Stroke affects around 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, it was substituted by a gas-tight multifunctional microfluidic system, a lab-on-a-chip, with an integrated patch-clamp micropipette. The system was combined with optical tweezers and optical spectroscopy. Laser tweezers were used to optically guide and steer single cells towards the fixed micropipette. Optical spectroscopy was used to investigate the biochemical composition of the sample. The designed, closed lab-on-a-chip 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 a series of experiments: optically trapped human red blood cells were steered to the fixed patch-clamp pipette within the microfluidic system. The oxygen content within the microfluidic channels was measured to 1 % compared to the usual 4-7 %. 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 optical 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. Similar investigations within a closed microfluidic system were initiated and showed promising results for further developments of a complete lab-on-a-chip multifunctional system for reliable patch-clamp 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 enable high volume production of scalable microchips 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.

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.

A new approach to perform patch-clamp experiments on living cells under controlled anoxic and normoxic conditions was developed and tested. To provide an optimal control over the oxygen content and the biochemical environment a patch-clamp recording micropipette was integrated within an oxygen tight poly-methyl methacrylate (PMMA) based microchip. The oxygen content within the microfluidic chamber surrounding patch-clamp micropipette was maintained at 0.5-1.5 % by a continuous flow of artificial extracellular solution purged with nitrogen. The nerve and glial cells acutely obtained from the male rat brain were trapped by the optical tweezers and steered towards the patch-clamp micropipette through the channels of the microchip in order to achieve a close contact between the pipette and the cellular membrane. The patch-clamp recordings revealed that optical tweezers did not affect the electrophysiological properties of the tested cells suggesting that optical trapping is a safe and non-traumatizing method to manipulate living cells in the microfluidic system. Thus, our approach of combining optical tweezers and a gas-tight microfluidic chamber may be applied in various electrophysiological investigations of single cells were optimal control of the experimental conditions and the sample in a closed environment are necessary.

We present a new approach of combining Lab-on-a-chip technologies with optical manipulation technique for accurate investigations in the field of cell biology. A general concept was to develop and combine different methods to perform advanced electrophysiological investigations of an individual living cell under optimal control of the surrounding environment. The conventional patch clamp technique was customized by modifying the open system with a gas-tight multifunctional microfluidics system and optical trapping technique (optical tweezers).The system offers possibilities to measure the electrical signaling and activity of the neuron under optimum conditions of hypoxia and anoxia while the oxygenation state is controlled optically by means of a spectroscopic technique. A cellbased microfluidics system with an integrated patch clamp pipette was developed successfully. Selectively, an individual neuron is manipulated within the microchannels of the microfluidic system under a sufficient control of the environment. Experiments were performed to manipulate single yeast cell and red blood cell (RBC) optically through the microfluidics system toward an integrated patch clamp pipette. An absorption spectrum of a single RCB was recorded which showed that laser light did not impinge on the spectroscopic spectrum of light. This is promising for further development of a complete lab-on-a-chip system for patch clamp measurements.

Introduktion: Syftet med studien är att studera enstaka nervcellers respons vid syrebrist i ett mikroflödessystem för att förstå nervcellens respons vid stroke. Målet med studien var att utveckla ett slutet mikroflödessystem som ger optimal kontroll av den omgivande miljön och samtidigt möjliggöra elektrofysiologiska undersökningar under kontrollerade syreförhållande. Material och metoder: Mikroflödescellen utvecklades för ett inverterat mikroskop, utrustad med en optisk pincett och optisk spektroskopi samt patch-clamp för elektrofysiologiska studier på en enstaka nervcell. Istället för att föra en pipett mot en cell i ett öppet system fångades en enskild cell optiskt i ett slutet mikroflödessystem och fördes mot en fixerad patch-clamp mikropipett. Cellen utsattes för olika syrehalter och övervakades av ett UV-Vis spektroskop medan cellens elektrofysiologiska aktivitet registreras med patch-clamp. Det slutna mikroflödessystemet med integrerad mikropipett, kopplades till ett pumpsystem för införandet av celler och buffert med olika kemiska egenskaper och syrehalter. I ett inverterat mikroskop integrerades optisk pincett, UV-Vis spektrometer och patch-clamp. Resultat och diskussion: För att pröva konceptet fångades och fördes en röd blodcell optiskt mot mikropipetten som befann sig på en fast position i mikroflödescellen. Cellens syrebindningstillstånd varierades genom att tillsätta syrefri eller syresatt buffert och registrerades med UV-Vis spektrometern. I ett vidare experiment manipulerades en nervcell optiskt i ett öppet system mot patch-clamp pipetten och elektrofysiologiska mätningar utfördes. Vi kunde verifiera att den optiska pincetten inte påverkade den elektrofysiologiska mätningen. För närvarandet utförs elektrofysiologiska mätningar i det slutna mikroflödessystemet för att se hur nervcellerna reagerar under varierande syrehalt. Genom mätningarna hoppas vi att få mer kunskap om försvarsmekanismerna som igångsätts av neuroner under syrefattiga förhållanden.

The response and the reaction of the brain system to hypoxia is a vital research subject that requires special instrumentation. With this research subject in focus, a new multifunctional lab-on-a-chip (LOC) system with control over the oxygen content for studies on biological cells was developed. The chip was designed to incorporate the patch clamp technique, optical tweezers and absorption spectroscopy. The performance of the LOC was tested by a series of experiments. The oxygen content within the channels of the LOC was monitored by an oxygen sensor and verified by simultaneously studying the oxygenation state of chicken red blood cells (RBCs) with absorption spectra. The chicken RBCs were manipulated optically and steered in three dimensions towards a patch-clamp micropipette in a closed microfluidic channel. The oxygen level within the channels could be changed from a normoxic value of 18% O 2 to an anoxic value of 0.0-0.5% O 2. A time series of 3 experiments were performed, showing that the spectral transfer from the oxygenated to the deoxygenated state occurred after about 227 ± 1 s and a fully developed deoxygenated spectrum was observed after 298 ± 1 s, a mean value of 3 experiments. The tightness of the chamber to oxygen diffusion was verified by stopping the flow into the channel system while continuously recording absorption spectra showing an unchanged deoxygenated state during 5400 ± 2 s. A transfer of the oxygenated absorption spectra was achieved after 426 ± 1 s when exposing the cell to normoxic buffer. This showed the long time viability of the investigated cells. Successful patching and sealing were established on a trapped RBC and the whole-cell access (Ra) and membrane (Rm) resistances were measured to be 5.033 ± 0.412 M Ω and 889.7 ± 1.74 M Ω respectively.

This paper reviews microfluidic technologies with emphasis on applications in the fields of pharmacy, biology, and tissue engineering. Design and fabrication of microfluidic systems are discussed with respect to specific biological concerns, such as biocompatibility and cell viability. Recent applications and developments on genetic analysis, cell culture, cell manipulation, biosensors, pathogen detection systems, diagnostic devices, high-throughput screening and biomaterial synthesis for tissue engineering are presented. The pros and cons of materials like polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), glass, and silicon are discussed in terms of biocompatibility and fabrication aspects. Microfluidic devices are widely used in life sciences. Here, commercialization and research trends of microfluidics as new, easy to use, and cost-effective measurement tools at the cell/tissue level are critically reviewed.

We present a new concept of integrating a micropipette within a closed microfluidic system equipped with optical tweezers and a UV-Vis spectrometer. A single red blood cell (RBC) was optically trapped and steered in three dimensions towards a micropipette that was integrated in the microfluidic system. Different oxygenation states of the RBC, triggered by altering the oxygen content in the microchannels through a pump system, were optically monitored by a UV-Vis spectrometer. The built setup is aimed to act as a multifunctional system where the biochemical content and the electrophysiological reaction of a single cell can be monitored simultaneously. The system can be used for other applications like single cell sorting, in vitro fertilization or electrophysiological experiments with precise environmental control of the gas-, and chemical content.

Hemoproteins usually supply cells and tissue with oxygen. A new hemoprotein mainly present in nerve cells called Neuroglobin was recently discovered. Enhanced expression of the protein has been shown to reduce hypoxic neural injury but the mechanism behind this function remains unknown. Methods enabling investigation of the protein in single functional neurons need to be developed. Here, we have studied how the electrical signaling capacity of a neuron was affected by hypoxic environments. Preliminary results show a trend of higher noise-level when a neuron is exposed to hypoxic compared to normoxic surroundings, which implies increased ion-channel activity. The setup used today shows shortages such as reduced control over the oxygen content due to leakage. Therefore, a gas-tight, multifunctional microfluidic system is under development which enables us to study influences of Neuroglobin concentrations on neuronal activity during hypoxia and anoxia. For electrophysiological recordings a patch-clamp micro pipette will be molded into the walls of the microfluidic system. A single biological cell is steered towards the pipette and attached there by means of optical tweezers. The Neuroglobin oxygen binding state will be studied using optical spectroscopy and the neuron environment will be manipulated by applying flows of varying oxygen content through the microfluidic system. This system will constitute a powerful tool in the investigation of the Neuroglobin mechanism of action.

På senare tid har aktuell forskning gett inblick i hur vår kropp fungerar på den biokemiska nivån. Det har lett till nya terapeutiska strategier för att förhindra eller bota neurologiska sjukdomar såsom hjärncancer, stroke, Alzheimers och Parkinsons. Framstegen beror till stor grad på utvecklandet av mätmetoder som ger kunskap om hur små organismer beter sig på den biomolekylära nivån. Vår tvärvetenskapliga forskning är inriktad på att utveckla metoder för att undersöka hur levande biologiska neuroner eller hjärnvävnad försvarar sig mot syrebrist eller andra stressituationer. Vi har inriktat oss på hemoproteiner då det nyligen har visat sig att de kan förhindra skador som uppstår vid syrebrist. Vi sammankopplar ett flertal mätmetoder och ett mikroflödessystem under ett mikroskop för att undersöka hemoproteiners verkan på enstaka neuroner i nära fysiologiska miljöer, det vill säga så likt det levande som möjligt. Det har visat sig vara viktigt att kunna studera nervcellernas elektriska signaler som är ett mått på stressnivån samtidigt som man mäter hur hemoproteiner samverkar med olika molekyler och proteiner. De elektriska signaleringsegenskaperna hos nervceller eller tunna hjärnsnitt mäts lämpligen genom patch-clamp teknik. Optisk Raman spektroskopi och UV-Vis spektroskopi fungerar väl för att studera haemoproteiner och de första Ramanmätningarna på tunna hjärnskivor visade att det går att skilja åt olika hemoproteiner. För att kunna kontrollera syrehalten krävs det en sluten gastät flödeskammare med möjlighet till patch-clamp där man snabbt kan flöda olika lösningar av varierande syre-, och salthalt. I detta system är patch-clamp pipetten fixerad till en position och cellerna förs till pipetten med hjälp av en optisk pincett som är en beröringsfri optisk manipuleringsmetod där biologiska celler fångas i ett starkt fokuserat laserljus som här förflyttas med hjälp av ett xyz-bord. Vi kommer att presentera de senaste framstegen i utvecklandet av mätsystemet där röda blodceller användes som modell.