@article{StankeWengerBieretal.2022, author = {Stanke, Sandra and Wenger, Christian and Bier, Frank Fabian and H{\"o}lzel, Ralph}, title = {AC electrokinetic immobilization of influenza virus}, series = {Electrophoresis : microfluids \& proteomics}, volume = {43}, journal = {Electrophoresis : microfluids \& proteomics}, number = {12}, publisher = {Wiley-Blackwell}, address = {Weinheim}, issn = {0173-0835}, doi = {10.1002/elps.202100324}, pages = {1309 -- 1321}, year = {2022}, abstract = {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.}, language = {en} } @article{AgarwalWarmtHenkeletal.2022, author = {Agarwal, Saloni and Warmt, Christian and Henkel, J{\"o}rg and Schrick, Livia and Nitsche, Andreas and Bier, Frank Fabian}, title = {Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use}, series = {Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry, Analusis and Quimica analitica}, volume = {414}, journal = {Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry, Analusis and Quimica analitica}, number = {10}, publisher = {Springer}, address = {Heidelberg}, issn = {1618-2642}, doi = {10.1007/s00216-022-03880-4}, pages = {3177 -- 3186}, year = {2022}, abstract = {The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65\%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66\%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.}, language = {en} } @article{PrueferWengerBieretal.2022, author = {Pr{\"u}fer, Mareike and Wenger, Christian and Bier, Frank Fabian and Laux, Eva-Maria and H{\"o}lzel, Ralph}, title = {Activity of AC electrokinetically immobilized horseradish peroxidase}, series = {Electrophoresis : microfluidics, nanoanalysis \& proteomics}, journal = {Electrophoresis : microfluidics, nanoanalysis \& proteomics}, publisher = {Wiley}, address = {Hoboken}, issn = {0173-0835}, doi = {10.1002/elps.202200073}, pages = {1920 -- 1933}, year = {2022}, abstract = {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.}, language = {en} } @article{BognarSupalaYarmanetal.2022, author = {Bogn{\´a}r, Zs{\´o}fia and Supala, Eszter and Yarman, Aysu and Zhang, Xiaorong and Bier, Frank Fabian and Scheller, Frieder W. and Gyurcsanyi, R{\´o}bert E.}, title = {Peptide epitope-imprinted polymer microarrays for selective protein recognition}, series = {Chemical science / RSC, Royal Society of Chemistry}, volume = {13}, journal = {Chemical science / RSC, Royal Society of Chemistry}, number = {5}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {2041-6539}, doi = {10.1039/d1sc04502d}, pages = {1263 -- 1269}, year = {2022}, abstract = {We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K-D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.}, language = {en} } @article{LauxWengerBieretal.2020, author = {Laux, Eva-Maria and Wenger, Christian and Bier, Frank Fabian and Hoelzel, Ralph}, title = {AC electrokinetic immobilization of organic dye molecules}, series = {Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry and Analusis}, volume = {412}, journal = {Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry and Analusis}, number = {16}, publisher = {Springer}, address = {Berlin}, issn = {1618-2642}, doi = {10.1007/s00216-020-02480-4}, pages = {3859 -- 3870}, year = {2020}, abstract = {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.}, language = {en} } @article{WarmtFenzelHenkeletal.2021, author = {Warmt, Christian and Fenzel, Carolin Kornelia and Henkel, J{\"o}rg and Bier, Frank Fabian}, title = {Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes}, series = {Scientific reports}, volume = {11}, journal = {Scientific reports}, number = {1}, publisher = {Macmillan Publishers Limited, part of Springer Nature}, address = {[London]}, issn = {2045-2322}, doi = {10.1038/s41598-021-99774-z}, pages = {9}, year = {2021}, abstract = {In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1\% Cy5-dUTP and 99\% unlabelled dTTP we were able to detect the bla(CTX-M15) gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.}, language = {en} } @article{FischbachLohBieretal.2017, author = {Fischbach, Jens and Loh, Qiuting and Bier, Frank Fabian and Lim, Theam Soon and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method}, series = {Scientific reports}, volume = {7}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/srep45085}, pages = {9}, year = {2017}, abstract = {We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.}, language = {en} } @misc{LauxDocoslisWengeretal.2017, author = {Laux, Eva-Maria and Docoslis, A. and Wenger, C. and Bier, Frank Fabian and H{\"o}lzel, Ralph}, title = {Combination of dielectrophoresis and SERS for bacteria detection and characterization}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {46}, journal = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S331 -- S331}, year = {2017}, language = {en} } @article{LauxBierHoelzel2018, author = {Laux, Eva-Maria and Bier, Frank Fabian and H{\"o}lzel, Ralph}, title = {Dielectrophoretic Stretching of DNA}, series = {DNA Nanotechnology}, journal = {DNA Nanotechnology}, edition = {2}, publisher = {Humana Press Inc.}, address = {New York}, isbn = {978-1-4939-8582-1}, issn = {1064-3745}, doi = {10.1007/978-1-4939-8582-1_14}, pages = {199 -- 208}, year = {2018}, abstract = {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.}, language = {en} } @article{LauxErmilovaPannwitzetal.2018, author = {Laux, Eva-Maria and Ermilova, Elena and Pannwitz, Daniel and Gibbons, Jessica and H{\"o}lzel, Ralph and Bier, Frank Fabian}, title = {Dielectric Spectroscopy of Biomolecules up to 110 GHz}, series = {Frequenz}, volume = {72}, journal = {Frequenz}, number = {3-4}, publisher = {De Gruyter}, address = {Berlin}, issn = {0016-1136}, doi = {10.1515/freq-2018-0010}, pages = {135 -- 140}, year = {2018}, abstract = {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.}, language = {en} }