57 research outputs found

    Miniaturized Protein Microarray with Internal Calibration as Point-of-Care Device for Diagnosis of Neonatal Sepsis

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    Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 μL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 μL patient samples are diluted with 36 μL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14%) quantification of serum proteins for the diagnosis of neonatal sepsis

    ELISA in the multiplex era: potentials and pitfalls

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    Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues

    Front Lines of Thoracic Surgery

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    Front Lines of Thoracic Surgery collects up-to-date contributions on some of the most debated topics in today's clinical practice of cardiac, aortic, and general thoracic surgery,and anesthesia as viewed by authors personally involved in their evolution. The strong and genuine enthusiasm of the authors was clearly perceptible in all their contributions and I'm sure that will further stimulate the reader to understand their messages. Moreover, the strict adhesion of the authors' original observations and findings to the evidence base proves that facts are the best guarantee of scientific value. This is not a standard textbook where the whole discipline is organically presented, but authors' contributions are simply listed in their pertaining subclasses of Thoracic Surgery. I'm sure that this original and very promising editorial format which has and free availability at its core further increases this book's value and it will be of interest to healthcare professionals and scientists dedicated to this field

    Detection of biomarkers using DNA biosensors in a fluorescent platform to improve current one-step point-of-care methodologies

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    Current methodologies for patient diagnosis require the use of skilled workers, expensive reagents, and instrumentation, and most importantly, time. As these methodologies encourage inconsequential follow-up between patients and their doctors, new methods must be developed. These methods should follow the WHO guidelines of ASSURED (Accessible to those in need, Sensitive to the clinical range of the target of interest, Specific to the target of interest with no false positives or negatives, User-friendly, Rapid, under one hour for results, Equipment free, and Delivered to those in need) to create point of care technologies available for all. To this end, my studies focus on the development of convenient calibration methods for use with fluorescent, aptamer-based biosensors (aptamer beacons). Specifically, I describe here the creation, characterization, and application of a one-tube method for the calibration of aptamer beacons using sensors for the detection of DNA, silver ions, and thrombin as my test bed.A significant hurdle in the point of care testing of biomarkers in complex solutions is that there is sample variation, requiring large volumes of the target of interest and complex and cumbersome calibration. In response, I have created a simple, three-step, one-tube method for the calibration of aptamer beacons supporting the direct quantification of specific target molecules within complex sample matrices. Specifically, I have shown that the sequential addition of two inexpensive and stable oligonucleotide-based reagents allows for the determination of the absolute minimum and maximum fluorescence produced by the sensor in each solution, providing the information to calibrate in the face of sample-to-sample variations in a simple, single-tube format. Using the three associated measurements together with the pre-determined disassociation constant of the aptamer I demonstrate the single-tube calibrated quantification of multiple targets directly in complex samples

    Protein Detection

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    The book explores distinct aspects of protein purification and characterization steps. It discusses solubility problems, resin selection tricks, and essential credentials in a purification process. In addition, the book examines aggregation and proteinopathy-related protein detection methods and reviews several essential protein detection and characterization methods in cancer for diagnosis, prognosis, and therapy, such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, western blot, mass spectrometry, and others

    A portable device for time-resolved fluorescence based on an array of CMOS SPADs with integrated microfluidics

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    [eng] Traditionally, molecular analysis is performed in laboratories equipped with desktop instruments operated by specialized technicians. This paradigm has been changing in recent decades, as biosensor technology has become as accurate as desktop instruments, providing results in much shorter periods and miniaturizing the instrumentation, moving the diagnostic tests gradually out of the central laboratory. However, despite the inherent advantages of time-resolved fluorescence spectroscopy applied to molecular diagnosis, it is only in the last decade that POC (Point Of Care) devices have begun to be developed based on the detection of fluorescence, due to the challenge of developing high-performance, portable and low-cost spectroscopic sensors. This thesis presents the development of a compact, robust and low-cost system for molecular diagnosis based on time-resolved fluorescence spectroscopy, which serves as a general-purpose platform for the optical detection of a variety of biomarkers, bridging the gap between the laboratory and the POC of the fluorescence lifetime based bioassays. In particular, two systems with different levels of integration have been developed that combine a one-dimensional array of SPAD (Single-Photon Avalanch Diode) pixels capable of detecting a single photon, with an interchangeable microfluidic cartridge used to insert the sample and a laser diode Pulsed low-cost UV as a source of excitation. The contact-oriented design of the binomial formed by the sensor and the microfluidic, together with the timed operation of the sensors, makes it possible to dispense with the use of lenses and filters. In turn, custom packaging of the sensor chip allows the microfluidic cartridge to be positioned directly on the sensor array without any alignment procedure. Both systems have been validated, determining the decomposition time of quantum dots in 20 nl of solution for different concentrations, emulating a molecular test in a POC device.[cat] Tradicionalment, l'anàlisi molecular es realitza en laboratoris equipats amb instruments de sobretaula operats per tècnics especialitzats. Aquest paradigma ha anat canviant en les últimes dècades, a mesura que la tecnologia de biosensor s'ha tornat tan precisa com els instruments de sobretaula, proporcionant resultats en períodes molt més curts de temps i miniaturitzant la instrumentació, permetent així, traslladar gradualment les proves de diagnòstic fora de laboratori central. No obstant això i malgrat els avantatges inherents de l'espectroscòpia de fluorescència resolta en el temps aplicada a la diagnosi molecular, no ha estat fins a l'última dècada que s'han començat a desenvolupar dispositius POC (Point Of Care) basats en la detecció de la fluorescència, degut al desafiament que suposa el desenvolupament de sensors espectroscòpics d'alt rendiment, portàtils i de baix cost. Aquesta tesi presenta el desenvolupament d'un sistema compacte, robust i de baix cost per al diagnòstic molecular basat en l'espectroscòpia de fluorescència resolta en el temps, que serveixi com a plataforma d'ús general per a la detecció òptica d'una varietat de biomarcadors, tancant la bretxa entre el laboratori i el POC dels bioassaigs basats en l'anàlisi de la pèrdua de la fluorescència. En particular, s'han desenvolupat dos sistemes amb diferents nivells d'integració que combinen una matriu unidimensional de píxels SPAD (Single-Photon Avalanch Diode) capaços de detectar un sol fotó, amb un cartutx microfluídic intercanviable emprat per inserir la mostra, així com un díode làser UV premut de baix cost com a font d'excitació. El disseny orientat a la detecció per contacte de l'binomi format pel sensor i la microfluídica, juntament amb l'operació temporitzada dels sensors, permet prescindir de l'ús de lents i filtres. Al seu torn, l'empaquetat a mida de l'xip sensor permet posicionar el cartutx microfluídic directament sobre la matriu de sensors sense cap procediment d'alineament. Tots dos sistemes han estat validats determinant el temps de descomposició de "quantum dots" en 20 nl de solució per a diferents concentracions, emulant així un assaig molecular en un dispositiu POC

    Advanced analyses of physiological signals and their role in Neonatal Intensive Care

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    Preterm infants admitted to the neonatal intensive care unit (NICU) face an array of life-threatening diseases requiring procedures such as resuscitation and invasive monitoring, and other risks related to exposure to the hospital environment, all of which may have lifelong implications. This thesis examined a range of applications for advanced signal analyses in the NICU, from identifying of physiological patterns associated with neonatal outcomes, to evaluating the impact of certain treatments on physiological variability. Firstly, the thesis examined the potential to identify infants at risk of developing intraventricular haemorrhage, often interrelated with factors leading to preterm birth, mechanical ventilation, hypoxia and prolonged apnoeas. This thesis then characterised the cardiovascular impact of caffeine therapy which is often administered to prevent and treat apnoea of prematurity, finding greater pulse pressure variability and enhanced responsiveness of the autonomic nervous system. Cerebral autoregulation maintains cerebral blood flow despite fluctuations in arterial blood pressure and is an important consideration for preterm infants who are especially vulnerable to brain injury. Using various time and frequency domain correlation techniques, the thesis found acute changes in cerebral autoregulation of preterm infants following caffeine therapy. Nutrition in early life may also affect neurodevelopment and morbidity in later life. This thesis developed models for identifying malnutrition risk using anthropometry and near-infrared interactance features. This thesis has presented a range of ways in which advanced analyses including time series analysis, feature selection and model development can be applied to neonatal intensive care. There is a clear role for such analyses in early detection of clinical outcomes, characterising the effects of relevant treatments or pathologies and identifying infants at risk of later morbidity

    Puesta a punto de sistemas de detección de proteína c-reactiva (crp) mediante reconocimiento molecular. Estudio de prestaciones

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    [ES] Según la OMS (Organización Mundial de la Salud), cerca de 17 millones de personas murieron en 2008 debido a enfermedades cardiovasculares (CVD) convirtiéndose en un problema serio para la salud pública. Debido a esto, muchos grupos de investigación centran sus esfuerzos en la caracterización de nuevos biomarcadores que permitan un diagnóstico rápido y fiable de las mismas. La proteína C-reactiva ha sido reconocida como uno de los biomarcadores de CVD y procesos inflamatorios. Así pues, dada su importancia clínica, actualmente es posible encontrar sistemas de detección de la misma en suero sanguíneo, principalmente mediante el inmunoensayo ELISA (Enzyme-Linked ImmunoSorbent Assay). Sin embargo, las técnicas tradicionales presentan inconvenientes como la necesidad de grandes cantidades de muestra para el diagnóstico o tiempos de detección largos. Es por eso que tecnologías alternativas, como son los biosensores, van ganando terreno en el campo del diagnóstico clínico ya que sus prestaciones son cada día mayores. Con el presente trabajo se pretende poner a punto un sistema de detección para la proteína C-reactiva en formato microarray. Para ello, se analizan y comparan los distintos parámetros críticos que componen un microarray: 1) formato del biosensor (inmunoensayo competitivo directo o indirecto); 2) tipo de bioreceptor (anti-CRP monoclonal o policlonal o CRP); y 3) inmovilización del bioreceptor (pasiva o covalente). Con este fin, se llevaron a cabo distintos experimentos sobre superficies funcionalizadas con distintos organosilanos para añadir a la misma grupos vinil, tiol y flúor, que fueron empleados para inmovilizar el bioreceptor por adsorción en el caso de los grupos vinil y mediante enlace covalente con los grupos tiol en superficies mixtas compuestas con flúor y tiol. Así, se obtuvo un sistema de detección de la CRP dentro del intervalo de interés clínico (1-3 g/mL), con porcentajes de recuperación entre 70 y 136%, mediante inmunoensayo directo competitivo en formato microarray usando superficies de vidrio funcionalizadas con una mezcla 1:1 en volumen de 1H, 1H, 2H, 2H Perfluorodecyl-trietoxisilano 97% y 3-Mercaptopropyl trimetoxisilano 85%. Finalmente, en el trabajo se plantea una alternativa al inmunoensayo, basándonos en la capacidad de CRP para unirse específicamente a la fosforilcolina y se plantea la puesta a punto de un sistema donde dicha molécula actúe como bioreceptor en una superficie de resina SU-8.[EN] According to the WHO (World Health Organization), around 17 million of people died in 2008 due to cardiovascular diseases (CDV), then, becoming a public health hazard. Due to this, many researching groups have already started to characterized new biomarkers or molecules present in the human fluids to diagnose CVDs in a reliable way. C-reactive protein is one of the already recognized biomarkers of CVD and inflammatory processes. Because of its clinic importance, it’s currently available detection system of it from human blood, mainly by ELISA immunoassay (Enzyme-Linked ImmunoSorbent Assay). However, traditional techniques have some disadvantages such as the requirement of high sample amounts or long detection times. Biosensors overcome those problems, that is the reason why their design is gaining ground in the diagnostic field. Therefore, the main objective of this thesis is the development of a system for CRP detection. For that, different critical parameters regarding to microarray design are analyzed and compared: 1) Biosensor format (direct or indirect competitive immunoassay); 2) type of bioreceptor (monoclonal or polyclonal antibody); 3) bioreceptor immobilization (passively or covalently). Regarding to this, glass surfaces were functionalized with different organosilanes in order to add vinil, thiol and Fluor groups to it, that were used further to immobilized the bioreceptor by either adsorption (with vinil groups) or covalent bounds (with thiol groups in mixed surfaces with thiol and fluor). Finally, it was developed a detection system for CRP within clinical range (1-3 mg/mL), with a recovery percentage around 70 and 136%, through a direct competitive immunoassay in a microarray printed in glass surfaces previously functionalized with a mixture 1:1 (in volume) of 1H, 1H, 2H, 2H Perfluorodecyl-triethoxysilane 97% and 3-Mercaptopropyl trimethoxysilane 85%. In addition, we suggested an alternative to the immunoassay. So, based on the CRP capability to bind specifically phosphorylcholine (PC) is possible to optimize a new detection system in microarray format where PC is immobilized in a functionalized SU-8 surface.Vargas Valderrama, A. (2014). Puesta a punto de sistemas de detección de proteína c-reactiva (crp) mediante reconocimiento molecular. Estudio de prestaciones. http://hdl.handle.net/10251/46140.Archivo delegad

    Lab-on-PCB Devices

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    Lab-on-PCB devices can be considered an emerging technology. In fact, most of the contributions have been published during the last 5 years. It is mainly focussed on both biomedical and electronic applications. The book includes an interesting guide for using the different layers of the Printed Circuit Boards for developing new devices; guidelines for fabricating PCB-based electrochemical biosensors, and an overview of fluid manipulation devices fabricated using Printed Circuit Boards. In addition, current PCB-based devices are reported, and studies for several aspects of research and development of lab-on-PCB devices are described

    Advances in Microfluidics Technology for Diagnostics and Detection

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    Microfluidics and lab-on-a-chip have, in recent years, come to the forefront in diagnostics and detection. At point-of-care, in the emergency room, and at the hospital bed or GP clinic, lab-on-a-chip offers the potential to rapidly detect time-critical and life-threatening diseases such as sepsis and bacterial meningitis. Furthermore, portable and user-friendly diagnostic platforms can enable disease diagnostics and detection in resource-poor settings where centralised laboratory facilities may not be available. At point-of-use, microfluidics and lab-on-chip can be applied in the field to rapidly identify plant pathogens, thus reducing the need for damaging broad spectrum pesticides while also reducing food losses. Microfluidics can also be applied to the continuous monitoring of water quality and can support policy-makers and protection agencies in protecting the environment. Perhaps most excitingly, microfluidics also offers the potential to enable entirely new diagnostic tests that cannot be implemented using conventional laboratory tools. Examples of microfluidics at the frontier of new medical diagnostic tests include early detection of cancers through circulating tumour cells (CTCs) and highly sensitive genetic tests using droplet-based digital PCR.This Special Issue on “Advances in Microfluidics Technology for Diagnostics and Detection” aims to gather outstanding research and to carry out comprehensive coverage of all aspects related to microfluidics in diagnostics and detection
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