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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.
Background: The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent Onchip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. Methods: HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the Onchip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. Results: We have successfully shown the OnChip-HDA and applied it for single- and duplex- detection of the pathogens N. gonorrhoeae and S. aureus. Conclusion: We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.
Isothermal amplification technologies are emerging on the horizon that could have the potential to pose as alternatives to PCR in terms of sensitivity and ease of use. One of the most recent isothermal technologies is helicase- dependent amplification (HDA). This technology uses the helicase's capability to disrupt the hydrogen bonds of a Watson-Crick base pair in order to separate dsDNA. A denaturation step, as is used in PCR, is no longer required. This gives rise to new, less expensive and less complicated designs for point-of-care devices and 'Lab on Chip' systems. Helicase-dependent OnChip-amplification (OnChip-HDA) is a further step into this direction as it integrates the HDA technology with microarray technology and its power of multiplexing. This special report will give an overview on the HDA and OnChip-HDA technology, and its potential for point-of-care diagnostics.
Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity
Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics
(2006)
Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.
Herein we present an efficient synthesis of a biomimetic probe with modular construction that can be specifically bound by the mannose binding FimH protein - a surface adhesion protein of E. coli bacteria. The synthesis combines the new and interesting DBD dye with the carbohydrate ligand mannose via a Click reaction. We demonstrate the binding to E. coli bacteria over a large concentration range and also present some special characteristics of those molecules that are of particular interest for the application as a biosensor. In particular, the mix-and-measure ability and the very good photo-stability should be highlighted here.
Amplifying bienzyme cycle-linked immunoassays for determination of 2,4- dichlorphenoxyacetic acid
(1996)
Ultrasensitive biosensors
(1996)
Nucleic acid based sensors
(1997)
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.