TY - JOUR A1 - Prüfer, Mareike A1 - Wenger, Christian A1 - Bier, Frank Fabian A1 - Laux, Eva-Maria A1 - Hölzel, Ralph T1 - Activity of AC electrokinetically immobilized horseradish peroxidase JF - Electrophoresis : microfluidics, nanoanalysis & proteomics N2 - Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H2O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications. KW - AC electrokinetics KW - dielectrophoresis KW - enzyme activity KW - immobilization; KW - nanoelectrodes Y1 - 2022 U6 - https://doi.org/10.1002/elps.202200073 SN - 0173-0835 SN - 1522-2683 SP - 1920 EP - 1933 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Wenger, Christian A1 - Bier, Frank Fabian A1 - Hoelzel, Ralph T1 - AC electrokinetic immobilization of organic dye molecules JF - Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry and Analusis N2 - The application of inhomogeneous AC electric fields for molecular immobilization is a very fast and simple method that does not require any adaptions to the molecule's functional groups or charges. Here, the method is applied to a completely new category of molecules: small organic fluorescence dyes, whose dimensions amount to only 1 nm or even less. The presented setup and the electric field parameters used allow immobilization of dye molecules on the whole electrode surface as opposed to pure dielectrophoretic applications, where molecules are attracted only to regions of high electric field gradients, i.e., to the electrode tips and edges. In addition to dielectrophoresis and AC electrokinetic flow, molecular scale interactions and electrophoresis at short time scales are discussed as further mechanisms leading to migration and immobilization of the molecules. KW - AC electrokinetics KW - AC electrophoresis KW - Molecular dielectrophoresis KW - Interdigitated electrodes KW - Organic dyes Y1 - 2020 U6 - https://doi.org/10.1007/s00216-020-02480-4 SN - 1618-2642 SN - 1618-2650 VL - 412 IS - 16 SP - 3859 EP - 3870 PB - Springer CY - Berlin ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Ermilova, Elena A1 - Pannwitz, Daniel A1 - Gibbons, Jessica A1 - Hölzel, Ralph A1 - Bier, Frank Fabian T1 - Dielectric Spectroscopy of Biomolecules up to 110 GHz JF - Frequenz N2 - Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spectra over 4 frequency decade up to 110 GHz. Routines for calibration and for data evaluation according to physicochemical interaction models have been developed. The frequency dependent permittivity and dielectric loss of some proteins and nucleic acids, the main classes of biomolecules, and of their sub-units have been determined. Dielectric spectra are presented for the amino acid alanine, the proteins lysozyme and haemoglobin, the nucleotides AMP and ATP, and for the plasmid pET-21, which has been produced by bacterial culture. Characterisation of a variety of biomolecules is envisaged, as is the application to studies on protein structure and function. KW - dielectric KW - spectroscopy KW - permittivity KW - protein KW - DNA KW - amino acid KW - plasmid Y1 - 2018 U6 - https://doi.org/10.1515/freq-2018-0010 SN - 0016-1136 SN - 2191-6349 VL - 72 IS - 3-4 SP - 135 EP - 140 PB - De Gruyter CY - Berlin ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Dielectrophoretic Stretching of DNA JF - DNA Nanotechnology N2 - The spatial control of DNA and of self-assembled DNA constructs is a prerequisite for the preparation of DNA-based nanostructures and microstructures and a useful tool for studies on single DNA molecules. Here we describe a protocol for the accumulation of dissolved lambda-DNA molecules between planar microelectrodes by the action of inhomogeneous radiofrequency electric fields. The resulting AC electrokinetic forces stretch the DNA molecules and align them parallel to the electric field. The electrode preparation from off-the-shelf electronic components is explained, and a detailed description of the electronic setup is given. The experimental procedure is controlled in real-time by fluorescence microscopy. KW - Alignment KW - Dielectrophoresis KW - DNA KW - Electrokinetics KW - Interdigitated electrodes KW - Stretching Y1 - 2018 SN - 978-1-4939-8582-1 SN - 978-1-4939-8581-4 U6 - https://doi.org/10.1007/978-1-4939-8582-1_14 SN - 1064-3745 SN - 1940-6029 SP - 199 EP - 208 PB - Humana Press Inc. CY - New York ET - 2 ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Electrode-based AC electrokinetics of proteins BT - a mini-review JF - Bioelectrochemistry : official journal of the Bioelectrochemical Society ; an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry N2 - Employing electric phenomena for the spatial manipulation of bioparticles from whole cells down to dissolved molecules has become a useful tool in biotechnology and analytics. AC electrokinetic effects like dielectrophoresis and AC electroosmosis are increasingly used to concentrate, separate and immobilize DNA and proteins. With the advance of photolithographical micro- and nanofabrication methods, novel or improved bioanalytical applications benefit from concentrating analytes, signal enhancement and locally controlled immobilization by AC electrokinetic effects. In this review of AC electrokinetics of proteins, the respective studies are classified according to their different electrode geometries: individual electrode pairs, interdigitated electrodes, quadrupole electrodes, and 3D configurations of electrode arrays. Known advantages and disadvantages of each layout are discussed. KW - AC electrokinetics KW - Dielectrophoresis KW - Electrodes KW - Electroosmosis KW - Proteins Y1 - 2017 U6 - https://doi.org/10.1016/j.bioelechem.2017.11.010 SN - 1567-5394 SN - 1878-562X VL - 120 SP - 76 EP - 82 PB - Elsevier B.V. CY - Amsterdam ER - TY - GEN A1 - Laux, Eva-Maria A1 - Gibbons, J. A1 - Ermilova, Elena A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Broadband dielectric spectroscopy of bovine serum albumin in the GHz range T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S347 EP - S347 PB - Springer CY - New York ER - TY - GEN A1 - Laux, Eva-Maria A1 - Knigge, Xenia A1 - Wenger, C. A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - AC electrokinetic manipulation of nanoparticles and molecules T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S189 EP - S189 PB - Springer CY - New York ER - TY - GEN A1 - Laux, Eva-Maria A1 - Docoslis, A. A1 - Wenger, C. A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Combination of dielectrophoresis and SERS for bacteria detection and characterization T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S331 EP - S331 PB - Springer CY - New York ER - TY - THES A1 - Laux, Eva-Maria T1 - Electric field-assisted immobilization and alignment of biomolecules T1 - Immobilisierung und Ausrichtung von Biomolekülen mit elektrischen Wechselfeldern N2 - 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. N2 - In dieser Doktorarbeit wurde eine Methode entwickelt, mit der Biomoleküle unter dem Einfluss von elektrischen Feldern auf Metallelektroden immobilisiert und ausgerichtet werden können. Für die Immobilisierung wurden weder Modifikationen an den Biomolekülen noch an den Elektroden benötigt. Zwei elektrokinetische Effekte, die Dielektrophorese und der AC-elektroosmotische Fluss, wurden als verantwortliche Effekte für die Molekülbewegung identifiziert. Mit einer neuen 3D Elektrodenkonfiguration wurde der AC-elektroosmotische Fluss minimiert. Damit konnte der Einfluss der experimentellen Parameter auf die Dielektrophoresekraft und deren Auswirkungen auf die Moleküle untersucht werden: Die permanente Immobilisierung von Proteinen wurde mit einem Rasterkraftmikroskop quantifiziert, indem die Volumina der immobilisierten Proteinablagerungen gemessen wurden, und daraus die maximal darin enthaltene Anzahl an Proteinen berechnet wurde. Diese Art der absoluten Quantifizierung war nur möglich, da die Proteine auch nach Abschalten des elektrischen Feldes auf den Wolframelektroden hafteten. Eine solche permanente Immobilisierung funktioneller Proteine auf Elektroden oder Oberflächen im Allgemeinen ist eine wichtige Voraussetzung für die Herstellung von Biosensoren. Des Weiteren muss die Biofunktion der Proteine nach der Immobilisierung erhalten bleiben. Da die Proteine durch die Immobilisierung chemisch oder physikalisch verändert werden, ist auch ihre Biofunktion häufig eingeschränkt. In dieser Arbeit wurde erstmals der Erhalt der Aktivität dielektrophoretisch immobilisierter Enzyme gezeigt. Hierfür wurde das Enzym Meerrettichperoxidase exemplarisch verwendet, dessen Aktivität über die Oxidation von Dihydrorhodamin 123, einem nicht-fluoreszentem Vorläufer des Fluoreszenzfarbstoffes Rhodamin 123, nachgewiesen wurde. Molekulare Ausrichtung und Immobilisierung – sowohl reversibel als auch permanent – wurde unter dem Einfluss inhomogener elektrischer Wechselfelder erreicht. Für die Bestimmung der Molekülausrichtung wurde mit ein Messaufbau entwickelt, der auf einem Fluoreszenzmikroskop basiert. Der Aufbau, das Messprotokoll und die Auswertungsmethode wurden mit einer selbst hergestellten Kontrollprobe, die aus ausgerichteten Acridinorangemolekülen in einem Flüssigkristall bestand, validiert. Lambda-DNA Doppelstränge wurden zwischen benachbarten Interdigitalelektroden gestreckt und temporär ausgerichtet. Die Ausrichtung von interkalierten PicoGreen-Molekülen im rechten Winkel zur Längsachse der Doppelstränge konnte hier gezeigt werden. Zudem konnte die ausgerichtete Immobilisierung des enhanced Green Fluorescent Protein nachgewiesen werden, indem die Fluoreszenz des Proteins und seine Struktureigenschaften ausgenutzt wurden. Aus den Messungen konnte der Winkel des Chromophors relativ zur Proteinlängsachse mit guter Übereinstimmung mit Röntgenkristallstrukturdaten bestimmt werden. Eine permanente Immobilisierung mit gleichzeitiger Ausrichtung der Proteine wurde entlang der Kanten, an den Spitzen und auf der Oberfläche von Interdigitalelektroden erzielt. Damit wurde zum ersten Mal eine ausgerichtete Immobilisierung von Proteinen mit elektrischen Wechselfeldern gezeigt. Diese Methode ist vielversprechend für die Immobilisierung von Antikörpern oder Enzymen mit einheitlicher Ausrichtung und dadurch verbessertem Zugang zu den aktiven Zentren, was nicht nur für die industrielle Biosensorherstellung von Interesse ist, sondern genauso für allgemeine Wechselwirkungsstudien von Proteinen. KW - dielectrophoresis KW - electrokinetics KW - proteins KW - immobilization KW - alignment KW - Dielektrophorese KW - elektrokinetische Effekte KW - Proteine KW - Immobilisierung KW - Ausrichtung Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-90271 ER -