Showing papers by "Stefan Seeger published in 1995"

New fluorescent dyes in the red region for biodiagnostics.

Abstract: The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

An All-Solid-State Flow Cytometer for Counting Fluorescent Microspheres

Abstract: Particle tracers are of interest in the investigation of fractured media. Because their transport behavior differs from that of solute tracers, they supply complementary information on fracture space. This information is crucial in the risk assessment of underground hazardous waste storage sites. An easy-to-handle particle tracer consists of fluorescent polystyrene spheres with diameters on the order of 1 μm. Up to now, detection and counting of these particles was performed by fluorescence microscopy of filtered samples. To allow faster and on-line continuous monitoring of single particles, a flow cytometer has been developed in the present work. The cytometer is built from solid-state optical components, resulting in a robust and transportable system suitable for field use. To maximize the fluorescence quantum efficiency, a new dye, JA22, was used. Use of an avalanche diode allowed the efficiency of photon detection to be maximized. For complete digital data acquisition and analysis, only a personal computer is required. The detection efficiency of the cytometer is >25%, the probe flow is 20 mL/h, and the present accuracy is <160 particles/mL.

One-step immobilization of immunoglobulin G and potential of the method for application in immunosensors

Abstract: Stable monolayers of antibodies incorporated in a polymer monolayer are prepared on the subphase of a Langmuir-Blodgett trough. Both copolyglutamate (Cpg) and photopolymerized phthalocyaninato-polysiloxane (Pcps) derivatives are ideal matrices for embedding immunoglobulin G (IgG). One-step immobilization of the biofilm onto several substrates is achieved by the vertical dipping technique. So far 30 optical waveguides can be coated within 10 min. Polymethyl methacrylate (PMMA) is found to be a suitable substrate for obtaining highly reproducible transfer ratios. Enzyme-linked immunosorbent assays are performed to determine the density and stability of active immobilized antibodies and to estimate specificity with regard to the suitable antigen. The specific binding capacity is comparable to that of the widely used silanization procedure via aminoalkyltrialkoxysilanes. First successful tests of the irradiation of the mixed photopolymer-immunoglobulin G layers are reported. Photochemical crosslinking is monitored by the film area decrease with irradiation time and both the density and stability of active immobilized antibodies are significantly enhanced in comparison with less or non-crosslinked films. Using an evanescent wave biosensor, laser diode-induced fluorescence emission is detected to observe the reaction of the immobilized antibodies with antigens linked to a fluorescent dye.

Use of diode lasers in an evanescent wave immunosensor for stationary and time-resolved detection of antigens

Abstract: A compact and inexpensive fiber-optic evanescent wave immunosensor is presented which uses a diode laser as excitation source both in continuous wave measurements as well as in time-resolved experiments. The continuous wave laser operates at 657 nm utilizing a commercially available dye-conjugated polyclonal antibody. Time-resolved experiments with a diode laser emitting at 674 nm were carried out with two fluorescent dyes, which have nearly identical absorption and emission wavelengths, but differ in their fluorescence lifetimes. This shows the possibility of detecting several molecules simultaneously, which are tagged with so-called ‘multiplex dyes’, by identifying the different fluorescence lifetimes. Using a new rhodamine dye a successful test of observing the time-resolved binding process between immobilized antibodies and dye-conjugated antigens is reported.

Ultrathin antibody networks for detection of antigens

Abstract: Most affinity sensor principles demand immobilized receptor molecules. A widely used multiple-step technique for covalent immobilization of proteins is based on a silanization procedure and a crosslinking agent, e.g., glutardialdehyde. In the presented work non- amphophilic Langmuir-Blodgett films based on phthalocy-aninato-polysiloxane derivatives or copolyglutamates are used to immobilize antibodies as monolayers. Due to a photopolymerization process of terminal C-C-double bonds at alkylic side chains of the non- amphophilic substance the stability of the films is improved compared to conventional LB films. Antibodies can be immobilized by a one-step-method subsequent to illumination. Polymethyl-methacrylate (PMMA) fibers are found to be ideal substrates for the antibody transfer. Enzyme-linked immunosorbent assays (ELISA) give quite good specificities and densities of active immobilized immunoglobulin (IgG). For antigen detection these films are combined with compact evanescent wave sensors. Antibodies were immobilized at the surface of a waveguide using the Langmuir-Blodgett (LB) technique. The light source is a diode laser, which results in a small and compact set-up and due to the red excitation wavelength the background fluorescence is reduced.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.