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الرئيسية > Design of a Multi-Sensor Platf...>الوصف

Design of a Multi-Sensor Platform for Integrating Extracellular Acidification Rate with Multi-Metabolite Flux Measurment for Small Biological Samples

المؤلف الرئيسي: Obeidat, Yusra Mahmoud (Author)
مؤلفين آخرين: Chen, Tom W. (Advisor)
التاريخ الميلادي: 2019
موقع: فورت كولنز
الصفحات: 1 - 140
رقم MD: 999296
نوع المحتوى: رسائل جامعية
اللغة: الإنجليزية
الدرجة العلمية: رسالة دكتوراه
الجامعة: Colorado State University
الكلية: The Graduate School
الدولة: الولايات المتحدة الأمريكية
قواعد المعلومات: +Dissertations
مواضيع:
الهندسة الكهربائية | تقنيات قياس الأكسجين | التمثيل الغذائي | المسح الكهروكيميائي المجهري
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المستخلص: Cell metabolism involves a set of cellular chemical reactions that are very important to cell development as well as its response to environmental changes around the cell. Understanding cell metabolism and the associated metabolic pathways has been the focus of many research efforts and it is gaining more attention recently. In assisted reproductive technology (ART), understanding metabolism of oocytes and embryos provides the possibility of selecting more viable embryos for transfer and reducing the number of embryos transferred in a given in vitro fertilization (IVF) cycle. Although stage-specific morphologic markers and grading systems have been developed and widely in use, this approach is unable to reliably assess the physiological status of the embryo and it is not only subjective but has a poor correlation with subsequent developmental competence. Therefore, there is an ever-increasing need for noninvasive quantitative markers of embryo viability. Analysis of metabolism has proved to be a valuable marker of embryo viability based on animal models. Through noninvasive analysis of metabolic markers, it will be feasible to identify those embryos with the highest probability of establishing a healthy pregnancy. Crucial to cell metabolic process is a set of analytes that can be used as indicators of cell metabolism. They include oxygen, glucose, and lactate. Rates of cellular oxygen consumption (OCR) and extracellular acidification (ECAR) are widely used proxies for mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic rate in cell metabolism studies. However, ECAR can result from both oxidative metabolism (carbonic acid formation) and glycolysis (lactate release), potentially leading to erroneous conclusions about metabolic substrate utilization. Co-measurement of extracellular glucose and lactate flux along with OCR and ECAR can improve the accuracy and provide better insight into cellular metabolic processes but is currently not feasible with any commercially available instrumentation.

Some techniques for measuring dissolved oxygen (DO), glucose and lactate rely on fluorescent labels. These techniques are incredibly labor intensive, and the pipet construction used is complex comparing with solid state and electrochemical methods. Injecting a cell with fluorescent label can also lead to experimental error, since biochemical mechanisms inside of the cell may interact with the label. Other techniques include the use of scanning electrochemical microscopy (SECM) for studying the metabolism of single cells, but it has its drawbacks including probe fouling, complex instrumentation, as well as calibration can also be challenging. Furthermore, electrochemical microphysiometers were used for monitoring changes in glucose and lactate concentrations in cell cultures, but these techniques need larger sample volumes and might need difficult calibration. Due to the lack of quantitative and real-time monitoring of cell metabolism, the success rate of in-vitro fertilization (IVF) is still low, with very low percentage of embryos transferred resulting in a term pregnancy [1-2]. Therefore, more work needs to be done for testing embryos metabolism in-vitro to improve the culture conditions and reduce the effect of environmental stresses and chose the media that balance all nutrients the cell needs during development. In this work, we present a miniaturized multi-sensor platform capable of real-time monitoring of OCR and ECAR along with extracellular lactate and glucose flux for small biological samples such as single equine and bovine embryos. This multiplexed approach enables validation of ECAR resulting from OXPHOS versus glycolysis, and expression of metabolic flux ratios that provide further insight into cellular substrate utilization. We demonstrate expected shifts in embryo metabolism during development and in response to OXPHOS inhibition as a model system for monitoring metabolic plasticity in very small biological samples.

In this work, DO was measured amperometrically using a three-electrode system of working (WE), counter (CE) and reference (RE) electrodes. Glucose and lactate were measured enzymatically by measuring the current generated from the oxidation of hydrogen peroxide generated from the catalysis of glucose or lactate at the WEs with their catalysis enzymes. pH was measured potentiometrically using two electrodes system of Indium Tin Oxide (ITO) WE and Au pseudo RE. A micro-chamber containing all four sensors was designed and manufactured to investigate single cell immersed in a respiration medium. The micro-chamber design is an important part of the platform that provides sufficient change of the target analytes in the micro-environment that enables the sensors to measure tiny changes of the target analytes due to cell respiration. This setup helps to measure the analytes with a change in concentration ranges from (0.001 to 30) fmol/s with high specificity which is comparable with what was published in literature. The specificity of our sensors was clearly determined by monitoring the switch in metabolism to glycolysis induced by adding oligomycin as an inhibitor for ATP-synthase. The ability to measure the extracellular acidification rate (ECAR) in addition to lactate production can help to differentiate the respiratory acid production from glycolytic acidification. The ability of the sensor to detect a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis was demonstrated in embryos by an ablation of oxygen consumption and an increase in lactate production as well as ECAR following addition of oligomycin. The increase in pH change rate after adding oligomycin and its slowdown after FCCP further indicates the dependence of cell on glycolysis and the increase of lactate production. The results of bovine or equine embryos show that the embryos metabolism change with development as expected and the amounts of glucose and oxygen uptakes and lactate production increase at later stages of developments, which match the existing biological knowledge of increasing the need for ATP production at later stages of development. Our system is capable to provide single-cell metabolism measurement with more complete panel than what commercially available devices such as Seahorse provides. Our results provide a clear insight into the mechanism of OXPHOS and glycolysis for single cells and a more complete analysis to include inter-sensor interference for improved accuracy. The capability of the platform is illustrated with measurements multi-metabolites of single-cell equine or bovine embryos for assisted reproduction technologies. However, this platform has a wide potential utility for analyzing small biological samples such as single cells and tumor biopsies for immunology and cancer research applications. Furthermore, we also present a preliminary interference analysis of the multi-sensor platform to allow better understanding of sensor interference in the proposed multi-sensor platform.

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