abstract: In this thesis, I present a lab-on-a-chip (LOC) that can separate and detect Escherichia Coli (E. coli) in simulated urine samples for Urinary Tract Infection (UTI) diagnosis. The LOC consists of two (concentration and sensing) chambers connected in series and an integrated impedance detector. The two-chamber approach is designed to reduce the non-specific absorption of proteins, e.g. albumin, that potentially co-exist with E. coli in urine. I directly separate E. coli K-12 from a urine cocktail in a concentration chamber containing micro-sized magnetic beads (5 µm in diameter) conjugated with anti-E. coli antibodies. The immobilized E. coli are transferred to a sensing chamber for the impedance measurement. The measurement at the concentration chamber suffers from non-specific absorption of albumin on the gold electrode, which may lead to a false positive response. By contrast, the measured impedance at the sensing chamber shows ~60 kÙ impedance change between 6.4x104 and 6.4x105 CFU/mL, covering the threshold of UTI (105 CFU/mL). The sensitivity of the LOC for detecting E. coli is characterized to be at least 3.4x104 CFU/mL. I also characterized the LOC for different age groups and white blood cell spiked samples. These preliminary data show promising potential for application in portable LOC devices for UTI detection. / Dissertation/Thesis / M.S. Electrical Engineering 2011

The wetting of a rough material is controlled by surface chemistry and morphology, the liquid phase, solutes, and surfactants that affect the surface tension with the gas phase, and environmental conditions such as temperature and pressure. Materials with high (>150˚) apparent contact angles are known as superhydrophobic and are very resistant to wetting. However, in complex biological mixtures eventually protein adsorbs, fouling the surface and facilitating wetting on time scales from seconds to months. The work here uses the partially-wetted (Cassie-Baxter) to fully-wetted (Wenzel) state transition to control drug delivery and to perform surfactant detection via surface tension using hydrophobic and superhydrophobic materials. First there is an overview of the physics of the non-wetting state and the transition to wetting. Then there is a review of how wetting can be controlled by outside stimuli and applications of these materials. Next there is work presented on controlling drug release using superhydrophobic materials with controlled wetting rates, with both in vitro and in vivo results. Then there is work on developing a sensor based on this wetting state transition and its applications toward detecting solute levels in biological fluids for point-of-care diagnosis. Finally, there is work presented on using these sensors for detecting the alcohol content in wine and spirits.

abstract: Measles is a contagious, vaccine-preventable disease that continues to be the leading cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins. Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects. Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23). These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker. / Dissertation/Thesis / Masters Thesis Biology 2016

Orientador: Lauro Tatsuo Kubota / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T23:55:15Z (GMT). No. of bitstreams: 1 Miranda_BarbaraSantosde_D.pdf: 3761472 bytes, checksum: d04db9862f7022a5bea49d756efbfdbc (MD5) Previous issue date: 2013 / Resumo: Este trabalho apresenta o desenvolvimento de dois sensores distintos que através da aplicação de nanomateriais em seus sistemas apresentam uma melhora no desempenho. Com o objetivo de estabilizar a fase alfa do hidróxido de níquel, foi proposta uma nova síntese que resultou em um novo material de hidróxido de níquel e nanopartículas de ouro. O do hidróxido de níquel se apresentou com a estrutura alfa desejada, a estrutura e morfologia das nanopartículas de ouro foram preservadas. O material híbrido se apresenta nanoestruturado e sem separação de fases. Sugere-se que este novo material seja formado como um nanocompósito onde as nanopartículas de ouro formam a matriz e o hidróxido de níquel recobre a superfície. A estabilidade do novo material é bem superior que a do hidróxido de níquel puro, quedas de corrente após 50 ciclos consecutivos de 6% contra 50% respectivamente. A atividade eletrocatalítica deste novo nanomaterial foi investigada frente à glicose e a constante eletrocatalítica obtida para o nanocompósito foi cerca de 7 vezes maior que para o hidróxido de níquel puro. A sensibilidade do eletrodo modificado com a-Ni(OH)2/AuNP se manteve similar aos da literatura. O segundo sensor estudado foi a aplicação de nanopartículas fluorescentes como marcadores do imunosensor para a detecção precoce do esporo do fungo da ferrugem asiática da soja. As nanopartículas fluorescentes permitiram alcancar um baixo limite de detecção, 2.2 ng/mL, o menor encontrado na literatura. Além disso, foi investigado outros parâmetros para o desenvolvimento de um dispositivo point-of-care, como o melhor substrato para reter o analito, a membrana de nitrocelulose, se apresentou com as melhores propriedades frente as alternativas. Ainda mais, o dispositivo foi construido para que possa ser utilizado por pessoas não treinadas no campo, em um tempo de análise menor que ELISA e PCR, sem o uso de equipamento caros e complicados, usando somente uma lâmpada de UV simples como fonte de excitação e a visualização do sinal a olho nu com um limite de detecção de apenas 300 esporos, o que e ainda dez vezes mais baixo que o atual teste comercial. / Abstract: This work presents the development of two distinct sensors that have shown great improvements in their performance trough nanoparticle application in their systems. The first study aims to stabilize the nickel hydroxide alpha structure for future carbohydrate detection. We proposed a new synthesis, which resulted in a nickel hydroxide and gold nanoparticle hybrid material. The new material has the expected nickel hydroxide alpha structure, maintains gold nanoparticle¿s structure and morphology and allows a simple electrode modification. We suggest that the new material is a nanocomposite where gold nanoparticles are the matrix while the nickel hydroxide cover the surface. The new material has higher stability properties than the pure nickel hydroxide, after 50 cycles the current decreased 6 and 50% respectively. In addition, an improvement was observed in the catalytic constant, approximately 7 times bigger than plain nickel hydroxide. Although the new material showed these good improvements, the electrode sensitivity did not differ from other studies in the literature. The second study presented comprehends the application of fluorescent nanoparticles as markers of an immunosensor for early diagnosis of Asian soybean rust spore. The use of these nanoparticles resulted in a very low limit detection sensor, 2.2 ng/mL, the lowest ever found in the literature. Moreover, others parameters were investigated in order to develop a point-of-care device, as the substrate to retain the analyte for the assay. Furthermore, the device was built in order to be used by the farmer himself, in loco, fast, without expensive equipment, a UV lamp as an excitation source and the signal is visible to naked eye. The visual detection limit is around 300 spores, which is tenfold lower than the available commercial kit. / Doutorado / Físico-Química / Doutora em Ciências

No description available.

Reliable access to the circulation is paramount in most medical and surgical emergencies. When venous access cannot be expediently established, intraosseous (IO) access is indicated. This method has a high success rate even in relatively inexperienced hands and there is considerable clinical experience of IO administration of drugs and fluids. There is however limited evidence on the use of IO samples for laboratory analysis. Also, uptake of drugs during shock has not been extensively studied. Further, there have been concerns that analysis of IO samples may damage laboratory equipment. We have studied, in a porcine model, the use of IO samples for point of care analysis of blood gases, acid base parameters and blood chemistries in stable circulation, in experimental septic shock, and in hypovolemia after major hemorrhage, comparing IO samples with arterial and venous samples, and comparing IO samples from different sites. We have also studied coagulation assays on IO samples in stable circulation and after major hemorrhage. Furthermore, we have compared IO and intravenous administration of antibiotics in experimental sepsis. Average differences between IO and arterial/venous samples varied between the studied analytes. During stable circulation, average IO levels of blood gases, acid-base parameters, hemoglobin/hematocrit and several blood chemistries approximated venous levels relatively well. Differences in acid-base and blood gas parameters, and lactate, were more pronounced in hypovolemia, as well as in sepsis. The dispersion of the differences was often relatively large, indicating limited precision. Average differences between two intraosseous sites were small. Intraosseous samples were clinically hypercoagulable with a strong tendency to clot in vitro, and thromboelastography demonstrated shortened reaction times compared with venous samples. Major bleeding and hemodilution moderately affected the studied coagulation parameters. In endotoxemic animals with circulatory instability, concentrations of cefotaxime and gentamicin in samples from the pulmonary artery were comparable at 5 minutes after intraosseous and intravenous administration, and during a 3 hour observation period. In summary, agreement between analytes in intraosseous and conventional blood samples was variable and often unpredictable, especially during circulatory compromise. Intraosseous samples clinically appeared hypercoagulable, and thromboelastography confirmed this. High and comparable concentrations of cefotaxime and gentamicin were found after intraosseous and intravenous administration of equivalent doses, suggesting that uptake is acceptable during septic instability.

Magister Public Health - MPH / Background: Point of Care Testing (POCT) refers to qualitative or quantitative tests done in health facilities where the patient is being attended to (on-site), and not in the conventional hospital laboratory setting. As a consequence of many developing countries not having access to conventional laboratory services (with trained laboratory personnel), diagnostic testing often relies on the availability of valid POC tests. All pregnant women attending antenatal care clinics in the Sedibeng District Primary Health Care (PHC) centres should be screened for syphilis, anaemia and HIV. This can be done by means of POC testing, which is easy to perform. These POC tests provide results promptly allowing treatment to be commenced immediately, if required. Despite this highly desirable benefit of POCT, there is circumstantial evidence which suggests that staff is choosing to send specimens to the laboratory for testing, instead of doing POCT themselves. The extent to which this happens and the factors contributing to this practice are not clear. Aim: The aim of this study was to assess the prevalence of screening for syphilis, anaemia, and HIV amongst pregnant women during their first antenatal care visit to PHC facilities in the Sedibeng District, and to establish the factors affecting the prevalence of appropriately using POCT for screening tests. Methodology: Study design: A quantitative, analytical, cross-sectional study was conducted. Study Population and Sample: Patient registers, staff expected to perform POCT and facility managers. 33 District’s health care workers expected to perform POCT on pregnant women during the first ANC visit and 30 facility managers from these facilities; 360 patient records (these were collected from a total of 7 200 patients’ records). The data was collected over a six month period (from 1st July 2012 to 31st December 2012). Data collection: Data was collected from 360 patient records to determine the rate, appropriateness and mechanism of screening for syphilis, anaemia and, HIV in pregnant women on their first antenatal visit. Interviewer-administered closed-ended questions was asked from 30 antenatal care clinic staff tasked with performing POC tests and from 30 PHC facility managers to determine the factors affecting the rate of conducting POCT. Data analysis: Data was analysed using univariate, bivariate and multivariate analyses. Ethical considerations: No harm was anticipated to anyone participating in the study or from the findings of the study. A major benefit of the study was that clarity on the factors affecting the rate of screening and the use of POCT was gained. This will hopefully facilitate the implementation of evidence–based interventions to improve POCT uptake if required.

Mobile computing devices are capable of changing how healthcare is delivered in the future, since they aim to merge and integrate all services into one device that is versatile, customisable, and portable. The aim of this study was to explore and describe the experiences of professional nurses with regard to accessing information at the point-of-care of the patient, in order to develop guidelines that could assist other professional nurses with implementing the mobile computing device for accessing information at the point-of-care of patients. To achieve the purpose of the study, a qualitative, explorative, descriptive, and contextual design was used to conduct this research – to gain an understanding of how the professional nurses experienced accessing information at the point-of-care via mobile computing devices. The study was conducted among the professional nurses employed at the public hospital, who were trained and provided with the mobile computing device for accessing information at the point-of-care for more than two years. In-depth interviewing was conducted to obtain the data. Data analysis was done using Tesch‘s method to make sense out of text and data. Four themes were identified, namely, the professional nurses‘ expression of various experiences regarding the training received; the need for support in implementing the mobile computing device; the accessing of information at the point-of-care as beneficial for educational purposes; and the accessing of information at the point-of-care as beneficial to patient care. Two main guidelines were developed. The study concludes with recommendations made with regard to the areas of nursing practice, education and research. Throughout the study, the researcher abided by the ethical considerations. The aspects of trustworthiness implemented in this study, included dependability, credibility, transferability and confirmability (Holloway & Wheeler, 2010:298).

Developing non-invasive and accurate diagnostics that are easily manufactured, robust and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment, particularly in the developing world. There is currently no rapid, low-cost and generic sensor fabrication technique capable of producing narrow-band, uniform, reversible colorimetric readouts with a high-tuneability range. This thesis aims to present a theoretical and experimental basis for the rapid fabrication, optimisation and testing of holographic sensors for the quantification of pH, organic solvents, metal cations, and glucose in solutions. The sensing mechanism was computationally modelled to optimise its optical characteristics and predict the readouts. A single pulse of a laser (6 ns, 532 nm, 350 mJ) in holographic “Denisyuk” reflection mode allowed rapid production of sensors through silver-halide chemistry, in situ particle size reduction and photopolymerisation. The fabricated sensors consisted of off-axis Bragg diffraction gratings of ordered silver nanoparticles and localised refractive index changes in poly(2-hydroxyethyl methacrylate) and polyacrylamide films. The sensors exhibited reversible Bragg peak shifts, and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 500-1100 nm. The application of the holographic sensors was demonstrated by sensing pH in artificial urine over the physiological range (4.5-9.0), with a sensitivity of 48 nm/pH unit between pH 5.0 and 6.0. For sensing metal cations, a porphyrin derivative was synthesised to act as the crosslinker, the light absorbing material, the component of a diffraction grating, as well as the cation chelating agent. The sensor allowed reversible quantification of Cu2+ and Fe2+ ions (50 mM - 1 M) with a response time within 50 s. Clinical trials of a glucose sensor in the urine samples of diabetic patients demonstrated that the glucose sensor has an improved performance compared to a commercial high-throughput urinalysis device. The experimental sensitivity of the glucose sensor exhibited a limit of detection of 90 µM, and permitted diagnosis of glucosuria up to 350 mM. The sensor response was achieved within 5 min and the sensor could be reused about 400 times without compromising its accuracy. Holographic sensors were also tested in flake form, and integrated with paper-iron oxide composites, dyed filter and chromatography papers, and nitrocellulose-based test strips. Finally, a generic smartphone application was developed and tested to quantify colorimetric tests for both Android and iOS operating systems. The developed sensing platform and the smartphone application have implications for the development of low-cost, reusable and equipment-free point-of-care diagnostic devices.

abstract: Cytometry is a method used to measure and collect the physical and chemical characteristics of a population of cells. In modern medical settings, the trend of precision and personalized medicines has imposed a need for rapid point-of-care diagnostic technologies. A rapid cytometric method, which aims at detecting and analyzing cells in direct patient samples, is therefore desirable. This dissertation presents the development of light-scattering-based imaging methods for detecting and analyzing cells and applies the technology in four applications. The first application is tracking phenotypic features of single particles, thereby differentiating bacterial cells from non-living particles in a label-free manner. The second application is a culture-free antimicrobial susceptibility test that rapidly tracks multiple, antimicrobial-induced phenotypic changes of bacterial cells with results obtained within 30 – 90 minutes. The third application is rapid antimicrobial susceptibility testing (AST) of bacterial cell growth directly in-patient urine samples, without a pre-culture step, within 90 min. This technology demonstrated rapid (90 min) detection of Escherichia coli in 24 clinical urine samples with 100% sensitivity and 83% specificity and rapid (90 min) AST in 12 urine samples with 87.5% categorical agreement with two antibiotics, ampicillin and ciprofloxacin. The fourth application is a multi-dimensional imaging cytometry system that integrates multiple light sources from different angles to simultaneously capture time-lapse, forward scattering and side scattering images of blood cells. The system has demonstrated capacity to detect red blood cell agglutination, assess red blood cell lysis, and differentiate red and white blood cells for potential implementation in clinical hematology analyses. These large-volume, light-scattering cytometric technologies can be used and applied in clinical and research settings to study, detect, and analyze cells. These studies developed rapid point-of-care diagnostic and imaging technologies for collectively advancing modern medicine and global health. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020