Waterborne or foodborne diseases are caused by consuming contaminated fluids or foods. The presence of pathogenic microorganisms can contaminate food or drinking water. These microorganisms can cause sickness even if they are present in minimal concentrations. The World Health Organization (WHO) has defined the standards for clean drinking water as the absence of E. coli in a 100 mL collected volume. Contaminated water or food can cause many diseases, and diarrhea is one of a prominent disease. Early detection of contamination in food or drinking water is critical. Conventional culture-based methods are time-consuming, labour intensive, and not suitable for on-site testing. Nucleic acid-based tests are sensitive and can rapidly detect pathogens. Microfluidic technology can play a significant role to develop low-cost, rapid, integrated, and portable nucleic acid-based detection devices. Microfluidic systems for isothermal amplification assays can be classified into two groups such as droplet-based and chamber-based systems. In this thesis, both droplet-based and chamber-based approaches were used to build the microfluidic methods for isothermal amplification assays. First, a simple electromechanical probe (tweezers) was developed that can manipulate a small aqueous droplet in a bi-layer oil phase. The tweezer consisted of two needles positioned close to each other and used polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. AC electric potential was applied to the two metal electrodes. Droplet acquired a charge from the high voltage electrode and consequently performed an oscillatory motion with the same electrode. This droplet motion was controlled using two parameters of electric potential and frequency of the applied signal. Initially, electrically actuated droplet (0.3 µL) motion was investigated for a range of applied potential (400-960 Volts) and frequencies (0.1-1000 Hz). The droplet motion with high voltage electrode was characterized into three modes such as detachment, oscillation, and attachment. Mechanical motion of tweezer was used to transport droplet to various positions. Consequently, operations such as transportation, extraction, and merging were demonstrated. First, droplet (5 µL) transportation was characterized under the applied potential of 2000 Volts at various frequencies (5 to 1000 Hz). The droplet was successfully transported to the speed of 15 mm/s at higher frequencies (100 or 1000 Hz). Droplets of various volumes (12-80 µL) were extracted by increasing applied electric potential, from 0 to 6000 Volts at 5 Hz. Then, the operation of droplets merging was demonstrated using operational conditions for electrical tweezer. Finally, electrical tweezer was used to prepare samples for isothermal amplification assays. Two droplets consisted of various reagents of isothermal amplification assays, were transported and merged using the electrical tweezer. Then, a merged droplet (25 µL) was transported and immobilized in the amplification zone. The temperature of the amplification zone (~65°C) was maintained using an in-situ heater. DNA amplification was verified by measuring the off-chip end-point fluorescence intensity of isothermal assays. Second, an integrated microfluidic device has been developed to prepare a sample for isothermal amplification assays. And in-situ real-time amplification assays were performed to detect bacteria. The device consisted of two chambers (lysis and amplification) connected through a microchannel. A low-cost fabrication method was introduced to embed two resistive wire heaters around both chambers. Initially, bacteria cells were thermally lysed in the lysis chamber at 92°C for 5 min. Then, DNA was electrophoretically transported from lysis to the amplification chamber. The electric potential of 10 Volts was applied for 10 min for DNA transportation. Next, transported DNA was amplified at 65°C and DNA amplification was detected by measuring in-situ fluorescence intensity in the real-time format. The operation of the integrated microfluidic device was demonstrated in three steps. 1) Operation of individual components. 2) Operation of two components in a coupled format. 3) Integrated operation of three components with measurement of fluorescent intensity in a real-time format. The bacteria samples with the concentration of 100 CFU/mL were detected in less than one hour. / Thesis / Doctor of Philosophy (PhD)

Yes / Acute kidney injury (AKI) can occur rarely in patients exposed to iodinated contrast and result in contrast-induced AKI (CI-AKI). A key risk factor is the presence of pre-existing chronic kidney disease (CKD), therefore it is important to assess patient risk and obtain kidney function measurement prior to administration. Point of care (PoC) testing provides an alternative strategy but there remains uncertainty, with respect to diagnostic accuracy and clinical utility. A device study compared three PoC analysers (Nova StatSensor, Abbott i-STAT, Radiometer ABL800 FLEX) with a reference laboratory standard (Roche Cobas 8000 series, enzymatic creatinine). Three hundred adult patients attending a UK hospital phlebotomy department were recruited to have additional blood samples for analysis on the PoC devices. The ABL800 FLEX had the strongest concordance with laboratory measured serum creatinine (mean bias=-0.86, 95% limits of agreement = -9.6 to 7.9) followed by the i-STAT (average bias=3.88, 95% limits of agreement = -8.8 to 16.6) and StatSensor (average bias=3.56, 95% limits of agreement = -27.7 to 34.8). In risk classification, the ABL800 FLEX and i-STAT identified all patients with an eGFR≤30, whereas the StatSensor resulted in a small number of missed high-risk cases (n=4/13) and also operated outside of the established performance goals. The screening of patients at risk of CI-AKI may be feasible with PoC technology. However in this study it was identified that the analyser concordance with the laboratory reference varies. It is proposed that further research exploring PoC implementation in imaging department pathways is needed. / Yorkshire and Humber Academic Health Science Network (Grant Number: YHP0318)

Dengue is a major public health problem that affects tens of millions of people annually in tropical and sub-tropical countries. This acute viral infection happens to be severe and even life threatening but there is still no available drug or vaccine. Previous studies have noted early higher viral burden in patients who develop more severe symptoms suggesting that administration of a potent and safe antiviral may prevent progression to severe dengue. To verify this hypothesis, we have conducted the first RCT directed towards reducing the viral burden in vivo by administrating chloroquine (CQ), a cheap and well-tolerated drug that inhibits DENV in vitro with concentrations achievable in vivo, to 307 Vietnamese adults with suspected dengue (257 of them were laboratory-confirmed cases). Unfortunately, we did not see an effect of CQ on the duration of infection. However in patients treated with CQ, we observed a trend towards a lower incidence of severe forms. We did not find any differences in the immune response that can explain this trend. We also found more adverse events, primarily vomiting, with CQ. In addition, we have explored the relationships between clinical features, antibody responses and virological markers in these patients. We found that the early magnitude of viremia is positively associated with disease severity and there are serotype dependent differences in infection kinetics. We found as well that DENV was cleared faster and earlier in patients with secondary infections. To complete this study, we have also evaluated 2 rapid lateral flow tests for the diagnosis of dengue in a panel of plasma samples from 245 RT-PCR confirmed dengue patients and 47 with other febrile illnesses. Our data suggest that the NS1 test component of these tests are highly specific and have similar levels of sensitivity (~60%). Both NS1 assays were significantly more sensitive for primary than secondary dengue. The IgM parameter in the SD Duo test improved overall test sensitivity without compromising specificity. All these findings are of major importance for further anti-viral drug testing.

A aplicação da tecnologia da informação na Saúde tem dado um suporte à prestação do cuidado ao paciente com mais qualidade e propiciado a adesão de processos mais lineares, eficientes e seguros. Apresenta-se, nesta Tese, o desenvolvimento e a avaliação de um módulo, denominado Sistematização da Assistência de Enfermagem (Módulo SAE) que, incorporado ao Sistema de Informação Hospitalar (SIH) do Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, seja capaz de automatizar as ações envolvidas no Processo de Enfermagem, identificando os benefícios e as limitações decorrentes da adoção de um sistema informatizado. A metodologia foi desenvolvida em duas etapas, uma para possibilitar o desenvolvimento e a implantação do Módulo SAE, outra para avaliar a contribuição para o enfermeiro e sua aplicabilidade no ponto de cuidado do paciente (PCP). A primeira etapa constou da organização das funcionalidades do Processo de Enfermagem no Módulo SAE. Como resultado, um conjunto de funcionalidades, estruturadas ou em texto livre, foi definido e compreende: coleta dos dados do paciente e exame físico, diagnóstico e evolução de enfermagem, intervenções e prescrição de enfermagem. O uso do Módulo SAE foi realizado em paralelo com a SAE manuscrita durante 6 meses e aprovado por 28 enfermeiros. Após esta fase, 50 outros enfermeiros foram treinados e utilizaram o Módulo SAE no seu trabalho diário, durante 15 meses, finalizando a primeira etapa com 78 enfermeiros treinados. A segunda etapa constou de uma pesquisa com os enfermeiros que responderam dois questionários para avaliar o uso do Módulo SAE, por meio de duas formas diferentes de acesso, uma delas por computador fixo no posto de enfermagem e a outra, um computador móvel desenvolvido no próprio hospital, para levar o sistema ao PCP. Para obter melhores resultados na pesquisa, as características pessoais desses enfermeiros foram analisadas e comparadas. Da homogeneidade dos resultados foram selecionados 42 profissionais aptos para usarem o computador móvel, dos quais, 25 participaram. Mediante os resultados analíticos e estatísticos, concluiu-se que o Módulo SAE incorporado no SIH contribuiu para o enfermeiro obter maior legibilidade e segurança no registro eletrônico, tanto por acesso fixo como por acesso móvel. A comparação entre as formas de acesso para a formulação do diagnóstico e da prescrição de enfermagem demonstrou resultado positivo a despeito das diferenças estatisticamente significantes (teste não paramétrico de Wilcoxon). Além disso, o registro eletrônico permitiu ao enfermeiro dispor de maior tempo para a assistência direta ao paciente (fixo 84% e móvel 60%). Quanto à facilidade do uso do computador móvel no PCP, as respostas positivas (84%) indicaram a diminuição do tempo gasto para a atualização da prescrição de enfermagem. / The application of information technology in health has given support to the patient care with more quality and provides more linear, efficient and safe processes. This thesis presents the development and evaluation of a module called Systematization of Nursing Care (SAE Module) that when incorporated into the Hospital Information System of the Heart Institute of the University of Sao Paulo Medical School is able to automate the actions involved in Nursing Process. The identification of the benefits and limitations resulting from the adoption of an information system are also described. The methodology was developed in two stages: the first involved the development and deployment of the SAE Module, and the second involved the assessment of the nursing computerized system to the nurse and its application at the point of patient care (PCP). The first step included the organization of the nursing process in the SAE Module. As a result, a set of features, structured or free texts were defined and comprised: collection of patient data and physical examination, nursing diagnosis and evolution, nursing interventions and prescription. The use of SAE Module was done in parallel with the SAE handwritten during 6 months and approved by 28 nurses. After this stage, 50 other nurses were trained and used it in their daily work for 15 months, finalizing the first step with 78 trained nurses. The second step involved the evaluation of nurses who replied two forms to evaluate SAE Module usage through two different ways of access, one using a fixed computer in the nursing counter and the other using a mobile computer, developed in the hospital in order to bring the system to the PCP. In order to obtain the best results from the evaluation, personal characteristics of these nurses were reviewed and compared. Fortytwo nurses were selected according to the homogeneity of the results, out of which 25 participated in the evaluation. The analytical and statistical results lead to the conclusion that the SAE Module incorporated into SIH contributed to the nurse to get more readability and security from patient electronic records for both fixed and mobile access. The comparison between the two forms of access to the formulation of nursing diagnosis and prescription has shown positive results despite statiscally significant differences (nonparametric Wilcoxon test). In addition, it was observed that the patient electronic record (fixed 84% and 60%) allowed an increase of the nurse´s time in the direct patient care. Regarding the use of a mobile computer in the PCP, the positive responses (84%) indicated a reduction in the time spent to update the nursing prescription.

<p>Point of care (POC) diagnosis is essential in personalized treatment to obtain effective clinical outcomes when the patient is on site. And Mass spectrometry (MS) system promotes the applications of rapid sampling ionization, which could be a tool for fast disease determination. In this thesis, a miniature MS system was firstly developed for POC tissue analysis. Lipid profile in rat organs were demonstrated. By coupling with online Paternò–Büchi (PB) reaction, fast determination of lipid C=C bond location isomers was realized. The system was applied to quantitatively analyze the change of lipid C=C location isomers between mouse normal and cancerous samples. The intensity ratio of fatty acid 18:1 (D9) and 18:1 (D11) in wild type breast tissue was calculated to be 2.881, while the ratio in tumor breast tissue was 0.667. The direct sampling-based miniature MS system is potential for POC analysis of lipid profiles and lipid biomarkers discovery.</p> <p>Secondly, an integration of paper-capillary spray and MS make it possible to analyze dried blood samples instantly in clinical laboratory and hospital. Quantitation of ratio between deuterate Phenylalanine and deuterate Tyrosine was achieved by using paper spray and paper-capillary spray MS directly, without any pretreatment of blood samples. Furthermore, these methods could generate calibration curves which enable the calculation of Phenylalanine concentration in whole human blood within 60 seconds. This disposable design is a promising application for point-of-care (POC) PKU analysis in newborn screening. </p> <p>At last, an increased in free fatty acids (FFAs) of cereals was observed during storage, and a simple and direct rice quality assessment was performed using nanoESI (Nano-Electrospray Ionization) mass spectrometry method. Six fatty acids including palmitic, oleic, and linoleic acids were compared between different rice species, growth regions and harvest years. 2D and 3D linear discriminant analysis (LDA) methods were deployed and a good sample separation was achieved. This direct sampling method of extracting FAs from rice surface combined with MS is suitable for industrial use in rapid identification for large number of samples.</p>

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

<p>The cardiac troponins are the biochemical markers of choice for the diagnosis of acute myocardial infarction (AMI) and risk prediction in patients with acute coronary syndrome (ACS). In this thesis, the role of early serial cardiac troponin I (cTnI) testing was assessed in fairly unselected patient populations admitted because of chest pain and participating in the FAST II-study (n=197) and the FASTER I-study (n=380). Additionally, the importance of cTnI testing in stable post-ACS patients from the FRISC II-study (n=1092) was studied.</p><p>The analyses in chest pain patients demonstrate that cTnI is very useful for early diagnostic and prognostic assessment. cTnI allowed already 2 hours after admission the reliable exclusion of AMI and the identification of low-risk patients when ECG findings and a renal marker such as cystatin C were added as conjuncts. Other biomarkers such as CK-MB, myoglobin, NT-pro BNP or CRP did not provide superior clinical information. However, myoglobin may be valuable in combination with cTnI results for the early prediction of an impending major AMI when used as input variable for an artificial neural network. Such an approach applying cTnI results only may also furthermore improve the early diagnosis of AMI.</p><p>Persistent cTnI elevation > 0.01 μg/L was detectable using a high-sensitive assay in 26% of the stable post-ACS patients from the FRISC II-study. NT-pro BNP levels at 6 months were the most important variable independently associated to persistent cTnI elevation besides male gender, indicating a relationship between adverse left ventricular remodeling processes and cTnI leakage. Patients with persistent cTnI elevation had a considerable risk for both mortality and AMI during 5 year follow-up. </p><p>These analyses thus, confirm the value of cTnI for early assessment of chest pain patients and provide new and unique evidence regarding the role of cTnI for risk prediction in post-ACS populations.</p>

The cardiac troponins are the biochemical markers of choice for the diagnosis of acute myocardial infarction (AMI) and risk prediction in patients with acute coronary syndrome (ACS). In this thesis, the role of early serial cardiac troponin I (cTnI) testing was assessed in fairly unselected patient populations admitted because of chest pain and participating in the FAST II-study (n=197) and the FASTER I-study (n=380). Additionally, the importance of cTnI testing in stable post-ACS patients from the FRISC II-study (n=1092) was studied. The analyses in chest pain patients demonstrate that cTnI is very useful for early diagnostic and prognostic assessment. cTnI allowed already 2 hours after admission the reliable exclusion of AMI and the identification of low-risk patients when ECG findings and a renal marker such as cystatin C were added as conjuncts. Other biomarkers such as CK-MB, myoglobin, NT-pro BNP or CRP did not provide superior clinical information. However, myoglobin may be valuable in combination with cTnI results for the early prediction of an impending major AMI when used as input variable for an artificial neural network. Such an approach applying cTnI results only may also furthermore improve the early diagnosis of AMI. Persistent cTnI elevation &gt; 0.01 μg/L was detectable using a high-sensitive assay in 26% of the stable post-ACS patients from the FRISC II-study. NT-pro BNP levels at 6 months were the most important variable independently associated to persistent cTnI elevation besides male gender, indicating a relationship between adverse left ventricular remodeling processes and cTnI leakage. Patients with persistent cTnI elevation had a considerable risk for both mortality and AMI during 5 year follow-up. These analyses thus, confirm the value of cTnI for early assessment of chest pain patients and provide new and unique evidence regarding the role of cTnI for risk prediction in post-ACS populations.

In der vorliegenden Arbeit wird eine integrierbare Pumpentechnologie für polymerbasierte mikrofluidische Systeme entwickelt. Ausgehend von den Anforderungen für die Durchführung molekulardiagnostischer Nachweise kommen dabei Fertigungsverfahren zum Einsatz, die sich auch für Einweg-Anwendungen eignen. Das genutzte Aktorprinzip für die integrierten Mikropumpen basiert auf der Elektrolyse von Wasser. Zur besseren technologischen Integrierbarkeit wird das Wasser in Form eines Hydrogels appliziert. Der Elektrolyt wird dabei mit einer Polymermembran mit geringer Wasserdampfdurchlässigkeit verschlossen. Die Membran wird in ihrem plastischen Verformbereich genutzt. Zur Dimensionierung der Mikropumpen und des mikrofluidischen Systems werden analytische und numerische Modelle entwickelt, die eine gute Übereinstimmung mit den Messwerten zeigen. Die Funktionsfähigkeit wird anhand zweier vollständig integriert ablaufender Immunoassays demonstriert. Dabei kommt ein polymerbasierter, optischer Biosensor zum Einsatz.

In this dissertation, three different microfluidic devices with bioanalytical applications are presented. From chapter to chapter, the bioanalytical focus will gradually become the development of a point-of-care sensor platform able to yield a reliable and quantitative response in the presence of the desired target. The first device consists of photolithographically-patterned gold on glass bipolar electrodes and PDMS Y-shaped microchannels for the controlled enrichment, separation from a mixture, and delivery of two charged dyes into separate receiving microchannels. The principle for the permanent separation of these dyes is based on the concept of bipolar electrochemistry and depended on the balancing/unbalancing of convective and electromigrating forces caused by the application of a potential bias, as well as the activation/deactivation of the bipolar electrodes. Two different bipolar electrode configurations are described and fluorescence is used to optimize their efficiency, speed, and cleanliness of delivery. The second device is a DNA sensor fabricated on paper by wax printing and folding to form 3D channels. DNA is detected by strand-displacement induced fluorescence of a single-stranded DNA. A multiplexed version of this sensor is also shown where the experiment results in “OR” and “AND” Boolean logic gate operations. In addition, the nonspecific adsorption of the reagents to cellulose is studied, demonstrating that significant reduction of nonspecific adsorption and increased sensitivity can be achieved by pre-treating the substrate with bovine serum albumin and by preparing all analyte solutions with spectator DNA. The third device, also made of paper, has a novel design and uses a versatile electrochemical detection method for the indirect detection of analytes via the direct detection of AgNP labels. A proof-of-concept experiment is shown where streptavidin-coated magnetic microbeads and biotin-coated AgNPs are used to form a composite model analyte. The paper device, called oSlip, and electrochemical method used are easily coupled so the resulting sensor has a simple user-device interface. LODs of 767 fM are achieved while retaining high reproducibility and efficiency. The fourth device is the updated version of the oSlip. In this case, the objective is to show the current progress and limitations in the detection of real analytes using the oSlip device. A sandwich-type immunoassay approach is used to detect human chorionic gonadotrophin (pregnancy hormone) present in human urine. Various optimization steps are performed to obtain the ideal reagent concentrations and incubation time necessary to form the immunocomposite in one step, that is, by mixing all reagents at the same time in the oSlip. Additionally, improvements to the electrochemical detection step are demonstrated. / text