- Switzerland
Stefan Seeger
University of Zurich, Switzerland, Chemistry, Faculty Member
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Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker... more
Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.
We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the... more
We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.
Research Interests: Biosensors and BioSensors
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Mesocrystals with the symmetry defying morphologies and highly ordered superstructures composed of primary units are of particular interest, but the fabrication has proved extremely challenging. A novel strategy based on biomineralization... more
Mesocrystals with the symmetry defying morphologies and highly ordered superstructures composed of primary units are of particular interest, but the fabrication has proved extremely challenging. A novel strategy based on biomineralization approach for the synthesis of hematite mesocrystals is developed by using silk fibroin as a biotemplate. The resultant hematite mesocrystals are uniform, highly crystalline, and porous nanostructures with tunable size and morphologies by simply varying the concentration of the silk fibroin and iron(III) chloride in this biomineralization system. In particular, we demonstrate a complex mesoscale biomineralization process induced by the silk fibroin for the formation of hematite mesocrystals. This biomimetic strategy features precisely tunable, high efficiency, and low-cost and opens up an avenue to access new novel functional mesocrystals with hierarchical structures in various practical applications.
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New fluorescent dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based on the concept of 'multiplex dyes', we have designed several rhodamine dyes with nearly identical... more
New fluorescent dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based on the concept of 'multiplex dyes', we have designed several rhodamine dyes with nearly identical absorption and emission spectral characteristics but different ...
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ABSTRACT
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Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as a donor and Alexa Fluor 350 (AF350) labeled avidin as an acceptor has been investigated by means of steady-state fluorescence and time-resolved... more
Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as a donor and Alexa Fluor 350 (AF350) labeled avidin as an acceptor has been investigated by means of steady-state fluorescence and time-resolved fluorescence spectroscopy. The donors are readily paired with acceptors through the well-established binding affinity of biotin and avidin. The fluorescence energy transfer efficiency was determined by the donor fluorescence emission and lifetime changes in the presence and absence of acceptor. The theoretical energy transfer efficiency and theoretical average distance between donor and acceptor after noncovalent binding was calculated by taking the distribution of tryptophan residues in βGAL and avidin as well as the location of AF350 in avidin into account, which agree with the experimental data. It is shown how information of the location of the acceptor can be obtained. Further, the fluorescence intensity image of AF350 on a biotinylated βGAL-coated quartz surface through UV FRET has been recorded using deep UV laser-based fluorescence lifetime microscopy. The results demonstrate that (a) deep UV laser-based fluorescence lifetime microscopy is a simple and useful method to study UV FRET of proteins using intrinsic fluorescence, (b) structural information even in complex multidonor systems can be obtained, and (c) FRET signals can be obtained to detect binding events using the native fluorescence of proteins as multidonor systems.
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Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use... more
Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the protein's diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.
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We demonstrate that a recently developed coating comprised of silicone nanofilaments can be selectively functionalized to yield well defined superhydrophobic, superhydrophilic, superoleophobic or superoleophilic domains on a single... more
We demonstrate that a recently developed coating comprised of silicone nanofilaments can be selectively functionalized to yield well defined superhydrophobic, superhydrophilic, superoleophobic or superoleophilic domains on a single substrate, constituting a simple and versatile ...
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In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that clusters consisting of up to several hundreds of protein monomers can form in solution even at low monomer concentrations and subsequently... more
In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that clusters consisting of up to several hundreds of protein monomers can form in solution even at low monomer concentrations and subsequently adsorb to the surface. Using FRET ...
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We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group... more
We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group containing silanes, silicone nanofilament templates were chemically modified to mimic anionic and cationic exchange resins. Investigations on the selectivity of the functionalized surfaces toward adsorption of charged model proteins were carried out by means of fluorescence techniques. Due to a high contact area resulting from the nanoroughness of the coating, excellent protein retention characteristics under various conditions were found. The surfaces were shown to be highly stable and reusable over several retention-elution cycles. Especially the full optical transparency and the possibility to use glass substrates as support material open new opportunities for the development of optical biosensors, open geometry microfluidics, or lab-on-a-chip devices.
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New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of "multiplex dyes," we have designed rhodamine dyes with nearly identical absorption and... more
New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of "multiplex dyes," we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.
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We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic... more
We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the waterglass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.
Research Interests: Biomedical Engineering, Microscopy, Fluorescence Microscopy, Biomedical Optics, Luminescence, and 10 moreInterfaces, Optical physics, Molecules, Optometry and Ophthalmology, Image Enhancement, Optics and Photonics, Reproducibility of Results, Sensitivity and Specificity, Equipment Design, and Equipment Failure Analysis
For a better understanding of the adsorption behaviour of biomolecules the surface tension parameters of surface-coatings for biological applications are determined by contact angle measurements. The correlation between surface tension... more
For a better understanding of the adsorption behaviour of biomolecules the surface tension parameters of surface-coatings for biological applications are determined by contact angle measurements. The correlation between surface tension parameters and chemical composition of thin films made of cellulose derivatives with varying functional groups and amino-functional thin films with varying backbone on glass cover slips is investigated. Calculations are
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We investigate nonspecific protein adsorption processes by comparing experimentally measured adsorption kinetics of beta-lactoglobulin with mathematical models. The adsorption and desorption behavior of this protein on a hydrophilic glass... more
We investigate nonspecific protein adsorption processes by comparing experimentally measured adsorption kinetics of beta-lactoglobulin with mathematical models. The adsorption and desorption behavior of this protein on a hydrophilic glass surface in citrate buffer (pH 3.0), monitored for a large set of different bulk concentrations (0.5x10(-8) M-1.5x10(-6) M) using a supercritical angle fluorescence (SAF) biosensor, is reported. Increasing adsorption rates and overshootings in the beginning of the adsorption are observed as well as a transition to an almost irreversibly bound state of the protein in the long term. Furthermore, rinsing experiments prove that adsorbed proteins abruptly change their desorption behavior from irreversible to reversible when a critical surface coverage theta(crit) is reached. Based on all experimental observations, a mathematical model composed of three adsorbed states differing in their surface affinity is proposed. Terms to account for lateral interactions between surface-bound proteins are included, which yield an excellent fit of the measured kinetics. For the first time, several phenomena that have been discussed in theoretical studies are confirmed by comparing experimental data with a single model.
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Research Interests: Analytical Chemistry, Biomedical Engineering, Water, Kinetics, Glass, and 14 moreNanotechnology, Optical Design, Real Time, Surface Properties, Reproducibility of Results, Microchemistry, Reaction Rate, Collection Efficiency, Sensitivity and Specificity, Equipment Design, Equipment Failure Analysis, Antigens, Refractometry, and Biosensing Techniques
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We have performed extensive studies on the long term chemical stability of a superhydrophobic coating comprised of silicone nanofilaments. Durability was tested by immersion in various liquid media over a period of 6 months. The coating... more
We have performed extensive studies on the long term chemical stability of a superhydrophobic coating comprised of silicone nanofilaments. Durability was tested by immersion in various liquid media over a period of 6 months. The coating properties were monitored by static contact angle and sliding angle measurements. Changes in surface topography were examined by scanning electron microscopy. The coatings show
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Particle tracers are of interest in the investigation of fractured media. Because their transport behavior Mers from that of solute tracers, they supply complementary information on fracture space. This infomation is crucial in the risk... more
Particle tracers are of interest in the investigation of fractured media. Because their transport behavior Mers from that of solute tracers, they supply complementary information on fracture space. This infomation is crucial in the risk assessment of underground hazardous waste ...
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Research Interests: Chemical Engineering, Analytical Chemistry, Fluorescence, Fluorescence Microscopy, Scanning Electron Microscopy, and 17 moreNanotechnology, Fluorescence Correlation Spectroscopy, Protein-Protein Interaction, Fluorescence Lifetime, Porosity, Lasers, Escherichia coli, Animals, Nanostructures, Analytical, Proteins, Tryptophan, Cattle, Chickens, Photons, Time Factors, and Protein Binding
An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle... more
An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).
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ABSTRACT
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