified optical disc drive (ODD), demonstrating the centrifugation based separation of plasma from whole blood in a low-cost FDM 3D-printed microfluidic LOAD device. A cholesterol assay and glucose assay was developed and optimised using cholesterol oxidase (ChOx) or glucose oxidase (GlOx) respectively and horseradish peroxidase (HRP) for the oxidative coupling of chromotropic acid (CTA) and 4-aminoantipyrine (AAP). This produced a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of cholesterol/glucose in solution. The colourimetric enzymatic cascade assays were developed for use within low-cost FDM 3D-printed microfluidic devices to demonstrate the capabilities and functionality of the devices. For comparison, the assay was run in standard 96 well plates with a commercial plate reader. The results demonstrated that the quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device had a performance comparable to a plate reader assay; glucose assay in whole blood samples R2 = 0.96.
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Ismail, Awale Nasteho. "Validation of a tetraplex assay for detection of antibodies in poultry serum using Luminex 200 platform." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-181920.
Full textAbstract:
Background: As a part of a national health control program, Statens VeterinärmedicinskaAnstalt performs diagnostics to screen flocks for certain pathogens causing high mortality,morbidity and/or serious economical losses. There are several viruses in the programincluding IBDV (infectious bursal disease virus), IBV (infectious bronchitis virus) and NDV(Newcastle disease virus). Method: 96 serum samples were collected from different poultryflocks in Sweden and analyzed by ELISA, which are currently used in the health controlprogram as well as by a commercial prototype of a multiplex immunoassay manufactured byLuminex Corp., which is currently under evaluation at the United States Department ofAgriculture USDA. This 4-plex assay detects antibodies for the three above-mentionedviruses as well as antibodies of avian reovirus. In the context of this study the ELISAs run inroutine diagnostics as well as a REO ELISA were used as the standard for comparison.Result: The antibody concentration in serum from vaccinated chickens was high while theantibody concentration level in serum from not vaccinated chickens was low. There was agood correlation between the multiplex assay and ELISA. Conclusion: The results obtainedin this study indicate that the Luminex multiplex assay tested has the potential to be usedroutinely for screening flocks.
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Cheow, Lih Feng. "Development of a concentration-enhanced mobility shift assay platform for aptamer-based biomarker detection and kinase profiling." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/71513.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
New methods to quantify rare biomarkers from patient samples are critical for developing point-of- care diagnostic platforms. To be compatible with resource limited settings, these assays have to provide fast and accurate results without sacrificing ease of use. Biosensing in homogeneous fashion is the preferred format which satisfies these criteria, but the lack of amplification method is a bottleneck that limits their use for sensitive applications. To address this issue, this thesis explores physical signal amplification means to increase the sensitivities of homogeneous assays. We identified several key applications where the use of these technologies could make a positive impact in improving medical diagnostics systems and advancing biological research. We first outline the use of electrokinetic concentration to realize a continuous signal amplification scheme that increases the sensitivity of homogeneous mobility shift assays. By simultaneously concentrating and separating reacted and unreacted species (with different mobilities) in this device, we can perform sensitive, quantitative and ratiometric measurement of target biomarkers. Using this platform, we improved the sensitivity of aptamer affinity probe capillary electrophoresis to achieve pM detection limit of IgE and HIV-RT in simple buffer and serum sample. This work is timely and impactful as it directly addresses the sensitivity shortcomings of using aptamers as low cost and robust substitutes for antibodies in point-of-care applications. Next, we presented a herringbone nanofilter array device which can perform continuous sizes-elective concentration of biomolecules based on their direct interaction with nanostructures with comparable critical dimensions. We demonstrated the use of this platform to perform a novel homogeneous immunoassay for detecting a cardiac biomarker, C-reactive protein, at clinically relevant concentrations. Finally, we demonstrated that the concentration-enhanced mobility shift assay platform is a powerful tool for probing biological activities such as cellular kinase activities. We have developed technology to isolate, grow and lyse single cells, and used our platform to measure kinase activities from single cells. Through rational design of peptide substrates and spacers, this platform has the ability to simultaneously concentrate and separate multiple analytes. This enables users to obtain simultaneous measurements of multiple cellular kinase activities that could reveal important information about their functional relationships.
by Lih Feng Cheow.
Ph.D.
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Bracht-Loman, Jillian Wilhelmina Paulina. "Validation of liquid biopsy-based analysis on the NanoString nCounter platform." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/672549.
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L'avaluació dels marcadors moleculars en teixit tumoral per predir el pronòstic del càncer i la resposta al tractament, també coneguda com a tractament personalitzat, ha transformat la pràctica clínica per a molts tipus de càncer. Es va descobrir que aquesta teràpia dirigida per genotipar, millora la supervivència del pacient i per tant s'han introduït diverses plataformes tècniques en els laboratoris clínics. No obstant això, no tots els tumors es poden biopsiar i, sovint, les quantitats de teixit són insuficients per a la caracterització del tumor. L'ARN, l'ADN i les proteïnes lliures circulants de biòpsies líquides, es poden extreure dels fluids corporals i poden reemplaçar o complementar les biòpsies de teixit. Les biòpsies líquides tenen diversos avantatges: la possibilitat de realització d’ estudis de serie o mínimament invasiva i permet analitzar l'heterogeneïtat tumoral. Malauradament, encara hi ha una gran bretxa entre la recerca bàsica i la implementació clínica de biòpsies líquides, principalment a causa de la manca de metodologies estandarditzades. A més, les plataformes tècniques que s'utilitzen actualment, no sempre són adequades per analitzar la baixa quantitat i qualitat de material del tumor derivat d'una biòpsia líquida. En conseqüència, la validació i implementació dels assajos de biomarcadors en biòpsies líquides en els laboratoris clínics, requereixen una plataforma tècnica estandarditzada que sigui sensible, ràpida, fàcil d'utilitzar, relativamente econòmica, flexible i amb poca quantitat de mostra.
La plataforma nCounter es pot utilitzar per analitzar tota classe de molècules, inclosos ARN, ADN i proteïnes. La hibridació de codis de barres codificats per colors pels objectius d'interès permet una lectura directa dels nivells d'expressió de gens i proteïnes o la detecció de mutacions. El desenvolupament d'assaigs de biomarcadors en teixits, usant el nCounter, va conduir a l'aprovació per la FDA de l'assaig Prosigna™ per a ús clínic en la tipificació del càncer de mama. Els esforços anteriors també han destacat el potencial d'aquesta plataforma per analitzar molècules derivades i amplificades de biòpsies líquides, encara que es necessiten estudis de validació en l'entorn clínic. En aquesta tesi validem l'ús de la plataforma NanoString nCounter per analitzar material de biòpsies líquides i desenvolupar assajos de biomarcadors clínicament rellevants.La evaluación de los marcadores moleculares en tejido tumoral para el pronóstico del cáncer y la predicción de respuesta al tratamiento (lo que habitualmente se conoce como tratamiento personalizado) ha transformado la práctica clínica a la hora de tratar muchos tipos de cáncer. Son numerosos los trabajos que desde hace tiempo respaldan el efecto que esta terapia dirigida por genotipo tiene sobre los pacientes oncológicos mejorando la supervivencia del paciente; consecuentemente, un amplio rango de plataformas técnicas han sido implementadas en los laboratorios clínicos en los últimos años. Sin embargo, no todos los tumores se pueden biopsiar y, a menudo, las cantidades de tejido son insuficientes para la caracterización del tumor. Las biopsias líquidas, como el ARN, el ADN o las proteínas circulantes tanto libres como encapsuladas en una membrana, pueden extraerse de los fluidos corporales reemplazando o complementando de este modo las tradicionales biopsias de tejido. Las biopsias líquidas tienen varias ventajas: ofrecen la posibilidad de realizar estudios seriados, son mínimamente invasivas y permiten analizar la heterogeneidad tumoral. Desafortunadamente, todavía existe una gran brecha entre la investigación básica y la implementación clínica de las biopsias líquidas, principalmente debido a la falta de metodologías estandarizadas. Además, las plataformas técnicas que se utilizan actualmente no siempre son adecuadas para analizar la baja cantidad y calidad de material del tumor procedente de una biopsia líquida. En consecuencia, la validación e implementación de los ensayos de biomarcadores en biopsias líquidas en los laboratorios clínicos requieren una plataforma técnica estandarizada que sea sensible, rápida, fácil de usar, viable económicamente, flexible y que requiera un aporte inicial de ácidos nucleicos bajo, debido a la baja concentración que normalmente se obtiene en las biopsias líquidas. La plataforma nCounter se puede utilizar para analizar todo tipo de moléculas, incluyendo ARN, ADN y proteínas. La hibridación de diferentes códigos formados por moléculas de colores siguiendo patrones específicos con secuencias de interés permite una lectura directa de los niveles de expresión de genes y proteínas o la detección de mutaciones. El desarrollo de ensayos de biomarcadores en tejidos usando nCounter condujo a la aprobación por la administración de fármacos y alimentos de los Estados Unidos (FDA) del ensayo Prosigna ™ para su uso clínico en la tipificación del cáncer de mama. Numerosos estudios han destacado el potencial de esta plataforma para analizar moléculas derivadas y amplificadas de biopsias líquidas, aunque estudios de validación en el entorno clínico aun son necesarios. El objeto de esta tesis es la validación del uso de la plataforma NanoString nCounter para analizar material de biopsias líquidas y desarrollar ensayos de biomarcadores clínicamente relevantes.
The assessment of predictive- and prognostic molecular markers in tumor tissue, also known as personalised treatment, has transformed clinical practice for many cancer types. This genotype-directed therapy was found to improve patient survival, and several technical platforms have been introduced in clinical laboratories since then. However, not all tumors can be biopsied and tissue quantities are often insufficient for tumor characterisation. Liquid biopsies, such as membrane-encapsulated- or circulating free RNA, DNA and proteins, can be derived from body fluids and can replace or complement tissue biopsies. They have several advantages, such as repeated sampling, a minimally invasive character and heterogeneous profiling. Unfortunately, there is still a big gap between basic research and clinical implementation of liquid biopsies, mainly due to the lack of standardised methodologies. In addition, currently used technical platforms are not always suitable to analyze the low quantity and quality of tumor-derived material that can be found in a liquid biopsy. In consequence, large-scale validation and clinical implementation of liquid biopsy-based biomarker assays requires a sensitive, quick, easy-to-use, relatively cheap, flexible and standardized technical platform with low input requirements. The nCounter platform can be used to analyze all types of molecules, including RNA, DNA and proteins. Binding of color coded barcodes to targets of interest allows for either a direct read-out of gene- or protein expression levels or the detection of mutations. Tissue-based biomarker assay development on nCounter led to the FDA approval of the Prosigna™ assay for clinical use in breast cancer subtyping. Previous efforts have also highlighted the potential of this platform to analyze amplified liquid biopsy-derived molecules, although validation studies in the clinical setting are needed. In this thesis we validated the use of the NanoString nCounter platform to analyze material from liquid biopsies and develop clinically relevant biomarker assays.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina
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Vasou, Andri. "Development of a novel cell-based screening platform to identify inhibitors of viral interferon antagonists from clinically important viruses." Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/8266.
Full textAbstract:
All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.
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Ngema, Xolani Terrance. "Metallic nanoparticles with polymeric shell: A multifunctional platform for application to biosensor." University of the Western Cape, 2018. http://hdl.handle.net/11394/6330.
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Philosophiae Doctor - PhD (Chemistry)Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (MTB) that usually affects the lungs leading to severe coughing, fever and chest pains. It was estimated that over 9.6 million people worldwide developed TB and 1.5 million died from the infectious disease of which 12 % were co-infected with human immunodeficiency virus (HIV) in the year 2015. In 2016 the statistics increased to a total of 1.7 million people reportedly died from TB with an estimated 10.4 million new cases of TB diagnosed worldwide. The development of the efficient point-of-care systems that are ultra-sensitive, cheap and readily available is essential in order to address and control the spread of the tuberculosis (TB) disease and multidrugresistant tuberculosis.
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Plank, Nicole [Verfasser], and A. [Akademischer Betreuer] Buschauer. "Dimeric histamine H2 receptor agonists as molecular tools and genetically engineered HEK293T cells as an assay platform to unravel signaling pathways of hH1R and hH2R / Nicole Plank. Betreuer: A. Buschauer." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1100276599/34.
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Datar, Akshata. "HIGH CONTENT IMAGING ASSAYS ON MICROARRAY CHIP BASED PLATFORM." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462795576.
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McMillan, Kay Seonaid. "Development of a microfluidic platform for multicellular tumour spheroid assays." Thesis, University of Strathclyde, 2016. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=27926.
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Microfluidics is a valuable technology for a variety of different biomedical applications. In particular, within cancer research, it can be used to improve upon currently used in vitro screening assays by facilitating the use of 3D cell culture models. One of these models is the multicellular tumour spheroid (MCTS), which provides a more accurate reflection of the tumour microenvironment in vivo by reproducing the cell to cell contact, the development of a nutritional gradient and the formation of a heterogeneous population of cells. Therefore, the MCTS provides a more physiologically relevant in vitro model for testing the efficacy of treatments at the preclinical level. Currently, methods for the formation and culture of spheroids have several limitations, including being labour intensive, being low throughput, producing shear stress towards cells and the hanging drop system being unstable to physical shocks. Recently, microfluidics (especially droplet microfluidics) has been employed for the culture and screening of spheroids, providing a high-throughput methodology which only requires small volumes of fluids and small numbers of cells. However, current issues with droplet microfluidics include complicated droplet gelation procedures and short cell culture times. In this thesis, the use of microfluidic technologies as an approach for spheroid formation and culture are investigated with the aim to create a platform for radiotherapeutic and chemotherapeutic treatment of spheroids using cell lines. Initially, the use of emulsion technology at the macro scale was evaluated to determine the best conditions for spheroid culture. Once this was achieved the spheroids were compared to spheroids using a traditional method and radiotherapeutic treatment was conducted. Subsequently, avenues for miniaturising the developed emulsion-based methods were studied to provide a microfluidic technology. Finally, along with identifying the optimal culture conditions using hydrogels, a microfluidic system that integrated both droplet and single phase microfluidics features was developed for the formation and culture of spheroids. Using the latter, proof of principle experiments were conducted to demonstrate the suitability of the platform for both chemotherapeutic and radiotherapeutic assays within the same device.
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Cary, ReJeana. "Sensing of Small Molecules, Biomarkers, and Pathogens using Unique Plasmonic Assay Platforms." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595848703283784.
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Taff, Brian M. 1978. "Microsystems platforms for array-based single-cell biological assays." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45879.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references (p. 157-164).
For much of the past century, plated cell cultures have served investigations regarding a variety of fundamental biological processes. Though this in vitro approach has been fruitful, for surveying topics including cell cycle effects3' 8, pro-survival4, 7, 9, 10 and apoptotic2' 11 signaling networks, gene regulationl2' 13, and stress dynamicsl4' 15 (among others), it caters best to harvesting the averaged responses from binned populations of cells and offers only limited avenues for tracking individual cell behaviors. Microsystems-based initiatives16-28 are beginning to aid this investigative shortcoming by offering a variety of strategies for handling individual cells. Such efforts, may ultimately serve studies of cross-population heterogeneity29-33, an effect often masked when tracking responses via averaged population-based means. As it is believed that small subpopulations of cells may be responsible or determining the fate of various diseases and developmental processes34-36, this new paradigm for probing cell function will likely offer key insights.In my dissertation, I offer a unique suite of microsystems-based tools37-42 for servicing novel biological assays centered on cross-population dynamics. This work leverages the investigative potential enabled by arrayed groupings of precisely-spaced single cells and presents innovations in active and passive cell trapping architectures, packaging design, and the use of novel materials for microfabrication41' 43. From proof-of-concept forays, where I discuss the first reported row/column-based electrically-addressable platform40 for trapping, imaging, and releasing collections of individual cells, to scaled implementations that employ frequency modulation to assign unique forcing effects to in-system constructs, I outlay fundamentals for designing, building, and evaluating dielectrophoresis-reliant (DEP) microsystems architectures.
(cont.) I further present matured platforms that, for the first time, parallelize single-cell manipulations within microfluidic devices by combining hydrodynamic weir-based cell capture with DEP-based actuation37-39. In progressions toward functional on-chip bioassays, I experimentally validate conditions for in-device cell viability and offer a novel means for tracking mitosis in individual cells. Ongoing work, related to the developments presented here, offers the hope of new empirical approaches to drug discovery, the assignment of gene function in the aftermath of the human genome project, and enhanced understandings of cell communication-linked dynamic responses.
by Brian M. Taff.
Ph.D.
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Chin, Vicki I. "A microfabricated platform for cell-based assays : applications to neural stem cells /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2004. http://wwwlib.umi.com/cr/ucsd/fullcit?p3153701.
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Romeo, Mariia Pilar Carreras. "The fabrication, integration and application of multiphase microfluidic assay platforms for high throughput experimentation." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499854.
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This thesis describes novel research into the generation and manipulation of aqueous droplets within a number of multiphase microfluidic platforms. Specific techniques for the mixing, dilution and sorting of droplets are reported, and the development of droplet based microreactor devices for the generation of alginate gel beads described. The alginate beads have been applied for the encapsulation of both yeast and leukemia cells, as a potential platform for the study of the behaviour of regular assemblies of cells. further modification of the platform has also demonstrated the feasibility of double layer alginate encapsulation.
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Dencker, Julia. "Effects of antibody labeling chemistry on assays developed for the Gyrolab immunoassay platform." Thesis, Uppsala universitet, Molekylär systembiologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-447207.
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The aim of this project was to make a comparison of the effects of antibody labeling chemistries on assays developed for the Gyrolab immunoassay platform. One of the labeling techniques was a heterogenous labeling technique targeting amino groups on the antibody. The other labeling technique was a site-specific labeling technique targeting the conserved Fc-glycan at the aspargine 297 residue on the IgG molecule. The site-specific labeling was performed using a kit from Genovis called GlyCLICK. The two labeling techniques were compared on four different assays developed for the Gyrolab platform. The assays tested in this project were two anti-drug antibody assays, a pharmacokinetics assay, a polyclonal antibody assay, and a monoclonal antibody assay. The drug tolerance was tested for the anti-drug antibody assays, resulting in better drug tolerance for reagents labeled with amino conjugation for the Humira assay with incubation overnight. A confirmatory analysis, testing the inhibition of negative control with addition of unlabeled drug in the Master Mix, was performed. This resulted in small differences in the inhibition between the different reagents, except for Keytruda on Gyrolab Bioaffy 200, for which the GlyCLICK labeled reagents led to a lower inhibition of the negative control. For all the assays the effects on signal to background ratio and limit of detection was investigated. The greatest advantages of GlyCLICK on the signal to background was observed for anti-drug antibody Keytruda assay and polyclonal antibody assay. For the polyclonal antibody assay, the results indicated potentially reduced need for the polishing step and for two wash solutions after addition of the detect reagent.
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Liu, Huiqing. "Characterization of two-component organothiol mixed monolayers on gold and quantification of nonspecific adsorption on mixed SAM biosensor platforms using electrochemical enzyme immunology." Auburn, Ala., 2007. http://repo.lib.auburn.edu/2007%20Spring%20Theses/LIU_HUIQING_29.pdf.
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Karimpour, Masoumeh. "Multi-platform metabolomics assays to study the responsiveness of the human plasma and lung lavage metabolome." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-120591.
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Metabolomics as a field has been used to track changes and perturbations in the human body by investigating metabolite profiles indicating the change of metabolite levels over time and in response to different challenges. In this thesis work, the main focus was on applying multiplatform-metabolomics to study the human metabolome following exposure to perturbations, such as diet (in the form of a challenge meal) and exhaust emissions (air pollution exposure in a controlled setting). The cutting-edge analytical platforms used for this purpose were nuclear magnetic resonance (NMR), as well as gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS). Each platform offered unique characterization features, allowing detection and identification of a specific range of metabolites. The use of multiplatform-metabolomics was found to enhance the metabolome coverage and to provide complementary findings that enabled a better understanding of the biochemical processes reflected by the metabolite profiles. Using non-targeted analysis, a wide range of unknown metabolites in plasma were identified during the postprandial stage after a well-defined challenge meal (in Paper I). In addition, a considerable number of metabolites were detected and identified in lung lavage fluid after biodiesel exhaust exposure compared to filtered air exposure (in Paper II). In parallel, using targeted analysis, both lung lavage and plasma fatty acid metabolites were detected and quantified in response to filtered air and biodiesel exhaust exposure (in Paper III and IV). Data processing of raw data followed by data analysis, using both univariate and multivariate methods, enabled changes occurring in metabolites levels to be screened and investigated. For the initial pilot postprandial study, the aim was to investigate the plasma metabolome response after a well-defined meal during the postprandial stage for two types of diet. It was found that independent of the background diet type, levels of metabolites returned to their baseline levels after three hours. This finding was taken into consideration for the biodiesel exhaust exposures studies, designed to limit the impact of dietary effects. Both targeted and non-targeted approaches resulted in important findings. For instance, different metabolite profiles were detected in bronchial wash (BW) compared to bronchoalveolar lavage (BAL) fluid with mainly NMR and LC-MS. Furthermore, biodiesel exhaust exposure resulted in different metabolite profiles as observed by GC-MS, especially in BAL. In addition, fatty acid metabolites in BW, BAL, and plasma were shown to be responsive to biodiesel exhaust exposure, as measured by a targeted LC-MS/MS protocol. In summary, the new analytical methods developed to investigate the responsiveness of the human plasma and lung lavage metabolome proved to be useful in an analytical perspective, and provided important biological findings. However, further studies are needed to validate these results.Metabolomik har använts för att spåra förändringar och störningar i kroppens funktioner genom undersökning av metabolit-profiler. I detta avhandlingasarbete har huvudfokus varit på tillämpning av flera olika analytiska plattformar för metabolomikstudier av det mänskliga metabolomet efter exponering för olika kost och avgasutsläpp från biodieselbränsle. De sofistikerade analytiska plattformarna som användes för detta ändamål var kärnmagnetisk resonans (NMR), samt gaskromatografi (GC) och vätskekromatografi (LC) kopplat till masspektrometri (MS). Varje plattform erbjöd unika karakteriseringsmöjligheter med detektion och identifiering av specifika grupper av metaboliter. Användningen av multipattformmetabolomik förbättrade täckningen av metabolomet och genererade kompletterande resultat som möjliggjorde en bättre förståelse av de biokemiska processer som reflekteras av metabolitprofilerna. Med hjälp av breda analyser har ett stort antal okända metaboliter i plasma identifierats under den postprandial fasen efter en väldefinerad måltid (i Paper I). Dessutom har ett stort antal metaboliter påvisats och identifierats i lungsköljvätska efter exponering av biodieselavgaser jämfört med kontollexponering med filtrerad luft (i Paper II). Parallellt med dessa breda analyser har också riktade analyser genomförts av både lungsköljvätska och plasma. Därigenom har bioaktiva lipider detekterats och kvantifieras efter avgasexponering och resultaten har jämförts med filtrerad luft som kontrollexponering (Paper III och IV). Processning av rådata följt av dataanalys, med både univariata och multivariata metoder möjliggjorde screening och fördjupad undersökning av förändringen i metabolitnivåer. I den första pilotstudien av postprandiala nivåer var syftet att undersöka responsen i plasmametabolomet efter en väldefinierad måltid under den postprandiala fasen vid två olika typer av kost. Resultaten visade att oberoende av kosten, så återvände metabolitnivåerna till sina baslinjenivåer tre timmar efter måltiden. Detta togs i beaktande vid exponeringsstudierna för biodieselavgaser, som designades så att dietens inverkan minimerades. Både breda och riktade analyser resulterade i viktiga resultat. Exempelvis så detekterades olika metabolitprofiler i bronkiell sköljvätska (BW) jämfört med bronkoalveolär sköljvätska (BAL), speciellt med NMR och LC-MS. Dessutom resulterade avgasexponering i förändrade metabolitprofiler, observerade med GC-MS, särskilt i BAL. Dessutom uppvisade fettsyrametaboliter i BW, BAL och plasma förändrade halter efter avgasexponering, uppmätt genom en riktad LC-MS/MS-analys. Sammanfattningsvis så visade sig de nya metoderna som utvecklats för att undersöka förändringar i metabolithalterna i plasma och lungsköljvätska fungera väl ur ett analytiskt perspektiv och resulterade i viktiga biologiska fynd. Fördjupade studier behövs dock för att validera resultaten.
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Ouyang, Wei Ph D. Massachusetts Institute of Technology. "Microfluidic platform for rapid biologics activity assessment using molecular charge modulation and electrokinetic concentration based receptor assays." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/106084.
Full textAbstract:
Thesis: S.M., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 86-91).
Biologics (biomolecule drugs) play a key role in modem medicine, yet assuring their quality and safety remains a major challenge for the biopharmaceutical industry. Developing a platform for rapid and reliable assessment of biologics activity is critical for quality control of biologics manufacturing and safety assurance. Herein we introduce a generally applicable platform for this purpose, using molecular charge modulation and electrokinetic concentration based receptor binding assays. This technology is homogeneous (immobilization-free) and overcomes many practical challenges of current immobilization-based binding assays. We developed various formats of assays for extracting the dissociation constants and dissociation rates of biologics toward their target receptors, which are directly related to the in vivo efficacy and duration of efficacy of biologics. We showcased the technology for rapid determination of biologics degradation, which is meaningful for assuring biologics safety. This work provides reliable biologics analysis comparable to state-of-the-art technologies with significantly less time (<1 h), reduced sample use, and simpler experimental setup, which holds promises as a platform for assuring the quality and safety of biologics at the point-of-care.
by Wei Ouyang.
S.M.
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Lund, Helen Louise. "A novel platform for creating digital PCR assays to detect genetic translocations and its application to the initial diagnosis of cancer." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57026.
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Chromosomal translocations can cause cancer, often through the formation of fusion genes that code for an unnatural tyrosine kinase that promotes constitutive activation of a signaling pathway controlling cell proliferation and differentiation. For example, the diagnostic hallmark of chronic myelogenous leukemia (CML) is an oncogene fusion formed from a reciprocal translocation (t(9;22)(q34.1;q11.2)) between chromosomes 9 and 22 that results in an altered chromosome 22q known as the Philadelphia chromosome. Approximately 95% of all CML patients harbor the gene fusion, BCR-ABL, which is formed via a double stranded break (DSB) within both the Abelson oncogene 1 (ABL) on chromosome 9q, which codes for a non-receptor tyrosine kinase (ABL), and the breakpoint cluster region gene (BCR) on chromosome 22q. BCR-ABL encodes a constitutively active tyrosine kinase BCR-ABL responsible for the uncontrolled proliferation associated with chronic myelogenous leukemia. The identification of these translocation events and/or associated fusion genes in clinical samples is critical to ensure the appropriate treatment for patients where the drug and related course of therapy target an activated fusion kinase. Clinical detection of complex chromosomal rearrangements is often conducted using fluorescence in situ hybridization (FISH). The FISH analysis, though effective, offers relatively poor sensitivity while being expensive, time-consuming and technically challenging to perform.
Here we have developed and validated a new general platform for creating assays against complex chromosomal rearrangements, including both reciprocal and non-reciprocal translocations. It utilizes droplet digital PCR (ddPCR) technology in lieu of FISH to quantify the rearrangement of proto-oncogenes that undergo rearrangement as part of the translocation event. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics or theranostics. The first provides a reliable and sensitive measure of DSBs within the major breakpoint region of BCR (M-BCR), permitting initial diagnosis of CML through unequivocal detection of the BCR-ABL fusion gene to a frequency of 0.25%. The second provides for the highly sensitive detection of DSBs in the anaplastic lymphoma kinase (ALK) gene that result in a non-reciprocal (inversion) translocation (inv(2)(p21;p23)) associated with an ALK-positive non-small cell lung cancer (NSCLC).Science, Faculty of
Graduate
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Carpenter, Sarah Elizabeth. "Enzyme linked spectroscopic assays for Glyoxylate the use of Peptidylglycine alpha-Amidating Monoxygenase for the discovery of Novel alpha-Amidated hormones /." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001415.
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Parić, Zlatko [Verfasser], and Joachim [Akademischer Betreuer] Wegener. "Development of a dual ECIS-SPR sensor platform for cell-based assays: Label-free analysis of g-protein coupled receptor signal transduction / Zlatko Parić ; Betreuer: Joachim Wegener." Regensburg : Universitätsbibliothek Regensburg, 2021. http://d-nb.info/1227039522/34.
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Aghebat, Rafat Ali [Verfasser], Friedrich C. [Akademischer Betreuer] Simmel, Tim [Gutachter] Liedl, and Friedrich C. [Gutachter] Simmel. "Programmable self-assembly of DNA origami arrays as a platform for multiplexed biomolecular and biochemical assays / Ali Aghebat Rafat ; Gutachter: Tim Liedl, Friedrich C. Simmel ; Betreuer: Friedrich C. Simmel." München : Universitätsbibliothek der TU München, 2020. http://d-nb.info/1222161753/34.
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Huang, Bo Yi, and 黃渤溢. "Development of an assay platform for screening Src kinase inhibitors using dot-blot assay." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/90642775013174705676.
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碩士國立清華大學
分子與細胞生物研究所
104
Src tyrosine kinase, known as a proto-oncogene, participates in many signal pathways to regulate various cellular functions. Abnormal activation of Src is also involved in cancer progression. Autophosphorylation of Src plays a crucial role in its activation. Herein, I have developed an assay platform to screen Src tyrosine kinase inhibitors by analyzing the autophosphorylation of Src. The autophosphorylation reaction was performed in a 96-well format thermocycler and the autophosphorylation of Src was analyzed by dot-blot assay. In this assay platform, the Z`-factor for each plate was greater than 0.49 and the acceptable tolerance of dimethyl sulfoxide was up to 1%. No drift or edge effects was observed. Known kinase inhibitors were used to validate this assay platform. All known Src tyrosine kinase inhibitors exhibited the inhibition of Src kinase activity in the confirmatory assay. The IC50 of Dasatinib and Saracatinib, two potent Src tyrosine kinase inhibitors, measured by this assay platform are 7.7 and 24.4 nM, respectively. Thus, this assay platform can be used to screen Src tyrosine kinase inhibitors for discovery of anti-cancer drug.
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Lin, Min-Hsuan, and 林旻萱. "Zebrafish in vivo electrocardiogram platform for drug assay and electrophysiology research." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/htxc74.
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博士國立清華大學
生物資訊與結構生物研究所
107
Zebrafish is a popular and favorable model organism for cardiovascular research, with an increasing number of studies implementing functional assays in the adult stage. For example, the application of electrocardiography (ECG) in adult zebrafish has emerged as an important tool for cardiac pathophysiology, toxicity, and chemical screen studies. However, few laboratories are able to perform such functional assay due to the high cost and limited availability of a convenient in vivo ECG recording system. In this study, an inexpensive ECG recording platform and operation protocol that has been optimized for adult zebrafish ECG research was developed. The core hardware includes integration of a ready-to-use portable ECG kit with a set of custom-made needle electrode probes. A combined anesthetic formula of MS-222 and isoflurane was first tested to determine the optimal assay conditions to minimize the interference to zebrafish cardiac physiology under sedation. For demonstration, we treated wild-type zebrafish with several pharmacological agents known to affect cardiac rhythms in humans, including isoproterenol, verapamil, quinidine, amiodarone and veratridine. Conserved electrophysiological responses to these drugs were induced in adult zebrafish and recorded in real time. The results show that zebrafish have highly similar physiological responses with human after most drug treatments. This economic ECG platform has the potential to facilitate teaching and training in cardiac electrophysiology with adult zebrafish and to promote future translational applications in cardiovascular medicine.
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Wu, Yen-Ta, and 巫衍達. "An Assay Platform to Identify Potential Antibiotics with Immobilized Penicillin-binding Proteins." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/46292592898528649839.
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碩士臺灣大學
生化科學研究所
95
The widespread use of antibiotics has generated serious drug resistance problems. These problems are more serious in hospitals and nursing home where the weak patients often need sustained treatment of antibiotics and thus are easily colonized with drug-resistant bacteria. Among them, methicillin-resistant Staphylococcus aueus, vancomycin-resistant enterococci, and Clostridium difficile, which are resistant to most antibiotics, have posted serious threats to public health. There is an unmet medical need to novel and new generation of antibiotics. Herein, we focused on penicillin-binding protein (PBPs) and developed a novel assay platform to facilitate identification of potential PBP inhibitors. Penicillin-binding proteins (PBPs) are essential for peptidoglycan (also called murein) biosynthesis and hence are one of the drug targets for antibiotics development. Class A PBPs are bifunctional proteins that have both transglycosylase and transpeptidase activity. Since the resistance against several β-lactam antibiotics that are targeting transpeptidase activity of class A PBPs has emerged, the transglycosylation represents a new target for potential therapeutics. As the first step, the PBPs from C. difficile and C. perfrigens were expressed and purified. The moenomycin binding activities to these two recombinant proteins were characterized using fluorescence polarization assay with fluorescent moenomycin. The transglysosylase activity of immobilized PBPs with fluorescent lipid II was analyzed by TLC analysis. A novel assay platform was designed so that immobilized PBPs is able to specifically ”fish out” transglycosylase inhibitors such as moenomycin or transglycosylase substrate, lipid II. The bound moenomycin can be easily identified using MALDI analysis and exerted their MIC activity in a 96-well microtiter plate. Thus this novel platform can be used to facilitate the identification of potential inhibitors for transglycosylase and transpeptidase. On the other hand, the biosynthesis of lipid II was realized and further purified by short pass chromatography, therefore milligram quantities of lipid II can be easily obtained. This can facilitate the development of lipid II-based assay of transglycosylase.
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Pearson, Brooke. "Development of a SERS Sandwich Assay Platform for Rapid Detection of Bacteria." 2017. https://scholarworks.umass.edu/masters_theses_2/529.
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The increased incidence of food pathogen outbreaks placed a new emphasis on the requirement of a rapid, sensitive, and reliable detection method for pathogens in food samples. Surface-enhanced Raman spectroscopy (SERS) is a technique that tremendously enhances the weak Raman scattering of an analyte by using a metallic nano-substrate. Herein, we developed an innovative SERS sandwich assay platform which is based on 3-mercaptophenylboronic acid (3-MPBA) or aptamer as a capturer, and 3-MPBA and silver nanoparticles (AgNPs) as the reporter for non-selective and selective detection of bacteria. Both optical and chemical (SERS mapping) imaging were used as mechanisms for bacterial detection and quantification. Using Salmonella enterica and Listeria monocytogenes as the model bacteria, we have identified a unique bacterial SERS signal upon the interaction between the captured bacteria, 3-MBPA and AgNPs, which was used as the base for reliable detection of bacteria using SERS mapping. The non-specific assay also possesses unique optical properties allowing for the enhanced visualization of bacteria at low microscope magnifications (10 and 20x objective lenses). Using 3-MBPA owe achieved sensitive detection and quantification of as low as 102 CFU/mL and a capture efficiency of 92.1% for nonselective detection of Salmonella. The capability of the assay method to detect specific bacteria using an aptamer was also demonstrated. Besides the SERS applications of this assay, it was discovered that the 3-MPBA coated gold chip developed for this assay enhances the visualization of bacteria under a light microscope allowing for facile and rapid detection and quantification. In anticipation for industrial applications, sample preparation methods and strategies were developed for simple and carbohydrate food matrices.
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許家豪. "An integrated zebrafish-based assay platform for the study of myocardial infarction and therapeutics development." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/11110999286628170278.
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Chen, Yen-Hao, and 陳彥豪. "Developing an Enzyme-Linked Immunosorbent Assay on the Centrifugal Platform with the Immiscible phase Filtration Technique." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/52h8p9.
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Ying, Li Chia, and 李佳頴. "Design and Optimization of an Enzyme-Linked Immunosorbent Assay on the Centrifugal Platform using Flow-Splitting Techniques." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/973zcv.
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碩士逢甲大學
化學工程學系
103
In this work, a centrifugal microfluidic platform was developed to replace microtiter plates for conducting enzyme-linked immunosorbent assays. The sequential flow control and flow-splitting were developed to reduce the labor required for sample and reagent loading. In addition, magnetic modules were developed to conduct both incubation and washing in automation. In addition, the factors which affect the sequential flow control of the capillary valves were investigated. Finally, the centrifugal microfluidic platform was used to conduct ELISA. The assay protocol can be finished within 38 minutes with the limit of detection (for hCG) around 0.15 mIU/mL
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Hu, Ben C. P., and 胡智棚. "Biological Application of a CMOS Chip Base Light Sensor System：Design of a Luminescent Assay Platform for Biochemicals." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/63280074593894356984.
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碩士國立交通大學
生物科技研究所
90
In order to solve a variety of biological problems, traditional biochemical examining instruments could not satisfy our needs, especially in healthy business nowadays. The worldwide market of in vitro diagnostics (IVD) is growing rapidly. The point-of-care (POC) and over-the-counter (OTC) testing requires a large quantity of small size healthy care instruments. Most of the clinical medical testing instruments are large auto analyzers that are expensive and very complex. The combination of biotechnology and semiconductor industry has great potential to substitute traditional biochemical examining instruments. For example, complementary meal oxide semiconductor (CMOS) photodiode chip is a small size, low cost and convenient using semiconductor sensor that can be manufactured through a 0.5μM standard process. We design CMOS chip as transducer for biochemical reactions and proceed with parallel test by standard and generally recognized fluorescence instrument (Hitachi F-4500) at the same time. The glucose oxidase (GOD) coupled horseradish peroxidase (HRP) -luminol-H2O2 system is used as an example to quantify the concentration of glucose. We have shown that it is feasible to use CMOS chip for biochemical examination. However, it still needs to improve and reform to become a practical one. For further improvement, we will minimize the amount of volume and increase the variety of measurements. Second, we will continue designing this new established system to become an automatized assay platform. In addition, we would research new biochips with those research institute of Electronics Engineering and research institute of Applied chemistry researchers by our available technology and experiences.
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Pagará, Beatriz Condeço Pinto. "Paper as a Colorimetric Biosensing Platform for Tetracyclines Detection in Milk." Master's thesis, 2018. http://hdl.handle.net/10362/61057.
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Tetracyclines are a type of antibiotic that exhibits activity against most gram-positive and gram-negative bacteria. These antibiotics are often added at subtherapeutic levels to feed to act as growth promoters. Due to its low bioavailability, only a fraction of the antibiotic is metabolized in animals, causing waste. These residues can enter human bodies through the food chain and lead to increased antimicrobial resistance, causing allergic or toxic reactions, which led several countries to implement a maximum level of residues for this type of antibiotic. Most methods of detecting TCs are time consuming or inadequate for field analysis. In this way, an inexpensive, easy-to-execute and fast analytical method is required.
In this sense, in the present work a colorimetric biosensor was developed in paper for the detection of four type of tetracyclines, presenting an alternative in the performance of point-of-care tests. Paper is low-cost, abundant, biodegradable and easy to dispose of by incineration.
The construction of the sensors was performed using Lab-on-Paper technology and is based on the synthesis of gold nanoparticles by reducing a gold salt in which tetracyclines constitute the reducing agent itself. Different concentrations of TCs result in the formation of different colour intensities.
Different concentrations of tetracyclines were tested and analysed using ImageJ software, allowing linear calibration lines to be obtained, that relate the concentration of antibiotics in a range between 0.1 and 10 μg/mL and the arithmetic mean of the RGB channels.
Validation tests of the sensors developed with TC in milk were also performed. It was observed that it is possible to detect this type of antibiotic in pre-treated milk, and four forms of milk treatment were studied.
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Ho, Nga T. "Multiplexed, affordable, and portable platform for real time quantification of counterfeit and substandard medicines." Thesis, 2016. https://hdl.handle.net/2144/17078.
Full textAbstract:
The World Health Organization estimates that about 10-30% of pharmaceuticals in the world are either substandard or counterfeit. The number is even higher in the developing countries. From a public health perspective, a key contributor to the development and proliferation drug resistant strains of infections, including tuberculosis (TB), malaria and other infections that are leading killers in resource limited settings is poor quality medicines. Most of the main causes are profit driven corruption in many pharmaceutical companies, the poor manufacture and quality control, and/or the inappropriate storage conditions. Poor quality drugs lead to loss of life, create morbidity, strain the financial structure of the health system and lead to long-term drug resistance that affects us all.
The current technology for screening poor quality drugs can be divided into 2 categories: the high end, precise and high cost technologies (such as High Performance Liquid Chromatography) and lower cost and qualitative technologies (such as Thin-Layered Chromatography). The high-end methods can give a precise measurement of active pharmaceutical ingredient (API) concentration and the presence of impurities in the tablets, but require trained personnel, advanced machine and lab set up, not suitable for field testing where most of poor quality pharmaceuticals have been found. The lower cost techniques require little training and simple equipment to operate at a relatively inexpensive price, but only gives qualitative results. In addition, most of current methods do not look at the dissolution profile of the tablets simultaneously with the concentration of API. Therefore, we propose to develop an assay that can quantify the concentrations of multiple APIs simultaneously and measure dissolution rates.
In order to address current gaps in knowledge, my research proposal has three main parts in the assay development: 1) Development of an fluorescent/luminescent assay for detection of counterfeit/substandard antimalarial using small-molecules-based methods and field testing in Ghana; 2) Development of a fluorescent assay for detection of water-soluble pharmaceuticals using SELEX; and 3) Design a detection platform using microfluidic chips for real time quantification of multiple active pharmaceutical ingredients. For proof-of-concept, an antimalarial drug (artesunate and amodiaquine) and antibacterial antibiotics (sulfamethoxazole and trimethoprim) are selected to demonstrate the probe development and test the chip performance. Overall, the assay will be rapid, robust, portable, inexpensive, multiplexed, quantitative, specific, and sensitive. At a big picture level, emphasizing drug quality and creating robust mechanisms of drug testing will improve health outcomes and enhance treatment efficacy in resource limited settings.
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"Modeling, Design, Fabrication, and Characterization of a Highly Sensitive Fluorescence-based Detection Platform for Point-of-Care Applications." Doctoral diss., 2018. http://hdl.handle.net/2286/R.I.51705.
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abstract: Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings.
This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV.
The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use.
The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost.
The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2018
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O'Neill, Adrian Thomas. "A microfluidic platform for human epidermal keratinocyte cytotoxicity assays." 2009. http://www.lib.ncsu.edu/theses/available/etd-12182008-164137/unrestricted/etd.pdf.
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Wang, Yu-Ting, and 王鈺婷. "Development of Point-of-Care Triglyceride Assays on the Centrifugal Platform." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/78301379850329469661.
Full textAbstract:
碩士逢甲大學
化學工程學所
100
This goal of this study is to develop point-of-care triglyceride assays on a centrifugal platform, which is portable, easy to use, low cost, while offering reasonable precision. The microfluidic disc analyzer is capable of actuating low volume samples and reagents through centrifugation. By integrating the disc spinning system and optical detection system through software interface, the testing results could be gathered from whole blood samples. Clinical samples were conducted by both the microfluidic disc analyzer and the instrument used in the hospital (Hitachi 7600), the test results showed good correlation and agreement.
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楊意楓. "Developing enzyme-linked immunosorbent assays on a centrifugal platform using aliquoting techniques." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/71170403058353940269.
Full textAbstract:
碩士逢甲大學
化學工程學系
101
In this work, an enzyme linked immunosorbent assay (ELISA) was successfully developed on a centrifugal platform. ELISA is an immunoassay which is capable of quantifying antibodies or antigens through immunoreactions. In the past, ELISA was conducted on microtiter plates. It required multiple reagent adding steps and long incubation time. It is both labor intensive and time consuming. Although the emerging of CD ELISA can potentially improve this process by automating the reagents adding process through sequential flow control, it is still labor-intensive in the reagent loading process. In order to solve this problem, we developed an innovated CD ELISA platform by applying the flow splitting techniques. In our improved design, each reagent only needs to be added once and it can be divided into equal amount and delivered into each reaction chamber within a few seconds. Therefore, to conduct 100 assays in parallel, only one reagent loading step is required. This has greatly improve the ease of use in conducting ELISA. In addition, the optimal operating conditions for the magnet arrangement, magnetic beads, assay chemistry, incubation time, were studied in this work. In our finalized design, an ELISA protocol can be completed within 70 minutes. The experimental results showed that the limit of detection (LOD) for human chorionic gonadotropin (hCG) performed by CD ELISA is about 1 mIU/mL, which is equivalent to the sensitivity performed on microtiter plate.
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Grumann, Markus [Verfasser]. "Readout of diagnostic assays on a centrifugal microfluidic platform / vorgelegt von Markus Grumann." 2006. http://d-nb.info/978031474/34.
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Bernacka-Wojcik, Iwona. "Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays." Doctoral thesis, 2014. http://hdl.handle.net/10362/13599.
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Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use.
Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area.
Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for
patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art.
Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the
output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide
polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation.
Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and
optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.Portuguese Science Foundation - (SFRH/BD/44258/2008), “SMART-EC” project
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Wang, Han. "Development of High-throughput and Robust Microfluidic Live Cell Assay Platforms for Combination Drug and Toxin Screening." Thesis, 2011. http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10517.
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Combination chemotherapies that introduce multi-agent treatments to target cancer cells are emerging as new paradigms to overcome chemotherapy resistance and side effects involved with conventional monotherapies. In environmental toxicology, characterizing effects of mixtures of toxins rather than simply analyzing the effect of single toxins are of significant interest. In order to determine such combination effects, it is necessary to systematically investigate interactions between different concentration-dependent components of a mixture. Conventional microtiter plate format based assays are efficient and cost-effective, however are not practical as the number of combinations increases drastically. Although robotic pipetting systems can overcome the labor-intensive and time-consuming limitations, they are too costly for general users. Microfluidic live cell screening platforms can allow precise control of cell culture microenvironments by applying accurate doses of biomolecular mixtures with specific mixing ratios generated through integrated on-chip microfluidic gradient generators.
This thesis first presents a live cell array platform with integrated microfluidic network-based gradient generator which enables generation and dosing of 64 unique combinations of two cancer drugs at different concentrations to an 8 by 8 cell culture chamber array. We have developed the system into a fully automated microfluidic live cell screening platform with uniform cell seeding capability and pair-wise gradient profile generation. This platform was utilized to investigate the gene expression regulation of colorectal cancer cells in response to combination cancer drug treatment. The resulting cell responses indicate that the two cancer drugs show additive effect when sequential drug treatment scheme is applied, demonstrating the utility of the microfluidic live cell assay platform.
However, large reagent consumption and difficulties of repeatedly generating the exact same concentrations and mixture profiles from batch to batch and device to device due to the fact that the generated gradient profiles or mixing ratios of chemicals have to rely on stable flow at optimized flow rate throughout the entire multi-day experiment limit the widespread use of this method. Moreover, producing three or more reagent mixtures require complicated microchannel structures and operating procedures when using traditional microfluidic network-based gradient generators. Therefore, an on-demand geometric metering-based mixture generator which facilitates robust, scalable, and accurate multi-reagent mixing in a high-throughput fashion has been developed and incorporated with a live cell array as a microfluidic screening platform for conducting combination drug or toxin assays. Integrated single cell trapping array allowed single cell resolution analysis of drugs and toxin effects. Reagent mixture generation and precise application of the mixtures to arrays of cell culture chambers repeatedly over time were successfully demonstrated, showing significantly improved repeatability and accuracy than those from conventional microfluidic network-based gradient generators. The influence of this improved repeatability and accuracy in generating concentration specified mixtures on obtaining more reliable and repeatable biological data sets were studied.
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Barbulovic-Nad, Irena. "New Microfluidic Platforms for Cell Studies." Thesis, 2009. http://hdl.handle.net/1807/26448.
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Biological cell manipulation and analysis is one of the most investigated applications of microfluidics. In the last decade, researchers have developed means to handle and sort cells, isolate and study single cells, assay whole and lysed cells, and transfect and electroporate in microchannels. Much of this work was motivated by the observation that many external forces and fields scale favorably in the micro-regime; this is especially the case for the electrical field. This dissertation investigates further integration of electrical forces with microfluidic devices, both channel- and droplet-based, in order to generate new, flexible and more efficient tools for studying cell biology.
The first part of the dissertation (Chapter 3) explores a new dielectrophoretic particle separation method in microchannels. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a direct current (DC) or low-frequency alternating current (AC) field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. Very effective separation at the low field strength suggests that this method can also be applied to a separation of biological cells that are not sensitive to low electric potential.
The second, larger part of the dissertation (Chapters 4 and 5) is focused on digital microfluidics (DMF), which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. It was demonstrated for the first time that DMF can be used as a method for cell culture and analysis. Several cell-based applications were implemented in DMF format including long-term culture, cell passaging, assaying and transfection. The data presented here suggest advanced performance of DMF techniques relative to standard macro-scale techniques. Cell analysis using DMF was found to be advantageous because of greatly reduced reagent and cell use, increased sensitivity, and the potential for multiplexing. Also, DMF technique for cell passaging demonstrated faster and more straightforward manipulation of cells than the standard techniques. In addition, no adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry.
The new DMF platform for long-term mammalian cell culture represents the first microfluidic implementation of any kind of all of the steps required for mammalian cell culture – cell seeding, growth, detachment, and re-seeding on a fresh surface. In addition, it is the first demonstration of long-term cell culture in nanoliter droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. We anticipate that the DMF cell culture and analysis techniques presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.
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DiCicco, Matthew. "Assessment of Novel Antimicrobial Therapy against Methicillin-resistant Staphylococcus pseudintermedius Biofilm with Conventional Assays and a Microfluidic Platform." Thesis, 2013. http://hdl.handle.net/10214/6663.
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This thesis is an investigation of methods to remediate methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilms through conventional and microfluidic-based in vitro assays. MRSP biofilm related infections are a major concern for veterinary clinicians as they may complicate remediation by the immune system or antimicrobials. Novel antimicrobials that have been found to reduce biofilm growth in other staphylococci were assessed in both mono- and combination therapy against MRSP biofilm. Quantitative assay results (p < 0.05) suggest fosfomycin alone and in combination with clarithromycin can significantly reduce biofilm formation. Morphological examination using scanning electron microscopy and atomic force microscopy further demonstrated the effectiveness of fosfomycin alone on biofilm formation on orthopaedic screws and mica sheets. Fabricated microfluidic assays were utilized to assess multiple concentrations of antimicrobial therapy against pre-formed biofilm under physiologically relevant conditions in a quick and repeatable manner. Results demonstrated the usefulness of microfluidic platforms in determining minimum biofilm eradication concentrations.
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(6863093), Li-Kai Lin. "Pollutant and Inflammation marker detection using low-cost and portable microfluidic platform, and flexible microelectronic platform." Thesis, 2019.
Find full textAbstract:
Existing methods for pathogen/pollutant detection or wound infection monitoring employ high-cost instruments that could only be operated by trained personnel, and costly device-based detection requires a time-consuming field-to-lab process. This expensive process with multiple prerequisites prolongs the time that patients must wait for a diagnosis. Therefore, improved methods for point-of-care biosensing are necessary. In this study, we aimed to develop a direct, easy-to-use, portable, low cost, highly sensitive and selective sensor platform with the goal of pollutant detection and wound infection/cancer migration monitoring. This study has two main parts, including microfluidic, electrical, and optical sensing platforms. The first part, including chapters 2, 3, and 4, focuses on Bisphenol A (BPA) lateral flow assay (LFA) detection; the second part, including chapter 5 focuses on the electrical sensing platform fabrication for one of the markers of inflammation, matrix metalloproteinases-9 (MMP-9), monitoring/detection. In chapters 2, 3, and 4, we found that the few lateral flow assays (LFAs) established for detecting the endocrine-disrupting chemical BPA have employed citrate-stabilized gold nanoparticles (GNPs), which have inevitable limitations and instability issues. To address these limitations, in chapter 2, a more stable and more sensitive biosensor is developed by designing strategies for modifying the surfaces of GNPs with polyethylene glycol and then testing their effectiveness and sensitivity toward BPA in an LFA. In chapter 3, we describe the development of a new range-extended bisphenol A (BPA) detection method that uses a surface enhanced Raman scattering lateral flow assay (SERS-LFA) binary system. In chapter 4, we examine advanced bisphenol A (BPA) lateral flow assays (LFAs) that use multiple nanosystems. The assays include three nanosystems, namely, gold nanostars, gold nanocubes, and gold nanorods, which are rarely applied in LFAs, compared with general gold nanoparticles. The developed LFAs show different performances in the detection of BPA. In chapter 5, a stable electrical sensing platform is developed for MMP-9 detection.
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Dahl, Andreas [Verfasser]. "Development of a miniaturised platform for PCR based assays with application in gene expression analysis and SNP genotyping / vorgelegt von Andreas Dahl." 2006. http://d-nb.info/988097540/34.
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