Development of an integrated opto-electric biosensor to dynamically examine cytometric proliferation and cytotoxicity

My doctoral research has focused on the development of microscale optical techniques for examining micro/bio fluidics. Preliminary work measured the velocity field in a microchannel, by optical slicing, using Confocal Laser Scanning Microscopy (CLSM). Next, Optical Serial Sectioning Microscopy (OSSM) was applied to examine thermometry by detecting the free Brownian motion of nano-particles suspended in mediums at different temperatures. An extension of this work used objective-based Total Internal Reflection Fluorescence Microscopy (TIRFM) to examine the hindered Brownian motion of nano-particles that were very close to a solid surface (within 1 mm). An optically transparent and electrically conductive Indium Tin Oxide (ITO) biosensor and an integrated dynamic live cell imaging system were developed to dynamically examine changes in cell coverage area, cell morphology, cell-substrate adhesion, and cell-cell interaction. To our knowledge this is the first sensor capable of conducting simultaneous optical and electrical measurements. This system consists of an incubator, which keeps cells viable by providing the necessary environmental conditions (37 °C temperature and 5 % CO2), and multiple microscopy techniques, including multispectrum Interference Reflection Microscopy (MS-IRM), TIRFM, Epi-fluorescence Microscopy, Phase Contrast Microscopy (PCM), and Differential Interference Contrast Microscopy (DICM). Along with investigations of cytometric proliferation including cellular barrier functions, in vitro cytotoxicity experiments were also conducted to examine the effect of a drug (cytochalasin D, a toxic agent) on cellular motility and cellular morphology. These cytotoxicity results give us a fundamental understanding of the cellular processes induced by the drug, which will be invaluable in the search for methods of preventing metastases. In this research, MS-IRM is used to examine the focal contacts and the gap morphology between cells and substrates, DICM is used to examine the coverage area of cells, and impedance measurements are used to correlate these two parameters. Advances in the understanding of vascular bio-transport in endothelial cells will have an impact on many aspects of cell biology, tissue engineering, and pharmacology. Particularly important will be the ability to test the popular hypothesis that the cell barrier function is regulated by specific cytoskeleton elements controlling intercellular and extracellular coupling

Quantitative Endothelial Cell Monolayer Impedance Sensing and Analysis

The electrical analysis of the biological material has been in existence since the turn of last century. A novel application of this technology to cellular monolayers was implemented by Giaever and Keese 20 years ago with their Electrical Cell-Substrate Impedance Sensing (ECIS) system. The capabilities of a real-time system for endothelial impedance measurement are of immense importance. The endothelium is typically the body’s first contact with stimuli and its reaction to medical conditions of inflammation, disease, and body response are of great significance to understanding the physiology of numerous conditions ranging from heart, lung, and renal disease, to intestinal diseases. It is the purpose of this Master’s thesis to analyze and optimize the ECIS system for making quantitative measurements of endothelial monolayer impedance, and accurately applying the results to a thoroughly reviewed analysis package in order to produce accurate cellular resistance parameters. The optimization of data acquisition (DAQ) is accomplished by systematic noise recognition, examination, and minimization; a task that has previously been unexplored in any studies using the ECIS system. Harmonic, 60 Hz, and Gaussian noise sources were well documented in unfiltered data and successfully minimized in the DAQ. Analog to digital (A/D) noise was found to be the lower limit of reducible noise and was properly documented and considered in analysis. Contamination of the electrode arrays from manufacturing processes and proper electrical connection were also found to be of concern to the proper functioning of the system. Analysis of the optimized acquired data was performed in the LabVIEW programming environment, as it offered a more flexible software package than that provided by the current commercially available ECIS system. The optimized system was applied to a further look into hand arm-vibration syndrome (HAVS) and it was concluded that the acceleration exposure dose, incorrectly calculated from the international standards, did not elicit an acute endothelial inflammation response by our measurements. The cumulative result of this study is that the ECIS system has been optimized and various unresolved sources of error were corrected for a more accurate real-time measurement of the endothelial monolayer barrier function in response to stimuli

Real-Time Monitoring and Quantification of Drug Induced Changes in Endothelial Cytoskeleton Filaments Using a Cellular Impedance Biosensor

The analysis of electrical signals originating from biological cells and tissues have yielded a large amount of useful information over the past years. Technologies have been developed, wherein we can monitor the health of the biological material of interest using these electrical signals. Instruments for the study of living cells have historically been of significant importance for such things as basic neuroscience, cell biology, pharmaceutical screening, environmental monitoring, and toxin detection. One of the practical realizations of these methods was successfully implemented by Giaver and Keese with their Electric Cell-Substrate Impedance Sensing (ECIS) system. The purpose of this Masters Thesis is to showcase the use of a Cellular Impedance Biosensor setup based on the ECIS system in real-time monitoring of endothelial barrier function and quantify the dynamic changes of the cytoskeleton filaments induced by different drugs. The filaments of the endothelial cell cytoskeleton play a critical role in cellular micro motion and the inflammatory response. The fact that the cellular cytoskeleton is the essential force behind all the motile activities of the cell is used in drug discovery methods to design ways so as to kill tumor cells and developing cell-based therapies for different cardiovascular pathologies. Cytochalasin D was chosen to study the actin filament response as the drug specifically inhibits the polymerization of actin filaments, which play a crucial role in the cellular mechanical strength and micromotion. Nocodazole was chosen to study microtubules, which play a pivotal part in the cellular mitotic activities and locomotion. These drugs were chosen as they have extreme effects on the cytoskeleton and would be ideal to showcase the use of the biosensor in tracking the intracellular dynamics. The biosensor system provides a simple interface for monitoring the electrical activity and impedance characteristics of populations of cultured cells over extended periods. The cell - sensor interface is created as cells attach to the gold electrode surface pre-coated with fibronectin. Using these impedance measurement techniques based on a simple cellular geometric model, we have been able to successfully monitor cellular adhesion, motility, proliferation and changes in the cellular cytoskeleton induced by different drugs as mentioned above. The kinetic response of the cellular cytoskeleton to different doses of these drugs is translated as changes in the impedance measured by the biosensor setup. The data obtained from the setup were quantified by correlating them with the images obtained by confocal microscopy. A wavelet transformation algorithm is applied to the acquired data in an attempt to capture the fluctuations and compare the cellular behavior before and after the addition of drugs. Study of the disruption of these filaments by toxins and pharmacological agents holds lot of promise to provide a model for studying their role in endothelial cell biomechanics and the pathology of cardiovascular, pulmonary, and renal disease. This thesis presents a cellular impedance biosensor setup that can carry out this study in real time with high sensitivity and reproducibility. Using these impedance measurement techniques, we have been able to successfully monitor cellular adhesion, motility, proliferation of different type of cells and their response to external stimuli

Sensors and Actuators for 2D and 3D Cell Culture Models based on Oxygen Sensitive Culture Substrates

Eukaryotic cells need oxygen to sustainably produce the metabolite ATP as their main source of energy. Next to glucose, oxygen is one of the key metabolites which can give us insight into cellular processes and conveys important information of parameters like cellular viability, metabolic activity and physiology. Thus, knowledge about cellular oxygen consumption is an essential parameter not only when studying pathophysiological conditions, such as metabolic disease, cancer or stroke, but also in drug and cytotoxicity screening. Methods to assess oxygenation levels and oxygen consumption of cells, spheroids and tissue range from electrochemical detection to the use of magnetic resonance and radioisotope techniques. However, in recent years the detection of oxygen by luminescence quenching of indicator dyes has emerged as a useful technique to study oxygenation of eukaryotic cells. Thus, one of the main projects in this work was to use planar oxygen sensitive culture substrates based on luminescence quenching as a novel means to estimate and image the oxygen consumption in 2D and 3D cell culture models. The substrates allowed the cultivation of adherent mammalian cells directly on the surface which provides the possibility to measure oxygen content directly beneath the cell monolayer. The combination of planar oxygen sensor foils with a suitable camera system enabled non-invasive, online monitoring of the spatial and the temporal oxygen distribution beneath the cell layer. The first project addressed the measurement of the oxygen consumption rate of adherent mammalian monolayer cells. Therefore, an imaging system based on microscopy optics (VisiSens TD mic) was developed in cooperation with PreSens GmbH, Regensburg. The system fits inside a standard cell incubator (48 l) and allows imaging the spatial and temporal distribution of oxygen with high resolution. Planar, polymer based oxygen sensitive foils (SF-RPSu4, PreSens GmbH) for a ratiometric fluorescence readout were used as growth substrates which allowed monitoring the oxygen content directly underneath the adherent cells. The sensor foils exhibited high photostability and biocompatibility which allowed their usage for long term cell experiments. The novel imaging system was used for first proof-of-concept studies to assess oxygen consumption rates in dependence on culture vessel volume and cell seeding density yielding highly reproducible results. Additionally, the novel ratiometric imaging system allowed the successful monitoring of the influence of medium composition, extracellular pH, drugs and toxins on respiratory activity of adherent cells. The high spatial resolution of the camera was used for long term imaging of oxygen gradients caused by a metabolically active spot of monolayer cells. Multiparametric monitoring of cells is a useful approach to obtain multiple information in one measurement. Thus, the second project focused on combining the optical oxygen sensing with impedimetric monitoring to obtain information about cell metabolic activity and cell morphology changes. Therefore, gold film electrodes were sputtered on planar, ratiometric oxygen sensor foils using a laser cut mask. A PDMS chamber was glued on top of the foil that served as cultivation chamber for the adherent mammalian cells. The dual ECIS-O2 sensor allowed simultaneous, non-invasive online recording of impedance and pO2 using a standard impedance analyzer and a commercial oxygen imaging system (VisiSens A1). Impedance changes were monitored for the cell population residing on the linear gold film electrodes, while oxygen levels were measured for those cells on the electrode-free sensor film. The dual sensor was characterized regarding its biocompatibility, signal stability and most sensitive impedimetric monitoring frequency. First studies conducted with the dual chip allowed simultaneous monitoring of the oxygen consumption and the impedance during cell adhesion. Moreover, the ECIS-O2 chip was used to investigate the influence of uncouplers and blockers of oxidative phosphorylation with impedance measurements providing information about changes in cell morphology and electrode coverage, while the ratiometric fluorescence readout reports on the cellular oxygen consumption rate. 3D tissue models such as multicellular tumor spheroids are considered useful in vitro tools in biomedical and clinical research. Due to their 3D structure, they close the gap between simple 2D monolayer cell culture and native tissue structures. Multicellular spheroids mimic physiological conditions found in tissue more closely compared to standard 2D cell culture, while handling and cultivation is easier compared to tissue. This makes multicellular tumor spheroids an attractive means in biomedical research and drug screening. Long-term studies on oxygenation in live spheroids are scarce because most assays are either endpoint-based or invasive such as microneedle or particle based approaches. Thus, the third project applied the novel imaging system VisiSens TD mic for quantitative monitoring of oxygenation levels within live MCF-7 tumor spheroids under standard growth conditions. Originally spherical spheroids were allowed to attach and spread on planar, biocompatible oxygen sensor foils forming half-spherical cell aggregates and oxygen content beneath the spheroid was monitored online with high spatio-temporal resolution. The imaging set-up allowed recording the detailed 2D distribution of oxygen gradients caused by metabolically active spheroids with higher resolution compared to commercially available imaging systems. Further studies were conducted investigating the effect of drugs blocking or uncoupling oxidative phosphorylation on oxygen gradients established in live and metabolically active MCF-7 spheroids. Wound healing assays are often used to study collective cell migration in vitro. The general concept of such assays is to introduce an artificial wound into a confluent layer of adherent cells and to study the migration of non-wounded cells from the periphery into the wound. The aim of the fourth project was the development of a novel wound healing assay based on oxygen sensitive culture substrates. The assay uses planar, polymer-based culture substrates with an embedded oxygen sensor dye as culture substrates for adherent cells. The functional layer was deposited on coverslips by spin-coating providing a transparent composite substrate that is addressable by light microscopy. Upon illumination with a suitable wavelength the dye molecules were excited. The 1O2 produced by luminescence quenching was used to selectively kill cells residing on areas of the functional coating that were exposed to the illumination light generating wounds with a highly reproducible geometry. The oxygen sensitive substrates designed for wound healing were analyzed regarding their biocompatibility and the influence of parameters such as light source, microscope filter settings and medium composition on wounding efficacy was tested. The novel wound healing assay was used to probe collective cell migration under various experimental conditions and it was compared to other wound healing assays such as scratch, barrier and ECIS® assay regarding parameters such as cost, throughput, reproducibility or wound morphology. Similar to the ECIS ® assay, wounds produced by the novel optical wound healing assay mimic the physiological conditions more closely, as dead cells remain as debris in the wound. Additional studies were performed investigating wound healing behavior of epithelial cells exposed to different extracellular pH values which resulted in reduced wound healing rates at low pH. Furthermore, it was shown that serum starvation and cytochalasin C reduced wound closure rate as well

Impedance-Based Analysis of the Cellular Response to Microparticles: Theory, Assay Development and Model Study

This thesis provides a model study on the information content of multimodal impedance-based assays to assess the impact of microscale particles on cell physiology of mammalian cells. In three main chapters, different approaches to this topic are presented and discussed: The first chapter focused on the simulation of several scenarios within cell-based assays. These simulations are all based on the so-called ECIS model, originally introduced by Giaever and Keese (1991), describing the impedance contribution of cell-covered gold-film electrodes. This theoretical part of the thesis should help to support the interpretation of impedance data. First, opening and closing of cell junctions (Rb) for different types of barrierforming cell layers were simulated and the accompanying changes in the complex impedance were extracted at various frequencies. The simulation data for some model epithelial and endothelial cell types showed that the relationship between resistance and barrier tightness may undergo inversion for frequencies above the cell-type specific threshold. Moreover, the influence of incomplete electrode coverage or inhomogeneity within the cell layer was studied systematically. For all experiments a good correlation between the simulated data and the experimental support was found. The aim of the second project was to establish a new opto-electrical assay to investigate the dye transfer via gap junctions into neighboring cells. The principle of this new assay was based on loading a selected cell population with Lucifer Yellow by in situ electroporation. The cell-type specific adjustment of the ac pulse parameters for a temporary permeabilization of the plasma membrane improved the incorporation of Lucifer Yellow into the cytoplasm without affecting NRK cell viability. The assay also required the optimization of the gold-film electrode layout which enabled the application of the ac pulses, the non-invasive impedance recordings before and after pulse application and the microscopic analysis of dye transfer from cells on the electrode into adjacent cells. The final electrode layout (8W4E-GJ) contained four “semi-elliptical” electrodes which were separated by a photopolymer-free gap to facilitate microscopic analysis without any interference from the red autofluorescence of the photopolymer. The development of an appropriate experimental protocol yielded on electroporation in Ca2+-free buffer and the application of two sequential ac pulses, as it was found to enhance the uptake efficiency into primary-loaded NRK cells. The opto-electrical assay was successfully applied to analyze the effect of the well-known gap junctional intercellular communication inhibitor 2-APB. The analysis of dye transfer via gap junctions was based on confocal fluorescence micrographs documenting dye transfer from the electrode into the photopolymer-free gap. The analysis was further improved by the application of the red-fluorescent TRITC dextran as co-electroporated reference dye, which was trapped in the cytoplasm of primary-loaded cells due to its molecular size. The image analysis of the position-dependent intensities of both dyes (TRITC dextran and Lucifer Yellow) allowed a quantification of gap junctional intercellular communication. The third chapter contains all sub-projects dealing with a multimodal and label-free analysis of the impact of micrometer-sized silica particles (Ø = 2 μm) on vitality, migration, proliferation and gap junctional intercellular communication of adherent NRK cells in vitro. A sequence of different impedimetric assays, all based on the well-established ECIS technique, was applied for the analysis of particle impact on cell physiology. Microscopic studies addressing the particle uptake revealed the presence of membrane-coated particles in the cytoplasm of NRK cells. Further evidence for particle uptake was gathered from ToFSIMS analysis that showed a densely-packed particle distribution around the cell nucleus in cells with intact plasma membranes. Time-resolved ECIS measurements revealed no acute cytotoxicity of silica particles as well as no influence on cell migration. Furthermore, the influence of silica particles on NRK proliferation was studied impedimetrically. No differences in the time-course of proliferation were found for particle-loaded or control cells. To study the influence of internalized particles on gap junctional intercellular communication the new optoelectrical assay was applied. Dye transfer to NRK cells in the periphery of the electrode was insignificantly different in absence and presence of silica particles. The results were supported by classical techniques, like FRAP analysis, scrape loading or parachute assay. Superior to other assays, the developed opto-electrical assay allowed for analysis of cell adhesion and cellular response to the presence of particle during an exposure time of 24 h prior to the dye transfer study. This enables the investigation of the impact of internalized particles on different cell-related parameters like viability, motility and gap junctional intercellular communication within one cell population

Development and evaluation of a calibration free exhaustive coulometric detection system for remote sensing.

Most quantitative analytical measurement techniques require calibration to correlate signal with the quantity of analyte. The purpose of this study was to employ exhaustive coulometry, an implementation of coulometric analysis in a stopped-flow, fixed-volume, thin-layer cell, to attain calibration-free measurements that would directly benefit intervention-free analysis systems designed for remote deployment. This technique capitalizes on the short diffusion lengths (\u3c 100 µm) to dramatically reduce the time for analysis (\u3c 30 sec). For this work, a thin-layer fluidic cell was designed in software, fabricated via CNC machining, and evaluated using Ferri/Ferrocyanide {Fe(CN)63-/4-} as a model analyte. The 2 µL fixed volume incorporated an oval, 8mm by 4 mm, thin-film gold electrode sensor with an integrated Ag|AgCl pseudo-reference electrode. The flow cell area matched the shape of the sensor, with a volume set by the thickness of a laser-cut silicone rubber gasket (~80 µm). A semi-permeable membrane isolated the working electrode and counter electrode chambers to prevent analyte diffusion. A miniaturized custom potentiostat was designed and built to measure reaction currents ranging from 10 mA to 0.1 nA. Software was developed to perform step voltammetry as well as cyclic voltammetry analysis for verifying electrode condition and optimal redox potential levels. Experimentally determined oxidation/reduction potentials of -100 mV and 400 mV, respectively, were applied to the working electrode for sample concentrations ranging from 50 µM to 10,000 µM. The current generated during the reactions was recorded and the total charge captured at each concentration was obtained by integrating the amperograms. When compared to the expected charge for a 2 µL sample, the total charge vs. concentration plots displayed a near perfect linearity over the full concentration range, and the expected charge (100 % converted) was reached within 20 seconds. The reaction currents ideally should have decayed to background levels, but exhibited constant offset values slightly larger than the background signal, a phenomenon assumed to be lingering residual flow from sample injection. After adding rigid tubing and external valves, the thin-layer cell was shown to remain within 6% of the theoretical charge after 200 seconds. Continued development of this system will offer the possibility of remote, calibration-free determinations of real-world analytes such mercury and lead

Photonic Biosensors: Detection, Analysis and Medical Diagnostics

The role of nanotechnologies in personalized medicine is rising remarkably in the last decade because of the ability of these new sensing systems to diagnose diseases from early stages and the availability of continuous screenings to characterize the efficiency of drugs and therapies for each single patient. Recent technological advancements are allowing the development of biosensors in low-cost and user-friendly platforms, thereby overcoming the last obstacle for these systems, represented by limiting costs and low yield, until now. In this context, photonic biosensors represent one of the main emerging sensing modalities because of their ability to combine high sensitivity and selectivity together with real-time operation, integrability, and compatibility with microfluidics and electric circuitry for the readout, which is fundamental for the realization of lab-on-chip systems. This book, “Photonic Biosensors: Detection, Analysis and Medical Diagnostics”, has been published thanks to the contributions of the authors and collects research articles, the content of which is expected to assume an important role in the outbreak of biosensors in the biomedical field, considering the variety of the topics that it covers, from the improvement of sensors’ performance to new, emerging applications and strategies for on-chip integrability, aiming at providing a general overview for readers on the current advancements in the biosensing field

Impedance-Based Real-Time Monitoring of Mammalian Cells upon Introduction of Xenobiotics into the Cytoplasm by In Situ Electroporation

Whole-cell biosensors are irreplaceable tool for studies of cellular mechanisms and behavior of the cell as a smallest living unit. Their development have progressed rapidly over past decades and nowadays we have powerful tools to study cell-based assays and to examine behavior of the cells exposed to different kinds of stimuli and challenges. Limited and selective permeability of the plasma membrane prevents the introduction of hydrophilic xenomolecules into the cytoplasm of mammalian cells. However, it is essential for many fields of cell biology, biomedicine or biotechnology to allow transport of such molecules (e.g. nucleic acids, antibodies, peptides or drugs) across the cell membrane. An ultimate goal of this thesis was to establish proof-of-principle assays for delivery of various bioactive molecules into adherent cells by in situ electroporation, and to monitor how these compounds influence cellular behavior, once they are internalized within the cell cytosol. Studies of in situ electroporation (ISE) were conducted using different types of mammalian cells (BAEC, CHO-K1/CHO-GFP, HaCaT, NRK and NIH-3T3) grown to confluence on small planar gold film electrodes. For every cell line individually, electric pulse parameters were optimized to achieve maximal loading efficiency, while keeping the invasiveness of the operation as low as possible. Impedance monitoring of in situ electroporation conducted with high time resolution showed biphasic changes of impedance signal after pulse application, indicating (i) fast recovery of the cell membrane integrity and (ii) relatively slow process of cell recovery after changes induced by membrane permeabilization. For the first time, release of intracellular material from the cells by ISE was studied using ECIS setup. Direct time-resolved imaging of NRK cells showed measurable efflux of fluorescence-labeled probes upon multiply applied electric pulses. In situ electroporation allowed transfer of second messenger (8-OH-)cAMP in the cell monolayers. Subsequent changes in impedance signal were in agreement with those observed after stimulation of the cells with membrane-permeable compounds CPT-cAMP and forskolin, as a consequence of triggering of the corresponding signaling cascades. Transport of nucleic acids into cytoplasm and nuclei of NRK and CHO-GFP cells was conducted in a highly-efficient manner. Besides fluorescent DNA aptamers, various types of siRNA molecules were successfully delivered into cells by in situ electroporation and their long-term sequence-specific silencing effect on cells was demonstrated and quantified by using microscopy and/ or impedance analysis. Transfection performance of ISE was compared with conventional and widespread delivery transfection method. In conclusion, this thesis demonstrated that in situ electroporation allows for highly efficient delivery of emerging types of molecules into monolayers of various types of cells