Lab on a chip for electrophysiological measurements with control of the oxygen content optical manipulation and spectroscopic analysis of biological cells

Detta är en avhandling från Luleå tekniska universitet

Sammanfattning: Stroke affects nearly 20 million people around the world every year. Clinically, stroke is a result of brain damage due to the shortage of oxygen delivered to the nerve cells. To minimize suffering and costs related to the disease, extensive research is performed on different levels. The focus of our research is to achieve fundamental understanding on how the lack of oxygen in brain tissue activates intrinsic biomolecular defense mechanisms that may reduce brain damage. More knowledge may hopefully lead to new therapeutic and preventive strategies on the molecular level for individuals in the risk zone for stroke or those who have just suffered a stroke. The area of study is based on the discovery of a hemoprotein called neuroglobin (Ngb), which is found in various regions in the brain, in the islets of Langerhans, and in the retina. Several studies have shown that Ngb seems to have a protective function against hypoxia-related damage. However, until now, it has not been understood how Ngb affects the nerve system and protects neurons from damage. The well-established patch-clamp technique is routinely used to measure and analyze the electrophysiological activity of individual biological cells. To perform accurate patchclamp experiments, it is important to create well-controlled physiological conditions, i.e. different oxygen levels and fast changes of nutrients and other biochemical substances. A promising approach is to apply lab on a chip technologies combined with optical manipulation techniques. These give optimal control over fast changing environmental conditions and enable multiple readouts. The conventional open patch-clamp configuration cannot provide adequate control of the oxygen content. Therefore, the aim of the thesis was to design and test a multifunctional microfluidic system, lab on a chip (LOC), that can achieve normoxic, anoxic and hypoxic conditions. The conventional patch clamp configuration was substituted by a gas-tight LOC system with an integrated patch-clamp micropipette. The system was combined with optical tweezers, optical sensor and optical spectroscopy. Optical tweezers were used to trap and guide single cells through the LOC microchannels towards the fixed micropipette. Optical spectroscopy was essential to investigate the biochemical composition of the biological samples. The developed, gas-tight LOC acted as a multifunctional system for simultaneous electrophysiological and spectroscopic experiments with good control over the oxygen content in the liquid perifusing the cells. The system was tested in series of experiments: optically trapped cells (red blood cells from human and chicken and nerve cells) were steered to the fixed patch-clamp pipette within the LOC system. The oxygen content within the microfluidic channels was measured to ∼ 1% compared to the usual 4-7% found in open system. The trapping dynamics were monitored in real-time while the spectroscopic measurements were performed simultaneously to acquire absorption spectra of the trapped cell under varying environments. To measure the effect of the laser tweezers on the sample, neurons from rats in a Petri dish were optically trapped and steered towards the patch-clamp micropipette where electrophysiological investigations were performed. The optical tweezers had no effect on the electrophysiological measurements. The future aim is to perform complete protocols of patch-clamp electrophysiological investigations while simultaneously monitoring the biochemical composition of the sample by optical spectroscopy. The straightforwardness and stability of the microfluidic chip have shown excellent potential to be applied for various biomedical applications. The subsequent ambition is to use this system as a mini laboratory that has benefits in cell sorting, patch-clamp and fertilization experiments where the gaseous and the biochemical content is of importance. The long-term goal is to study the response of individual neurons and defense mechanisms under hypoxic conditions that may establish new ways to understand cell behavior related to Ngb for various diseases such as stroke, Alzheimer’s and Parkinson’s.

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