Biomedical subjects

Thomas Ruckstuhl

Publications and source records attributed to Thomas Ruckstuhl.

11 recordsLinked to original sources

PubMed2006

Conformational reorientation of immunoglobulin G during nonspecific interaction with surfaces.

To achieve a better understanding of the nonspecific adsorption process of proteins on solid surfaces, the mechanism of this interaction was investigated by a model system comprising the structurally flexible ("soft") protein goat anti-rabbit immunoglobulin G and a set of chemically defined surfaces. The thermodynamic properties of both protein and surfaces were derived from contact angle measurements by applying the Lifshitz-van der Waals acid-base approach, and the Gibbs free enthalpy of interaction was calculated. The protein shows two conformational states, one hydrophobic and the other hydrophilic. The interaction energy indicates that the hydrophobic conformation favorably adsorbs onto the surfaces. With real-time binding kinetics, measured by a supercritical angle fluorescence biosensor, we show that during the nonspecific adsorption the protein performs a reorientation in its three-dimensional amino acid structure from a hydrophilic to a hydrophobic molecular structure. Unlike the rates of adsorption and desorption, the transition rate is independent of the type of surface and only influenced by the structural reorganization of the protein.

Adsorption↗

PubMed2005

Towards single-molecule DNA sequencing: assays with low nonspecific adsorption.

New DNA sequencing techniques are currently being developed using single-molecule fluorescence-based detection of enzymatic double-strand synthesis. Such application requires surface architectures on which single-stranded templates can be immobilized. A further important attribute is a very low tendency to attract fluorescently labeled bases nonspecifically. On this account, the adsorption behaviour of Cy5-dNTPs on a variety of surface coatings was studied by performing real-time measurements of the DNA synthesis using a supercritical angle fluorescence biosensor. It is demonstrated that polyacrylic acid coatings are an excellent choice to minimize the nonspecific binding of the bases.

Adsorption↗

PubMed2005

Microscope objective for large-angle fluorescence used for rapid detection of single nucleotide polymorphisms in DNA hybridization.

A new type of microscope objective is used for the rapid detection of sequence-dependent affinity variations in DNA hybridization. We demonstrate that by performing probe/target hybridization on coverslips at room temperature terminal SNPs (single nucleotide polymorphisms) can be detected within seconds. The study of weak pair interaction, such as the association of very short DNA oligomers, requires the use of high analyte concentrations of both partners to generate a detectable amount of associated pairs. The background of high concentrations of unbound fluorescing analyte can easily hide the low signal of a weakly affine reaction and makes association extremely difficult to detect. Fluorescence detection is a powerful approach to analyze minute amounts of material, even single molecules, but it is usually limited to rather low concentrations. This limitation is now overcome due to the new type of microscope objective, which produces an extremely small detection volume at a water/glass interface.

Carbocyanines↗

PubMed2005

Confocal reader for biochip screening and fluorescence microscopy.

We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.

Biosensing Techniques↗

PubMed2004

Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence.

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

Base Pair Mismatch↗

PubMed2004

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

Biochemistry↗

PubMed2004

Real-time detection of polymerase activity using supercritical angle fluorescence.

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

Biosensing Techniques↗

PubMed2003

Sizing of single fluorescently stained DNA fragments by scanning microscopy.

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-L-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

Algorithms↗

PubMed2003

Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument.

We present a new optical biosensor for probing molecular binding to a water/glass interface. The system is designed to measure the kinetics of surface reactions down to low analyte concentrations straightforwardly. The selective detection of surface bound fluorescence is achieved by collecting supercritical angle fluorescence (SAF) emission of surface bound molecules into the glass. Thereby the expansion of the detection volume into the aqueous probe is reduced to about one sixth of the fluorescence wavelength, consequently bulk fluorescence from the solution is rejected successfully. The SAF-signal is captured by a parabolic glass lens, which leads to high spatial collection efficiency and detection sensitivity. The sensor has an inverted optical design and is compatible with common glass cover slips, which strongly facilitates operation for the user working in the biological and biochemical fields. The performance of the system is demonstrated by real time measurements of antibody-antigen reactions. Rate constants of the reaction were extracted. Antigen concentrations were detected down to 10(-13) mol/l.

Antigen-Antibody Complex↗

PubMed2003

Real-time detection of nucleotide incorporation during complementary DNA strand synthesis.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.

Base Sequence↗

PubMed2003

Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens.

We present a theoretical study of a new total-internal-reflection fluorescence microscope for the detection of fluorescence at a water-glass interface. The system is designed for confocal imaging and spectroscopy of nanoparticles and single molecules. Focusing and fluorescence collection through standard glass coverslips is accomplished by a parabolic mirror lens. The large aperture of the element is used to excite fluorescence within the evanescent field of a diffraction-limited focus and to collect focal emission with high efficiency. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below that of an attoliter (10(-18) L), which can be advantageous for monitoring surface binding of single molecules without interference from fluorescence of the unbound bulk. Calculations of the electric fields in the focus region and simulated confocal imaging demonstrate the performance of the system.

Journal Article↗