1,720,978 research outputs found
Selective Exhaled Breath Condensate Collection and Competitive Fluorescent Biosensor for Non-Invasive Glucose Detection
No full textTwo thirds of patients with diabetes avoid regularly monitoring their blood glucose levels because of the painful and invasive nature of current blood glucose detection. As an alternative to blood sample collection, exhaled breath condensate (EBC) has emerged as a promising noninvasive sample from which to monitor glucose levels. However, the inconsistency in the methods used to collect EBC significantly impacts the reliability of reported analyte concentrations in EBC. Furthermore, this dilute sample matrix requires a highly sensitive glucose biosensor to enable robust and accurate glucose detection at the point-of-care. Together, a reliable collection method and sensitive detection system can enable accurate modeling of glucose transport from blood to breath that is reflective of airway glucose homeostasis.I address this research gap by simultaneously designing a standardized EBC collection method that allows for separation of dead space and alveolar air and developing a competitive fluorescent biosensor that can resolve micromolar glucose concentrations changes. First, I develop a low-cost, automated condenser that selectively collects exhaled breath that has been exchanged with lung fluid based on the detection of higher breath temperatures that are characteristic of the lower respiratory regions. Using this device, I investigate the relationship between blood and EBC glucose in diabetic and normoglycemic human subjects. Next, I engineer the exquisitely sensitive E. coli glucose binding protein (GBP) with a chemo-enzymatic tag to selectively conjugate it to highly photostable quantum dots (QDs). Finally, I take advantage of the competitive binding of glucose (KD=0.35 µM) and galactose (KD=1.4 µM) to GBP to develop a fluorescent glucose biosensor using the GBP-QD conjugate
Assay Optimization for Point of Care Detection of HIV
No full textAs of 2021, 38.4 million people worldwide are living with Human Immunodeficiency virus (HIV), with eastern and southern Africa having the highest prevalence. The efficacy of treatment is determined by identifying acute HIV infections (AHI) and prompting early antiretroviral therapy (ART) initiation to achieve viral suppression and reduce the risk of transmission. Existing rapid tests that detect host antibodies are affected by long seroconversions which allow the viruses to remain undetected until long after infection. On the contrary, highly sensitive nucleic acid amplification test (NAAT) based assays, serving as the gold standard for detection are restricted by their long turnaround time and high cost of implementation thus, restricting their use in low resource settings. Further, drug resistance cases and patient non-compliance to treatment may lead to HIV progression to aids; therefore, effective viral load monitoring is a critical component in the HIV care continuum. To address the gaps in viral load monitoring and early HIV detection, I propose to develop assays for handheld self-test platforms to detect low concentrations of HIV via two different approaches: 1) I will optimize an existing NAAT - based assay to semi-quantitatively detect HIV particles that were spiked in clinical samples and 2) I will Investigate the binding kinetics between HIV p24 antigen and Anti-HIV-1 p24 Antibody using the principle of Bio-layer Interferometry. Thus, I will lay the foundation for the development of a novel and highly sensitive p24 detection assay. Overall, this work will enable detection of ahi detection as well as support people living with HIV (PLHIV) management, all while remaining connected to healthcare and provider support
Portable Platforms for Molecular-Based Detection of Pathogens in Complex Sample Matrices
No full textPathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use
Characterization and Development of Lateral Flow Assays for Automated Multi-Step Processes and Point-Of-Care Cervical Cancer Detection
No full textPaper-fluidic devices are a popular platform for point-of-care diagnostics due to their low cost, ease of use, and equipment-free detection of target molecules. The most common example is the lateral flow assay, in which samples are added to a paper membrane and a colorimetric indicator provides a binary signal indicating whether the molecule of interest is present. A novel lateral flow assay was developed to detect a protein biomarker for early stage cervical cancer. Cervical cancer can be cured if detected and treated at an early stage, but approximately 90% of cervical cancer deaths occur in low and middle-income countries due to lack of accessible testing. Methods for detecting the biomarker, valosin-containing protein (VCP), were optimized using enzymatic and gold nanoparticle dot blots, then lateral flow assays were developed and validated using purified VCP and cervical cancer HeLa cells. Future validation with patient tissue samples will permit translation of this device to testing clinics in low-resource areas.Despite advantages for use in resource limited settings, lateral flow assays are limited by their inability to perform more complex or multi-step processes, such as nucleic acid amplification or enzymatic signal enhancement. Thermally actuated wax valves are one mechanism that provides complete control over fluid obstruction and release. To better understand how wax valves can be used in fully automated, self-contained lateral flow assays, different sizes and geometries of valves were tested to investigate their effects on actuation time, flow rate, and flow pattern. Another limitation in the understanding of lateral flow assays is the lack of experimental data describing the microscale flow within the pores of the paper membrane that drives the biophysical reactions in the assay. Mathematical models can be designed to explain macroscopic phenomena, but so far, no literature has compared microfluidic models to microfluidic data. To quantify microfluidic properties within lateral flow assays, fluorescent nanoparticles were imaged flowing through different areas of the membrane and their velocity was quantified using micro-particle image velocimetry (µPIV). Scanning electron microscope images were used to verify that this experimental model was reasonable for describing microfluidic properties of the lateral flow assay. Altogether, this document investigates how developing lateral flow assays for cervical cancer detection can save lives by improving the accessibility of an early diagnosis, and how more robust lateral flow assay characterization can expand their applicability to a broad range of detection processes
Stimuli-Responsive Valving Mechanisms for Paper-Based Diagnostics
No full textRapid identification of disease-causing pathogens at the point-of-care enables immediate treatment and infection control. However, existing rapid diagnostic devices fail to detect the low concentrations of pathogens present in the early stages of infection, causing delayed and even incorrect treatment. A delay in antibiotic treatment of as few as 24 hours after infection onset will drastically decrease a patient’s chance of survival. The transport of a patient’s sample to a centralized testing laboratory can contribute hours to this delay. For instance, the most sensitive assay, nucleic acid detection, can only be performed at centralized laboratories. The multistep sample preparation and costly instrumentation required to analyze samples has prohibited nucleic acid detection assays from reaching the point-of-care. There remains a critical need to bring rapid and sensitive pathogen identification technologies out of the laboratory to ensure effective treatment.Paper-based devices have emerged as a portable platform for nucleic acid detection but are limited by their imperfect control of reagent incubation and false positive results. Here, I have developed mechanisms to specifically and automatically detect the nucleic acids of pathogens on paper-based devices. First, I characterize wax-ink valves that enable controlled incubation and delivery of reagents through device stages. Next, I implement toe-hold mediated strand displacement reactions to increase the specificity of nucleic acid detection with paper-based devices. Lastly, I functionalize polymers with nucleic acid probes and explore their potential integration into paper-based devices as bio-responsive valves. I demonstrate how such novel valving mechanisms enable the automatic and multi-step analysis of bacteria and viruses on paperbased platforms, improving the detection of infectious diseases at patients’ point-of-care
Molecular Point-of-Care diagnostic for Treponema pallidum subsp. pertenue (yaws)
No full textThe eradication of yaws a neglected tropical disease caused by Treponema pallidum subsp. pertenue, which affects children living in very deprived hard to reach rural communities is constrained by the lack of rapid, accurate diagnosis. I sought to develop a molecular point-of-care test for the diagnosis of yaws. A Loop-mediated isothermal amplification (LAMP) assay with primers targeting the conserved gene, tp0967, with visual detection by lateral flow test strip was developed and optimized. The limit of detection was evaluated while 63 samples from clinical cases of yaws and 5 samples with PCR-confirmed syphilis were used to determine the sensitivity and specificity of the assay compared to the current molecular testing protocol. Reagents were dried in tubes and tested up to 14 days. The developed LAMP assay was found to be optimal when run at 65oC in a water bath for 30 minutes. The limit of detection was 2.7*104 DNA copies/ml. The sensitivity of the LAMP assay using unextracted and DNA extracted samples were 0.67 and 1.00 respectively. None of the syphilis samples tested positive in any of the assays. We show the development of a fast and sensitive LAMP assay for yaws detected by lateral flow test strip. Using extracted DNA, the assay sensitivity is at par with gold standard detection. The assay can be adapted to minimal sample processing required for in-field detection without DNA extraction.</div
Design and Fabrication of Soft Biosensors and Actuators
No full textSoft materials have gained increasing prominence in science and technology over the last few decades. This shift from traditional rigid materials to soft, compliant materials have led to the emergence of a new class of devices which can interact with humans safely, as well as reduce the disparity in mechanical compliance at the interface of soft human tissue and rigid devices.One of the largest application of soft materials has been in the field of flexible electronics, especially in wearable sensors. While wearable sensors for physical attributes such as strain, temperature, etc. have been popular, they lack applications and significance from a healthcare perspective. Point-of-care (POC) devices, on the other hand, provide exceptional healthcare value, bringing useful diagnostic tests to the bedside of the patient. POC devices, however, have been developed for only a limited number of health attributes. In this dissertation I propose and demonstrate wireless, wearable POC devices to measure and communicate the level of various analytes in and the properties of multiple biofluids: blood, urine, wound exudate, and sweat.Along with sensors, another prominent area of soft materials application has been in actuators and robots which mimic biological systems not only in their action but also in their soft structure and actuation mechanisms. In this dissertation I develop design strategies to improve upon current soft robots by programming the storage of elastic strain energy. This strategy enables us to fabricate soft actuators capable of programmable and low energy consuming, yet high speed motion. Collectively, this dissertation demonstrates the use of soft compliant materials as the foundation for developing new sensors and actuators for human use and interaction
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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