Refine
Year of publication
Document Type
- Article (74)
- Other (5)
- Postprint (2)
- Monograph/Edited Volume (1)
- Conference Proceeding (1)
- Doctoral Thesis (1)
- Preprint (1)
- Review (1)
Is part of the Bibliography
- yes (86)
Keywords
- AC electrokinetics (4)
- Dielectrophoresis (3)
- DNA (2)
- Interdigitated electrodes (2)
- RPA (2)
- Surface plasmon resonance (2)
- dielectrophoresis (2)
- nanoelectrodes (2)
- protein (2)
- surface plasmon resonance (2)
Institute
- Institut für Biochemie und Biologie (86) (remove)
Amplifying bienzyme cycle-linked immunoassays for determination of 2,4- dichlorphenoxyacetic acid
(1996)
Ultrasensitive biosensors
(1996)
Nucleic acid based sensors
(1997)
Trends in der Bioanalytik
(2002)
The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immonoassay principle. The concentration of the progesterone antibody was kept at 1 µg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.
Biosensoren
(2003)
Microarrays are new analytical devices that allow the parallel and simultaneous detection of thousands of target compounds. Microarrays, also called DNA chips, are widely used in gene expression, the genotyping of individuals, point mutations, detection of single nucleotide polymorphisms, and short tandem repeats. Microarrays have highly specific base-pair interactions with labeled complementary strands, which makes this technology to a powerful analytical device for monitoring whole genomes. In this article, we provide a survey of the common microarray manufacturing methods, from the selection of support material to surface structuring, immobilization and hybridization, and finally the detection with labeled complementary strands. Special attention is given to the immobilization of single strands, since fast chemical reactions, the creation of homogeneous surface functionalities as well as an oriented coupling are crucial pre-conditions for a good spot morphology and microarrays of high quality
An artificial gene was constructed combining the T7 promoter and terminator with the EGFP-gene from the plasmid pEGFP. The functionality of the construct was shown by in vitro translation. The gene-construct was immobilised on a planar glass surface. The transcription was performed on the immobilised gene and mRNA was determined by RT-PCR. Multiple use of the immobilised gene was demonstrated
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.
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.
Enzymatic isothermal rolling circle amplification (RCA) produces long concatemeric single-stranded DNA (ssDNA) molecules if a small circular ssDNA molecule is applied as the template. A method is presented here in which the RCA reaction is carried out in a flow-through system, starting from isolated surface-tethered DNA primers. This approach combines gentle fluidic handling of the single-stranded RCA products, such as staining or stretching via a receding meniscus, with the option of simultaneous (fluorescence) microscopic observation. It is shown that the stretched and surface-attached RCA products are accessible for hybridization of complementary oligonucleotides, which demonstrates their addressability by complementary base pairing. The long RCA products should be well suited to bridge the gap between biomolecular nanoscale building-blocks and structures at the micro- and macroscale, especially at the single- molecule level presented here.
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.
This work describes a cell-based assay that does not depend on radioactivity or laboratory animals for the detection of ligands of angiotensin II type 1 receptor (AT(1)R). The assay makes use of stable transfected Chinese hamster ovary cells (CHO-AT(1)R) expressing the AT(1)R. A sequential saturation assay principle was used in which receptor binding sites of the CHO-AT(1)R cells are blocked by the analyte in a concentration-dependent manner. Afterwards, TAMRA-angiotensin II, a fluorescence-labeled ligand, was added to bind to the remaining free binding sites of the receptor. In consequence, the fluorescence signal determined is inversely proportional to the concentration of the analyte.
This work describes a method for surface regeneration of microfluidic microarray printheads through plasma techniques. Modification procedures were chosen in a way to obtain high reproducibility with a minimum of time consumption. The idea behind this is a complete regeneration of a microarray printhead before or after usage to achieve best printing results over a typical print job. A sequence of low-pressure oxygen-plasma and plasma polymerization with hexamethyldisiloxane (HMDSO) was used to regenerate printheads. Proof of the concept is given through quality control performed with a spotter implemented CCD camera, contact angle measurements and a typical hybridization experiment. Stable printing results were obtained over 3000 activations showing that the presented method is suitable for treatment of microarray printheads.
For the investigation of alternating current electrokinetic effects, a system is presented that allows for the simultaneous observation of fluid flow above and around microelectrodes in all three directions in space. Beside the usual microscopical view from top, lateral observation through the same objective is made possible by two small mirrors that are placed next to the electrodes. Fluid flow and movement of fluorescent nanoparticles above interdigitated electrodes are monitored by fluorescence microscopy and digital imaging and are further analysed by image processing. Field frequencies are varied from 10 Hz to 1 GHz at up to 10V(rms). Electrical conductivity of the fluid is monitored in situ in the actual measuring chamber.
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.
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.
Biodetection formats, such as DNA and antibody microarrays, are valuable tools in the life sciences, but for some applications, the detection limits are insufficient. A straightforward strategy to obtain signal amplification is the rolling circle amplification (RCA), an easy, isothermal, and enzymatic nucleic acid synthesis that has already been employed successfully to increase the signal yield for several single-analyte and multiplexing assays in conjunction with hybridization probes. Here, we systematically investigated the parameters responsible for the RCA driven signal amplification with fluorescent labels, such as the type of fluorophore chosen, labeling strategy, composition of reaction solution, and number of handling steps. In labeling strategies, post-synthetic labeling via a Cy3-hybridization probe was compared to the direct incorporation of fluorescent Cy3-dUTP and DY-555-dUTP into the nascent strand during synthesis. With our direct labeling protocol, the assay's runtime and handling steps could be reduced while the signal yield was increased. These features are very attractive for many detection formats but especially for point-of-care diagnostic kits that need to be simple enough to be performed by scientifically untrained personnel.
Background: Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.
Results: Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.
Conclusions: The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.zeige weniger
Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.