The understanding of processes occurring at the interface between two media are of prior importance in various fields of research, from material sciences to biology. A custom-made microscope objective based on the supercritical angle technique was developed in our group, allowing to probe these interfacial events by carrying out surface-sensitive and low invasive spectroscopy of aqueous samples. A biological example of particular interest is the comprehension of neurodegenerative diseases which seem caused by the interaction of specific peptides with the membrane of the neurons. Taking advantage of our optical setup, we used supercritical angle fluorescence spectroscopy to specifically monitor the interaction between a supported lipid bilayer (SLB) and the Amyloid β peptide, notably responsible of the Alzheimer disease. Different forms of the peptide (40 and 42 amino acids composition) were tested and the interfacial fluorescence measured to get information about the lipid integrity and mobility. The adsorption of the peptide was also characterized in terms of kinetic and affinity.
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 water/glass 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.
Total internal reflection fluorescence (TIRF) microscopy is a powerful technique to investigate surface bound emitters exclusively, i.e. with low interference of the fluorescing bulk in the adjacent aqueous solution. Confocal microscopy is a powerful technique to detect low emission intensities, like the emission of a single molecule, with high temporal resolution and high signal-to-noise ratio. We present a confocal total internal fluorescence microscope, which combines the virtues of diffraction limited confocal imaging and TIRF. Annular supercritical focusing and fluorescence collection through standard glass cover slips is accomplished by a parabolic mirror lens. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below attoliters. Beyond, the large aperture of the element leads to a high collection efficiency of surface bound emitters (~50%). The system is characterized in detail by calculations of the electric fields in the focus region and simulated confocal imaging. Single molecule experiments demonstrate the performance of the microscope in practice.
In recent years, laser induced fluorescence detection and spectroscopy of single molecules (SMD) has seen a tremendous development. Besides fundamental research on individual molecular systems, practical applications of this sensitive detection technique for analytical and diagnostic purposes are becoming more and more important. For a wider applicability of the SMD technique it is desirable to detect not only the presence or absence of a molecule within a given detection volume,, but also t be able to quantify this fluorescence absolutely. Modified flow cytometry system for SMD that are ensuring such a quantifiable fluorescence detection of single molecules are already successfully applied for e.g. DNA fragment sizing. In our talk, we present a modified confocal detection set-up for performing SMD on surfaces that aims at a quantified detection of single molecule fluorescence. First experimental results presented and emerging problems are discussed.
It is shown here that an optical tweezer can successfully be employed to study interactions between coated microbeads and either biological surfaces, viz. membranes of neuronal cells, or artificial surfaces derivatized with amino acids. For biological applications, polystyrene beads of a diameter of 3 micrometer were coated with different proteins of the Extracellular Matrix (ECM) and brought into contact with glial cells and neurons. By comparing interaction forces, effects of bacterial phospholipase C, formation of membrane threads and surfing distances of bound ECM protein coated beads on cells we demonstrate a different anchorage of the tenascin-C receptor in primary neurons and glia. To study interactions at artificial surfaces with optical tweezers, polystyrene beads with a diameter of 10 micrometer were coated with different amino acids. Friction coefficients were measured between these beads and glass surfaces comprising amino acids coupled to well-defined layers of cellulose derivatives produced by the Langmuir-Blodgett technique. The technique provides an easy method for friction measurements and the characterization and quality control of surfaces. We conclude that optical tweezers are a powerful tool for the characterization of biological and artificial surfaces.
We present a comparison between two basically different optical detection systems: a confocal epifluorescence microscope, and a new evanescent wave detection system employing a parabolic optical element. In a microscope set-up, fluorescence light is collected within a cone around the optical axis, whereas in the evanescent light detector, fluorescence light is collected mainly at angles larger than the so-called critical angle of total internal reflection. Based on a thorough theoretical modeling of both experimental set-ups, comparison between the two detection systems is made Particularly, the optical detection efficiency is compared. 5
We describe a new optical system for the detection of laser- induced fluorescence of surface bound molecules. The system consists of two optical elements: a paraboloid glass segment, and a spherical long focal lens. The function of the paraboloid glass segment is twofold: First, due to jump in refractive index at the water-glass interface, surface bound molecules emit the major part of their fluorescence into the paraboloid. Second, the parabolic shape of the segment services as a collecting mirror. We demonstrate that with this simple system an extremely high light collection efficiency can be achieved, without using sophisticated and expensive objectives with high numerical aperture. Ray- tracing calculations show that the system is able of confocal imaging, which is very important for efficient suppression of scattered excitation light. Experimental result showing the exquisite performance and sensitivity are presented.
Recognition molecules play important regulatory roles during pattern formation of the nervous system. We here show that optical tweezers can successfully be used to functionally and quantitatively compare neuron interactions with glycoproteins of the extracellular matrix. We measured the forces of interactions between tenascin-C, laminin-1 and fibronectin coated polystyrol beads and the cell membranes of different nervous cell-types. This was achieved by applying the optical tweezers as picotensometer to pull the adhering beads with varying axial forces. To this aim, the minimal laser powers for capturing polystyrene beads in different solutions were measured. With the corresponding Archimedes Forces the acting forces in the range of pN were calculated. The glycoproteins showed significantly different behavior. Specific binding was shown by antibody experiments, in which the binding forces were significantly reduced for the corresponding antigens but not for the other glycoproteins. Together with experiments concerning the moving within the plane of the membrane and the cleavage with glycosyl-phosphatidyl-inositol-anchors specific phospholipase-C we suggest that these glycoproteins interact with neurons by different mechanisms.
The fluorescence-based detection of single dye labeled protein molecules at interfaces is presented. Glass substrates with covalent immobilized antibodies serve for capturing matching antigens from volume concentrations between 10-12 and 10-17 mol/l. The unspecific binding at the interface has been reduced to a level down to 0.1% of the maximum signal level. At concentrations lower than 10-13 mol/l we observe single antibody-antigen complexes at the surface. We developed a scanning method for counting single antibody- antigen complexes. The counting results are used for calibrating the volume concentration dependency. AT the present stage, the detection limit of this molecule counting process is of the order of 10-17 mol/l, and the dynamic range detectable antigen concentration is more than 8 orders of magnitude, without reaching a limiting value. The instrumental set-up is similar to that of a confocal microscope. A diode laser is used as an excitation source. As an first application in early-stage-diagnostics, we investigated the detection of a single cardiac-actin molecule in human plasma.
Non specific localization of photosensitizers after application in vivo limits progress in PDT. Relatively selective distribution of photosensitizers in malignant tissues is crucial for a successful treatment. The target specificity of photosensitizers may be improved by linking photosensitizers with monoclonal antibodies. In this approach, a high specific monoclonal antibody, BM-2 which is directed against epitopes of the mucine glycoprotein TAG-12 was used. This antibody shows reactivity with 96% of all primary breast carcinomas. BM-2 was conjugated with a second generation phthalocyanine photosensitizer which is only weakly phototoxic to human T-47D tumor cells without conjugation in vitro. With respect to future clinical application, illumination times from 25 to 100 minutes and a powerful diode laser with an emission of 690 nm was chosen, which provides deeper tissue penetration in vivo. We observed phototoxicity towards T-47D human breast carcinoma cells at concentrations ranging from 0.25 to 6 micromol/L and light doses from 6 to 24 J/cm2. The immunoconjugates discriminated mucine-positive and mucine-negative tumor cells and showed high photocytotoxic selectivity towards mucine-positive T-47D cells in vitro. The conjugate showed no dark toxicity. In vivo experiments will follow.
The immobilization techniques presented represent an innovative method for the highly specific determination of antigens and other proteins in a very short time and may be a useful tool for the industrial preparation of highly sensitive biosensors. Non amphiphilic alkylated cellulose films are used as matrices to reduce non-specific interactions with biomolecules. These films are transferred onto waveguides by using the Langmuir-Blodgett- technique. After photochemical stabilization the cellulose films serve as excellent matrices for the immobilization of proteins at high density. Furthermore, one-step immobilization of a mixed photopolymer antibody film with a continuously operating LB-trough is demonstrated. The activity and specificity of immobilized antibodies and streptavidin is controlled by an enzyme-linked immunosorbent assay (ELISA) and an evanescent wave immunosensor.
Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.
Optical sensors based on the utilization of the evanescent field arising at the interface between two media in the case of total internal reflection are an excellent tool for the reduction of time consuming and complex chemical analysis. We developed a fiber-optic based set-up with visible diode lasers as excitation sources. As recognition element an optical fiber covered with a photopolymerized antibody monolayer was used. Beside the commercially available cyanine fluorescent dye Cy 5, newly developed fluorescent dyes in the red spectral region were coupled to antibodies. In order to test the set-up in a clinically relevant system the antibodies BM-2 and BM-7 were chosen. With this antibody system the tumor marker mucine in a sandwich immunoassay was investigated. This protein shows increased concentrations in serum and ascites in the case of breast cancer. The combination of semiconductor devices and ultrathin antibody layers together with an antibody system directed against mucine offers the possibility of an on-line detection of the tumor marker.
For the production of recognition elements for evanescent wave immunosensors optical waveguides have to be coated with ultrathin stable antibody films. In the present work non amphiphilic alkylated cellulose and copolyglutamate films are tested as monolayer matrices for the antibody immobilization using the Langmuir-Blodgett technique. These films are transferred onto optical waveguides and serve as excellent matrices for the immobilization of antibodies in high density and specificity. In addition to the multi-step immobilization of immunoglobulin G(IgG) on photochemically crosslinked and oxidized polymer films, the direct one-step transfer of mixed antibody-polymer films is performed. Both planar waveguides and optical fibers are suitable substrates for the immobilization. The activity and specificity of immobilized antibodies is controlled by the enzyme-linked immunosorbent assay (ELISA) technique. As a result reduced non-specific interactions between antigens and the substrate surface are observed if cinnamoylbutyether-cellulose is used as the film matrix for the antibody immobilization. Using the evanescent wave senor (EWS) technology immunosensor assays are performed in order to determine both the non-specific adsorption of different coated polymethylmethacrylat (PMMA) fibers and the long-term stability of the antibody films. Specificities of one-step transferred IgG-cellulose films are drastically enhanced compared to IgG-copolyglutamate films. Cellulose IgG films are used in enzymatic sandwich assays using mucine as a clinical relevant antigen that is recognized by the antibodies BM2 and BM7. A mucine calibration measurement is recorded. So far the observed detection limit for mucine is about 8 ng/ml.
The detection of several substances in a mixture can be realized by using luminescent markers and discriminating them by their luminescence lifetimes. The number of simultaneously detectable analytes can be increased by extending the time region of the lifetimes of the dyes. The fluorescence lifetimes of commercial available organic dyes do not exceed 100 ns. Longer decays are found among the complexes of transition metals. Therefore the photophysical properties of a number of luminescent ruthenium(II) tris(alpha-diimine) complexes in air equilibrated aqueous buffer solution were measured at room temperature. Their application as luminescence markers in diagnostics and analytics is discussed. The possibility of emission enhancement by addition of detergents to reduce water quenching and by addition of sodiumsulfite to reduce oxygen quenching was investigated. Some of the ruthenium compounds were encapsulated in carboxylated polystyrol beads. Resulting changes of the luminescence decays are presented. The possibility of increasing sensitivity by multilabeling with dyed beads in analytical applications is discussed.
Evanescent wave sensors are an interesting tool for the fast detection of reactions between biomolecules. By using small and inexpensive diode lasers together with fluorescent dyes in the red spectral region, it was possible to construct a highly specific sensor. This was due to the use of the immobilization of receptor molecules via the Langmuir-Blodgett technique which enhances the specificity drastically. As a clinically relevant system an immunoassay of the tumor marker mucine was investigated. Mucine could be detected with a sandwich test using the antibody system BM-2/BM-7. In order to detect several analytes at one time, there are two possible ways: time-resolved detection using multiplex-dyes and the parallelization of the sensor by using several fibers simultaneously. A position-resolved evanescent wave sensor using a CCD camera is described.
Information on the pH directly on surfaces of dental enamel is an important aspect in research on tooth decay. As an alternative to pH-electrodes our approach to the problem is the optical determination of pH by pH sensitive fluorescent dyes immobilized to tooth surfaces. In this study a model for measuring pH either on aminated cellulose substrates or on enamel (in vitro) with a fluorescein type dye is presented. The experimental realization is a fiber optic sensor with a nitrogen-pumped dye laser system and photodiode for the detection of the emitted fluorescence light. The surface pH values in the range between 4 and 7 were derived from the ratios of the excitation bands at 490 nm and 460 nm.
A drug screening assay based on patterned cells was developed. The patterning was performed by detaching single lymphocytes with laser tweezers from a poly-ethylene oxide (PEO) surface and immobilizing them on a surface coated with Cell-TakR, a strong cellular adhesive. The detachment force of the cells from the PEO-surface was determined on a single cell base with the laser tweezers to be between 1.5 and 4.5 pN. Different lymphocyte classes were loaded with the fluorescent calcium indicator Fluo-3 and the pH indicator BCECF. Specific stimulation of the immobilized cells is monitored.
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.
Markus Sauer, Kyung-Tae Han, Volker Ebert, Ralph Mueller, Andreas Schulz, Stefan Seeger, Juergen Wolfrum, Jutta Arden-Jacob, Gerhard Deltau, Nicolaas Marx, Karl-Heinz Drexhage
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 fluorescence lifetimes. In order to influence the excited state lifetime without changing the spectral characteristics we modified rhodamines with non- conjugated substituents that promote non-radiative transitions. First investigations with covalently coupled biomolecules show the potential of multiplex dyes in DNA sequencing and antigen detection. The biomolecules are identified through the intrinsic fluorescence lifetime of the dye. This principle offers the possibility to make use of different fluorescence lifetimes in each wavelength region. Therefore the number of discernable tags is greatly enhanced. 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 absorption and emission beyond 600 nm and have a high fluorescence quantum efficiency, even in aqueous buffer systems. Time-resolved and intensity measurements of the dyes covalently coupled to a synthetic oligonucleotide are presented. The results obtained in different capillary systems using laser diodes as excitation sources show the potential of these dyes in the red region of the spectrum.
A new bioanalytical concept that uses the characteristic fluorescence lifetimes of new multiplex dyes as an identification parameter is presented. Several dyes can be distinguished at the same wavelength by recognition of the fluorescence lifetimes. It is shown that the identification can be achieved in a capillary gel electrophoresis system in a few milliseconds with a misclassification rate of 10-4. As an example the simultaneous detection of two antibodies at one wavelength is presented.
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