TY - JOUR A1 - Agarwal, Saloni A1 - Hamidizadeh, Mojdeh A1 - Bier, Frank Fabian T1 - Detection of reverse transcriptase LAMP-amplified nucleic acid from oropharyngeal viral swab samples using biotinylated DNA probes through a lateral flow assay JF - Biosensors : open access journal N2 - This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing. KW - RT-LAMP KW - LFA KW - NAAT-LFA KW - semi-quantitative KW - surveillance-based diagnostics Y1 - 2023 U6 - https://doi.org/10.3390/bios13110988 SN - 2079-6374 VL - 13 IS - 11 PB - MDPI CY - Basel ER - TY - JOUR A1 - Bognár, Zsófia A1 - Supala, Eszter A1 - Yarman, Aysu A1 - Zhang, Xiaorong A1 - Bier, Frank Fabian A1 - Scheller, Frieder W. A1 - Gyurcsanyi, Róbert E. T1 - Peptide epitope-imprinted polymer microarrays for selective protein recognition BT - application for SARS-CoV-2 RBD protein JF - Chemical science / RSC, Royal Society of Chemistry N2 - 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. Y1 - 2021 U6 - https://doi.org/10.1039/d1sc04502d SN - 2041-6539 VL - 13 IS - 5 SP - 1263 EP - 1269 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Agarwal, Saloni A1 - Warmt, Christian A1 - Henkel, Jörg A1 - Schrick, Livia A1 - Nitsche, Andreas A1 - Bier, Frank Fabian T1 - Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use JF - Analytical and bioanalytical chemistry : a merger of Fresenius' journal of analytical chemistry, Analusis and Quimica analitica N2 - 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. KW - Point of care testing (POCT) KW - Lateral flow assay (LFA) KW - COVID-19 KW - Reverse transcription loop-mediated isothermal amplification (RT-LAMP); KW - SARS-CoV-2 N-gene Y1 - 2022 U6 - https://doi.org/10.1007/s00216-022-03880-4 SN - 1618-2642 SN - 1618-2650 VL - 414 IS - 10 SP - 3177 EP - 3186 PB - Springer CY - Heidelberg ER - 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 - 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 - TY - JOUR A1 - Warmt, Christian A1 - Fenzel, Carolin Kornelia A1 - Henkel, Jörg A1 - Bier, Frank Fabian T1 - Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes JF - Scientific reports N2 - 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. Y1 - 2021 U6 - https://doi.org/10.1038/s41598-021-99774-z SN - 2045-2322 VL - 11 IS - 1 PB - Macmillan Publishers Limited, part of Springer Nature CY - [London] 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 - Kagel, Heike A1 - Bier, Frank Fabian A1 - Frohme, Marcus A1 - Glökler, Jörn F. T1 - A Novel Optical Method To Reversibly Control Enzymatic Activity Based On Photoacids JF - Scientific reports N2 - Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report a novel optical approach to reversibly control a typical biochemical reaction by changing the pH and using acid phosphatase as a model enzyme. The reversible photoacid G-acid functions as a proton donor, changing the pH rapidly and reversibly by using high power UV LEDs as an illumination source in our experimental setup. The reaction can be tightly controlled by simply switching the light on and off and should be applicable to a wide range of other enzymatic reactions, thus enabling miniaturization and parallelization through non-invasive optical means. Y1 - 2019 U6 - https://doi.org/10.1038/s41598-019-50867-w SN - 2045-2322 VL - 9 PB - Nature Publishing Group CY - London ER - TY - JOUR A1 - Kersting, Sebastian A1 - Rausch, Valentina A1 - Bier, Frank Fabian A1 - von Nickisch-Rosenegk, Markus T1 - A recombinase polymerase amplification assay for the diagnosis of atypical pneumonia JF - Analytical biochemistry : methods in the biological sciences N2 - Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and symptoms are nonspecific and do not differ from other causes of pneumonia. Therefore, a rapid diagnosis within a clinically relevant time is essential for a fast onset of the proper treatment. Although methods based on polymerase chain reaction significantly improved the identification of pathogens, they are difficult to conduct and need specialized equipment. We describe a rapid and sensitive test using isothermal recombinase polymerase amplification and detection on a disposable test strip. This method does not require any special instrumentation and can be performed in less than 20 min. The analytical sensitivity in the multiplex assay amplifying specific regions of S. pneumoniae and L. pneumophila simultaneously was 10 CFUs of genomic DNA per reaction. In cross detection studies with closely related strains and other bacterial agents the specificity of the RPA was confirmed. The presented method is applicable for near patient and field testing with a rather simple routine and the possibility for a read out with the naked eye. Y1 - 2018 U6 - https://doi.org/10.1016/j.ab.2018.04.014 SN - 0003-2697 SN - 1096-0309 VL - 550 SP - 54 EP - 60 PB - Elsevier CY - San Diego 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 -