Ewald Terpetschnig - Academia.edu (original) (raw)
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Papers by Ewald Terpetschnig
Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the beh... more Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluo-rescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macro-molecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.
Current Pharmaceutical Biotechnology, 2010
We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-6... more We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-665 on a plasmonic platform of self-assembled colloidal structures (SACS) of silver prepared on a semitransparent silver film. A SeTau-665 immunoassay was performed on this platform and a control glass slide. The fluorescence properties of this label substantially change due to plasmonic interactions. While the average brightness increase of SeTau 665 in ensemble measurements was about 70-fold, fluorescence enhancements up to four-hundred times were observed on certain "hot spots" for single molecule measurements. The intensity increase is strongly correlated with a simultaneous decrease in fluorescence lifetime in these "hot spots". The large increase in brightness allows the reduction of the excitation power resulting in a reduced background and increased photostability. The remarkable fluorescence enhancements observed for SeTau 665 on our plasmonic platform should allow to substantially improve single molecule detection and to reduce the detection limits in sensing devices.
Bioconjugate Chemistry, Sep 1, 2009
We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR... more We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR) dye, and its use as a fluorescent label to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter. In a model assay, we demonstrate that a biotinylated Seta-633 tracer binds to antibiotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 1.8fold upon binding to a specific antibody (antibiotin, MW ) 160 kDa), while the titration with bovine serum albumin (BSA) or nonspecific antibody does not result in a noticeable change in lifetime. This behavior is contrary to that of fluorescent tracers like Cy5 or Alexa 647, which typically exhibit much smaller lifetime changes upon binding to antibodies.
ABSTRACT Various methods for forming dyed microspheres are provided. One method includes activati... more ABSTRACT Various methods for forming dyed microspheres are provided. One method includes activating a chemical structure coupled to a dye using heat or light to form a reaction intermediate in the presence of a microsphere. The reaction intermediate covalently attaches to a polymer of the microsphere thereby coupling the dye to the polymer and forming the dyed microsphere. Additional methods are provided for forming a dyed microsphere coupled to a molecule. These methods include dyeing the microspheres as described above in addition to synthesizing the molecule on an outer surface of the dyed microspheres. A population of dyed microspheres is also provided. Each of the dyed microspheres of the population includes a dye attached to a polymer of each of the dyed microspheres by a chemical structure. A coefficient of variation in dye characteristics of the population of dyed microspheres attributable to the dye is less than about 10%.
Analytical Biochemistry, 1995
Monatsh Chem, 1988
... 1-Butyl-5,10-dihydro-4,IOa-diphenyl-pyrrolidinoE2,3-bJE1,5Jbenzodiazepin~2,3~ dion (7 b) 0.34... more ... 1-Butyl-5,10-dihydro-4,IOa-diphenyl-pyrrolidinoE2,3-bJE1,5Jbenzodiazepin~2,3~ dion (7 b) 0.34 g (1 retool) 6 b und 0.12 g (1.11 retool) o-Phenylendiamin ergeben wie bei der Herstellung yon 7 a ... Wiley, New York, p 4 [11] Kollenz G, Penn G, Ott W, Peter's K, Peters E~M, yon ...
ABSTRACT The present invention relates to improved covalent coupling of two or more entities such... more ABSTRACT The present invention relates to improved covalent coupling of two or more entities such as biomolecules, polymer compositions, organic/inorganic molecules/materials, and the like, including their combinations, through one or more novel reactive groups attached to linker groups of 2–1000 atoms length. The present invention also contemplates the use of bifunctional bridge molecules to link two or more entities, wherein the functional groups of the bridge molecules are the novel reactive groups of the present invention.
The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the ar... more The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation.
Fresenius' Journal of Analytical Chemistry, 1999
The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic st... more The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic stationary phases has been used for the detection and quantification of ligand-polymer binding events. A blank polymer and an L-phenylalaninamide-imprinted polymer were prepared using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as a crosslinker. The MIP is first loaded with dye, and a solution of the dye in the eluent is passed through the MIP. If analyte is injected into the dye solution in the eluent, part of the dye is competitively replaced by the analyte from the MIP. Specifically, the competitive displacement of rhodamine B by amino acids and phenylalaninamide (Phe-NH 2 ), respectively, has been studied under polar and hydrophobic elution conditions. Enantioselective binding of Phe and Phe-NH 2 to the imprinted polymer was shown to occur in the micromolar concentration range. It is proposed that the displacement of non-specific dyes from MIPs be used for the development of multisensors based upon these highly specific and stable materials, which provide promising alternatives to the use of biological macromolecules in sensor technology.
Methods in Enzymology, 1997
Various methods for forming dyed microspheres are provided. One method includes activating a chem... more Various methods for forming dyed microspheres are provided. One method includes activating a chemical structure coupled to a dye using heat or light to form a reaction intermediate in the presence of a microsphere. The reaction intermediate covalently attaches to a polymer of the microsphere thereby coupling the dye to the polymer and forming the dyed microsphere. Additional methods are provided for forming a dyed microsphere coupled to a molecule. These methods include dyeing the microspheres as described above in addition to synthesizing the molecule on an outer surface of the dyed microspheres. A population of dyed microspheres is also provided. Each of the dyed microspheres of the population includes a dye attached to a polymer of each of the dyed microspheres by a chemical structure. A coefficient of variation in dye characteristics of the population of dyed microspheres attributable to the dye is less than about 10%.
Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the beh... more Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluo-rescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macro-molecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.
Current Pharmaceutical Biotechnology, 2010
We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-6... more We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-665 on a plasmonic platform of self-assembled colloidal structures (SACS) of silver prepared on a semitransparent silver film. A SeTau-665 immunoassay was performed on this platform and a control glass slide. The fluorescence properties of this label substantially change due to plasmonic interactions. While the average brightness increase of SeTau 665 in ensemble measurements was about 70-fold, fluorescence enhancements up to four-hundred times were observed on certain "hot spots" for single molecule measurements. The intensity increase is strongly correlated with a simultaneous decrease in fluorescence lifetime in these "hot spots". The large increase in brightness allows the reduction of the excitation power resulting in a reduced background and increased photostability. The remarkable fluorescence enhancements observed for SeTau 665 on our plasmonic platform should allow to substantially improve single molecule detection and to reduce the detection limits in sensing devices.
Bioconjugate Chemistry, Sep 1, 2009
We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR... more We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR) dye, and its use as a fluorescent label to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter. In a model assay, we demonstrate that a biotinylated Seta-633 tracer binds to antibiotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 1.8fold upon binding to a specific antibody (antibiotin, MW ) 160 kDa), while the titration with bovine serum albumin (BSA) or nonspecific antibody does not result in a noticeable change in lifetime. This behavior is contrary to that of fluorescent tracers like Cy5 or Alexa 647, which typically exhibit much smaller lifetime changes upon binding to antibodies.
ABSTRACT Various methods for forming dyed microspheres are provided. One method includes activati... more ABSTRACT Various methods for forming dyed microspheres are provided. One method includes activating a chemical structure coupled to a dye using heat or light to form a reaction intermediate in the presence of a microsphere. The reaction intermediate covalently attaches to a polymer of the microsphere thereby coupling the dye to the polymer and forming the dyed microsphere. Additional methods are provided for forming a dyed microsphere coupled to a molecule. These methods include dyeing the microspheres as described above in addition to synthesizing the molecule on an outer surface of the dyed microspheres. A population of dyed microspheres is also provided. Each of the dyed microspheres of the population includes a dye attached to a polymer of each of the dyed microspheres by a chemical structure. A coefficient of variation in dye characteristics of the population of dyed microspheres attributable to the dye is less than about 10%.
Analytical Biochemistry, 1995
Monatsh Chem, 1988
... 1-Butyl-5,10-dihydro-4,IOa-diphenyl-pyrrolidinoE2,3-bJE1,5Jbenzodiazepin~2,3~ dion (7 b) 0.34... more ... 1-Butyl-5,10-dihydro-4,IOa-diphenyl-pyrrolidinoE2,3-bJE1,5Jbenzodiazepin~2,3~ dion (7 b) 0.34 g (1 retool) 6 b und 0.12 g (1.11 retool) o-Phenylendiamin ergeben wie bei der Herstellung yon 7 a ... Wiley, New York, p 4 [11] Kollenz G, Penn G, Ott W, Peter's K, Peters E~M, yon ...
ABSTRACT The present invention relates to improved covalent coupling of two or more entities such... more ABSTRACT The present invention relates to improved covalent coupling of two or more entities such as biomolecules, polymer compositions, organic/inorganic molecules/materials, and the like, including their combinations, through one or more novel reactive groups attached to linker groups of 2–1000 atoms length. The present invention also contemplates the use of bifunctional bridge molecules to link two or more entities, wherein the functional groups of the bridge molecules are the novel reactive groups of the present invention.
The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the ar... more The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation.
Fresenius' Journal of Analytical Chemistry, 1999
The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic st... more The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic stationary phases has been used for the detection and quantification of ligand-polymer binding events. A blank polymer and an L-phenylalaninamide-imprinted polymer were prepared using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as a crosslinker. The MIP is first loaded with dye, and a solution of the dye in the eluent is passed through the MIP. If analyte is injected into the dye solution in the eluent, part of the dye is competitively replaced by the analyte from the MIP. Specifically, the competitive displacement of rhodamine B by amino acids and phenylalaninamide (Phe-NH 2 ), respectively, has been studied under polar and hydrophobic elution conditions. Enantioselective binding of Phe and Phe-NH 2 to the imprinted polymer was shown to occur in the micromolar concentration range. It is proposed that the displacement of non-specific dyes from MIPs be used for the development of multisensors based upon these highly specific and stable materials, which provide promising alternatives to the use of biological macromolecules in sensor technology.
Methods in Enzymology, 1997
Various methods for forming dyed microspheres are provided. One method includes activating a chem... more Various methods for forming dyed microspheres are provided. One method includes activating a chemical structure coupled to a dye using heat or light to form a reaction intermediate in the presence of a microsphere. The reaction intermediate covalently attaches to a polymer of the microsphere thereby coupling the dye to the polymer and forming the dyed microsphere. Additional methods are provided for forming a dyed microsphere coupled to a molecule. These methods include dyeing the microspheres as described above in addition to synthesizing the molecule on an outer surface of the dyed microspheres. A population of dyed microspheres is also provided. Each of the dyed microspheres of the population includes a dye attached to a polymer of each of the dyed microspheres by a chemical structure. A coefficient of variation in dye characteristics of the population of dyed microspheres attributable to the dye is less than about 10%.