@phdthesis{Stanke2023,
  author    = {Stanke, Sandra},
  title     = {AC electrokinetic immobilization of influenza viruses and antibodies on nanoelectrode arrays for on-chip immunoassays},
  doi       = {10.25932/publishup-61716},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-617165},
  school      = {Universit{\"a}t Potsdam},
  pages     = {x, 115},
  year      = {2023},
  abstract  = {In the present thesis, AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, were demonstrated as a simple and fast method to functionalize the surface of nanoelectrodes with submicrometer sized biological objects. These nanoelectrodes have a cylindrical shape with a diameter of 500 nm arranged in an array of 6256 electrodes. Due to its medical relevance influenza virus as well as anti-influenza antibodies were chosen as a model organism. Common methods to bring antibodies or proteins to biosensor surfaces are complex and time-consuming. In the present work, it was demonstrated that by applying AC electric fields influenza viruses and antibodies can be immobilized onto the nanoelectrodes within seconds without any prior chemical modification of neither the surface nor the immobilized biological object. The distribution of these immobilized objects is not uniform over the entire array, it exhibits a decreasing gradient from the outer row to the inner ones. Different causes for this gradient have been discussed, such as the vortex-shaped fluid motion above the nanoelectrodes generated by, among others, electrothermal fluid flow. It was demonstrated that parts of the accumulated material are permanently immobilized to the electrodes. This is a unique characteristic of the presented system since in the literature the AC electrokinetic immobilization is almost entirely presented as a method just for temporary immobilization. The spatial distribution of the immobilized viral material or the anti-influenza antibodies at the electrodes was observed by either the combination of fluorescence microscopy and deconvolution or by super-resolution microscopy (STED). On-chip immunoassays were performed to examine the suitability of the functionalized electrodes as a potential affinity-based biosensor. Two approaches were pursued: A) the influenza virus as the bio-receptor or B) the influenza virus as the analyte. Different sources of error were eliminated by ELISA and passivation experiments. Hence, the activity of the immobilized object was inspected by incubation with the analyte. This resulted in the successful detection of anti-influenza antibodies by the immobilized viral material. On the other hand, a detection of influenza virus particles by the immobilized anti-influenza antibodies was not possible. The latter might be due to lost activity or wrong orientation of the antibodies. Thus, further examinations on the activity of by AC electric fields immobilized antibodies should follow. When combined with microfluidics and an electrical read-out system, the functionalized chips possess the potential to serve as a rapid, portable, and cost-effective point-of-care (POC) device. This device can be utilized as a basis for diverse applications in diagnosing and treating influenza, as well as various other pathogens.},
  language  = {en}
}
@phdthesis{Pruefer2023,
  author    = {Pr{\"u}fer, Mareike},
  title     = {Charakterisierung und wechselfeldgest{\"u}tzte Herstellung von Enzym-Nanoarrays},
  doi       = {10.25932/publishup-61232},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612329},
  school      = {Universit{\"a}t Potsdam},
  pages     = {104},
  year      = {2023},
  abstract  = {Dielektrophorese ist die Manipulation polarisierbarer Partikel durch inhomogene elektrische Wechselfelder. In dieser Arbeit wurden drei verschiedene Enzyme durch Dielektrophorese immobilisiert und anschließend hinsichtlich ihrer katalytischen Aktivit{\"a}t untersucht: Meerrettichperoxidase, Cholinoxidase aus Alcaligenes sp. und Glucoseoxidase aus Aspergillus niger. Die Immobilisierung erfolgte durch Dielektrophorese auf nano-Elektrodenarrays aus Wolfram-Zylindern mit 500 nm Durchmesser oder aus Titannitrid-Ringen mit 20 nm Breite. Die Immobilisierung der Enzyme konnte fluoreszenzmikroskopisch entweder anhand der intrinsischen Fluoreszenz oder aufgrund einer Fluoreszenzmarkierung vor oder nach der Immobilisierung f{\"u}r alle getesteten Enzyme nachgewiesen werden. Die Messung der Enzymaktivit{\"a}t erfolgte quantitativ durch den direkten oder indirekten Nachweis des gebildeten Produktes oder, im Falle der Cholinoxidase, durch Beobachtung der intrinsischen Fluoreszenz des Cofaktors FAD, die vom Oxidationszustand dieses Enzyms abh{\"a}ngt. F{\"u}r die Meerrettichperoxidase konnte so eine hohe erhaltene Enzymaktivit{\"a}t nach der Immobilisierung nachgewiesen werden. Die Aktivit{\"a}t der permanent immobilisierten Fraktion der Meerrettichperoxidase entsprach bis zu 47 \% der h{\"o}chstm{\"o}glichen Aktivit{\"a}t einer Monolage dieses Enzyms auf den Elektroden des Chips. Diese Aktivit{\"a}t kann als aktive, aber zuf{\"a}llig gegen{\"u}ber der Oberfl{\"a}che ausgerichtete Enzymschicht interpretiert werden. F{\"u}r die permanent immobilisierte Glucoseoxidase wurde nur eine Aktivit{\"a}t entsprechend <1,3 \% der Aktivit{\"a}t einer solchen Enzymschicht detektiert, w{\"a}hrend f{\"u}r die immobilisierte Cholinoxidase gar keine Aktivit{\"a}t nachgewiesen werden konnte. Die Aktivit{\"a}t der durch DEP immobilisierten Enzyme konnte somit quantitativ bestimmt werden. Der Anteil an erhaltener Aktivit{\"a}t h{\"a}ngt dabei stark vom verwendeten Enzym ab.},
  language  = {de}
}
@phdthesis{Gerling2022,
  author    = {Gerling, Marten Tobias},
  title     = {A microfluidic system for high-precision image-based live cell sorting using dielectrophoretic forces},
  doi       = {10.25932/publishup-58742},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-587421},
  school      = {Universit{\"a}t Potsdam},
  pages     = {vii, 87, VI},
  year      = {2022},
  abstract  = {An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods. Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality. In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly. With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95\% and about 85\% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system. The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.},
  language  = {en}
}
@phdthesis{Knigge2020,
  author    = {Knigge, Xenia},
  title     = {Einzelmolek{\"u}l-Manipulation mittels Nano-Elektroden und Dielektrophorese},
  doi       = {10.25932/publishup-44313},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-443137},
  school      = {Universit{\"a}t Potsdam},
  pages     = {106, xxxii},
  year      = {2020},
  abstract  = {In dieser Arbeit wurden Nano-Elektroden-Arrays zur Einzel-Objekt-Immobilisierung mittels Dielektrophorese verwendet. Hierbei wurden fluoreszenzmarkierte Nano-Sph{\"a}ren als Modellsystem untersucht und die gewonnenen Ergebnisse auf biologische Proben {\"u}bertragen. Die Untersuchungen in Kombination mit verschiedenen Elektrodenlayouts f{\"u}hrten zu einer deterministischen Vereinzelung der Nano-Sph{\"a}ren ab einem festen Gr{\"o}ßenverh{\"a}ltnis zwischen Nano-Sph{\"a}re und Durchmesser der Elektrodenspitzen. An den Proteinen BSA und R-PE konnte eine dielektrophoretische Immobilisierung ebenfalls demonstriert und R-PE Molek{\"u}le zur Vereinzelung gebracht werden. Hierf{\"u}r war neben einem optimierten Elektrodenlayout, das durch Feldsimulationen den Feldgradienten betreffend gesucht wurde, eine Optimierung der Feldparameter, insbesondere von Spannung und Frequenz, erforderlich. Neben der Dielektrophorese erfolgten auch Beobachtungen anderer Effekte des elektrischen Feldes, wie z.B. Elektrolyse an Nano-Elektroden und Str{\"o}mungen {\"u}ber dem Elektroden-Array, hervorgerufen durch Joulesche W{\"a}rme und AC-elektroosmotischen Fluss. Zudem konnte Dielektrophorese an Silberpartikeln beobachtet werden und mittels Fluoreszenz-, Atom-Kraft-, Raster-Elektronen-Mikroskopie und energiedispersiver R{\"o}ntgenspektroskopie untersucht werden. Schließlich wurden die verwendeten Objektive und Kameras auf ihre Lichtempfindlichkeit hin analysiert, so dass die Vereinzelung von Biomolek{\"u}len an Nano-Elektroden nachweisbar war. Festzuhalten bleibt also, dass die Vereinzelung von Nano-Objekten und Biomolek{\"u}len an Nano-Elektroden-Arrays gelungen ist. Durch den parallelen Ansatz erlaubt dies, Aussagen {\"u}ber das Verhalten von Einzelmolek{\"u}len mit guter Statistik zu treffen.},
  language  = {de}
}
@phdthesis{Laux2016,
  author    = {Laux, Eva-Maria},
  title     = {Electric field-assisted immobilization and alignment of biomolecules},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-90271},
  school      = {Universit{\"a}t Potsdam},
  pages     = {IX, 120},
  year      = {2016},
  abstract  = {In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors. Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123. Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal. Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields. Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins.},
  language  = {en}
}
@phdthesis{Kirschbaum2009,
  author    = {Kirschbaum, Michael},
  title     = {A microfluidic approach for the initiation and investigation of surface-mediated signal transduction processes on a single-cell level},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-39576},
  school      = {Universit{\"a}t Potsdam},
  year      = {2009},
  abstract  = {For the elucidation of the dynamics of signal transduction processes that are induced by cellular interactions, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed and then also correlated with each other. This cannot be achieved by ensemble measurements because averaging biological data ignores the variability in timing and response patterns of individual cells and leads to highly blurred results. Instead, only a multi-parameter analysis at a single-cell level is able to exploit the information that is crucially needed for deducing the signaling pathways involved. The aim of this work was to develop a process line that allows the initiation of cell-cell or cell-particle interactions while at the same time the induced cellular reactions can be analyzed at various stages along the signal transduction cascade and correlated with each other. As this approach requires the gentle management of individually addressable cells, a dielectrophoresis (DEP)-based microfluidic system was employed that provides the manipulation of microscale objects with very high spatiotemporal precision and without the need of contacting the cell membrane. The system offers a high potential for automation and parallelization. This is essential for achieving a high level of robustness and reproducibility, which are key requirements in order to qualify this approach for a biomedical application. As an example process for intercellular communication, T cell activation has been chosen. The activation of the single T cells was triggered by contacting them individually with microbeads that were coated with antibodies directed against specific cell surface proteins, like the T cell receptor-associated kinase CD3 and the costimulatory molecule CD28 (CD; cluster of differentiation). The stimulation of the cells with the functionalized beads led to a rapid rise of their cytosolic Ca2+ concentration which was analyzed by a dual-wavelength ratiometric fluorescence measurement of the Ca2+-sensitive dye Fura-2. After Ca2+ imaging, the cells were isolated individually from the microfluidic system and cultivated further. Cell division and expression of the marker molecule CD69 as a late activation event of great significance were analyzed the following day and correlated with the previously recorded Ca2+ traces for each individual cell. It turned out such that the temporal profile of the Ca2+ traces between both activated and non-activated cells as well as dividing and non-dividing cells differed significantly. This shows that the pattern of Ca2+ signals in T cells can provide early information about a later reaction of the cell. As isolated cells are highly delicate objects, a precondition for these experiments was the successful adaptation of the system to maintain the vitality of single cells during and after manipulation. In this context, the influences of the microfluidic environment as well as the applied electric fields on the vitality of the cells and the cytosolic Ca2+ concentration as crucially important physiological parameters were thoroughly investigated. While a short-term DEP manipulation did not affect the vitality of the cells, they showed irregular Ca2+ transients upon exposure to the DEP field only. The rate and the strength of these Ca2+ signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, experimental conditions were identified that caused the least interference with the physiology of the cell. The possibility to precisely control the exact time point of stimulus application, to simultaneously analyze short-term reactions and to correlate them with later events of the signal transduction cascade on the level of individual cells makes this approach unique among previously described applications and offers new possibilities to unravel the mechanisms underlying intercellular communication.},
  language  = {en}
}