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 - 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 - GEN A1 - Stanke, S. A1 - Wenger, C. A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - Dielectrophoretic functionalization of nanoelectrode arrays for the detection of influenza viruses T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S337 EP - S337 PB - Springer CY - New York ER - TY - GEN A1 - Knigge, Xenia A1 - Wenger, C. A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - AC electrokinetic immobilisation of nanoobjects as individual singles in regular arrays T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2017 SN - 0175-7571 SN - 1432-1017 VL - 46 SP - S187 EP - S187 PB - Springer CY - New York 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 - 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 - 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 - 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 - JOUR A1 - Breitenstein, Michael A1 - Nielsen, Peter E. A1 - Hölzel, Ralph A1 - Bier, Frank Fabian T1 - DNA-nanostructure-assembly by sequential spotting JF - Journal of nanobiotechnology N2 - Background: The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results: For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures. Conclusions: The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element. Y1 - 2011 U6 - https://doi.org/10.1186/1477-3155-9-54 SN - 1477-3155 VL - 9 IS - 11 PB - BioMed Central CY - London ER - TY - GEN A1 - Breitenstein, Michael A1 - Nielsen, Peter E. A1 - Hölzel, Ralph A1 - Bier, Frank Fabian T1 - DNA-nanostructure-assembly by sequential spotting T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background: The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results: For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures. Conclusions: The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1027 KW - atomic force microscope KW - peptide nucleic acid KW - persistence length KW - adapter oligonucleotide KW - high fluorescence signal Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431108 SN - 1866-8372 IS - 1027 ER - TY - GEN A1 - Breitenstein, Michael A1 - Hölzel, Ralph A1 - Bier, Frank Fabian T1 - Immobilization of different biomolecules by atomic force microscopy T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Background Micrometer resolution placement and immobilization of probe molecules is an important step in the preparation of biochips and a wide range of lab-on-chip systems. Most known methods for such a deposition of several different substances are costly and only suitable for a limited number of probes. In this article we present a flexible procedure for simultaneous spatially controlled immobilization of functional biomolecules by molecular ink lithography. Results For the bottom-up fabrication of surface bound nanostructures a universal method is presented that allows the immobilization of different types of biomolecules with micrometer resolution. A supporting surface is biotinylated and streptavidin molecules are deposited with an AFM (atomic force microscope) tip at distinct positions. Subsequent incubation with a biotinylated molecule species leads to binding only at these positions. After washing streptavidin is deposited a second time with the same AFM tip and then a second biotinylated molecule species is coupled by incubation. This procedure can be repeated several times. Here we show how to immobilize different types of biomolecules in an arbitrary arrangement whereas most common methods can deposit only one type of molecules. The presented method works on transparent as well as on opaque substrates. The spatial resolution is better than 400 nm and is limited only by the AFM's positional accuracy after repeated z-cycles since all steps are performed in situ without moving the supporting surface. The principle is demonstrated by hybridization to different immobilized DNA oligomers and was validated by fluorescence microscopy. Conclusions The immobilization of different types of biomolecules in high-density microarrays is a challenging task for biotechnology. The method presented here not only allows for the deposition of DNA at submicrometer resolution but also for proteins and other molecules of biological relevance that can be coupled to biotin. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 872 KW - Atomic Force Microscope KW - Immobilization KW - Cross Contamination KW - Roth GmbH KW - Microcontact Printing Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-435075 SN - 1866-8372 IS - 872 ER - TY - JOUR A1 - Stanke, Sandra A1 - Wenger, Christian A1 - Bier, Frank Fabian A1 - Hölzel, Ralph T1 - AC electrokinetic immobilization of influenza virus JF - Electrophoresis : microfluids & proteomics N2 - The use of alternating current (AC) electrokinetic forces, like dielectrophoresis and AC electroosmosis, as a simple and fast method to immobilize sub-micrometer objects onto nanoelectrode arrays is presented. Due to its medical relevance, the influenza virus is chosen as a model organism. One of the outstanding features is that the immobilization of viral material to the electrodes can be achieved permanently, allowing subsequent handling independently from the electrical setup. Thus, by using merely electric fields, we demonstrate that the need of prior chemical surface modification could become obsolete. The accumulation of viral material over time is observed by fluorescence microscopy. The influences of side effects like electrothermal fluid flow, causing a fluid motion above the electrodes and causing an intensity gradient within the electrode array, are discussed. Due to the improved resolution by combining fluorescence microscopy with deconvolution, it is shown that the viral material is mainly drawn to the electrode edge and to a lesser extent to the electrode surface. Finally, areas of application for this functionalization technique are presented. KW - AC electrokinetics KW - AC electroosmosis KW - dielectrophoresis KW - influenza virus KW - nanoelectrodes Y1 - 2022 U6 - https://doi.org/10.1002/elps.202100324 SN - 0173-0835 SN - 1522-2683 VL - 43 IS - 12 SP - 1309 EP - 1321 PB - Wiley-Blackwell CY - Weinheim ER -