Biomedical subjects

Ulrich J Krull

Publications and source records attributed to Ulrich J Krull.

13 recordsLinked to original sources

PubMed2006

Adsorption and hybridization of oligonucleotides on mercaptoacetic acid-capped CdSe/ZnS quantum dots and quantum dot-oligonucleotide conjugates.

Interest in the unique optical properties of quantum dots (QDs) has resulted in the development QD-bioconjugates for imaging and diagnostics. Although these applications are numerous, considerably less is known about the interactions between QDs and biomolecules. In this work, we describe hydrogen-bonding interactions between oligonucleotides and CdSe/ZnS quantum dots capped with mercaptoacetic acid ligands. The strength of the interactions can be modulated by changes in the pH and ionic strength, the addition of formamide, and differences between ssDNA and dsDNA. Fluorescence resonance energy transfer experiments have shown that conjugated oligonucleotides adopt a conformation that lies across the surface of the QD. The hydrogen-bonding interactions also affect the kinetics of hybridization with QD-DNA conjugates and the thermal stability of QD-conjugated dsDNA. The former is analogous to conventional solid-phase hybridization, where stronger oligonucleotide adsorption leads to faster kinetics. With respect to the latter, interactions with the QD surface can sharpen the melt transition and alter the melt temperature of dsDNA. These effects are largely absent when adsorptive interactions are minimized.

Adsorption↗

PubMed2006

Fluorescence resonance energy transfer and complex formation between thiazole orange and various dye-DNA conjugates: implications in signaling nucleic acid hybridization.

Fluorescence resonance energy transfer (FRET) was investigated between the intercalating dye thiazole orange (TO), and the dyes Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black FQ (IabFQ), and Iowa Black RQ (IabRQ), which were covalently immobilized at the end of dsDNA oligonucleotides. In addition to determining that TO was an effective energy donor, FRET efficiency data obtained from fluorescence lifetime measurements indicated that TO intercalated near the middle of the 19mer oligonucleotide sequence that was used in this study. Discrepancies in FRET efficiencies obtained from intensity and lifetime measurements led to the investigation of non-fluorescent complex formation between TAMRA and modified TO. The hydrophobicity of TO was modified by the addition of either an alkyl or polyethylene glycol (PEG) side-chain to study effects of dimer and aggregate formation. It was found that at stoichiometric excesses of modified TO, fluorescence quenching of TAMRA was observed, and that this could be correlated to the hydrophobicity of a TO-chain species. The TAMRA:TO-chain association constant for the TO-alkyl system was 0.043+/-0.002 M(-1), while that obtained for the TO-PEG was 0.037+/-0.002 M(-1). From the perspective of method development for the transduction of hybridization events, we present and evaluate a variety of schemes based on energy transfer between TO and an acceptor dye, and discuss the implications of complex formation in such schemes.

Biosensing Techniques↗

PubMed2005

Surface characterization of 3-glycidoxypropyltrimethoxysilane films on silicon-based substrates.

Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF-SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50-100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.

DNA Probes↗

PubMed2005

Electrokinetically based approach for single-nucleotide polymorphism discrimination using a microfluidic device.

In this work, we describe and implement an electrokinetic approach for single-nucleotide polymorphism (SNP) discrimination using a PDMS/glass-based microfluidic chip. The technique takes advantage of precise control of the coupled thermal (Joule heating), shear (electroosmosis), and electrical (electrophoresis) energies present at an array of probes afforded by the application of external electrical potentials. Temperature controllers and embedded thermal devices are not required. The chips can be easily and inexpensively fabricated using standard microarray printing methods combined with soft-lithography patterned PDMS fluidics, making these systems easily adaptable to applications using higher density arrays. Extensive numerical simulations of the coupled flow and thermal properties and microscale thermometry experiments are described and used to characterize the in-channel conditions. It was found that optimal conditions for SNP detection occur at a lower temperature on-chip than for typical microarray experiments, thereby revealing the importance of the electrical and shear forces to the overall process. To demonstrate the clinical utility of the technique, the detection of single-base pair mutations in the survival motor neuron gene, associated with the childhood disease spinal muscular atrophy, is conducted.

Cyclic AMP Response Element-Binding Protein↗

PubMed2005

Synthesis and fluorescence studies of thiazole orange tethered onto oligonucleotide: development of a self-contained DNA biosensor on a fiber optic surface.

Thiazole orange dyes were derivatized with ethylene glycol linkers of various lengths, and were covalently linked to the 5' end of the oligonucleotides after solid-phase synthesis. The labeled oligonucleotides exhibited enhanced fluorescence upon hybridization to complementary DNA sequences at the surfaces of optical fibers, providing for a self-contained labeling strategy. It was determined that the melt temperatures of DNA hybrids using one mixed polypyrimidine base oligonucleotide sequence were dependent on the length of the tethers, and that the melt temperature could be shifted by more than 10 degrees C when tethers were introduced.

Base Sequence↗

PubMed2005

Trends in the development of nucleic acid biosensors for medical diagnostics.

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.

Biosensing Techniques↗

PubMed2004

The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors.

Contamination of food and water supplies by microorganisms such as Escherichia coli, the need for point-of-care bedside analysis of biological samples, and concerns about terrorist attacks using biological organisms, have made the development of fast, reliable, and sensitive analytical methodologies for use in monitoring of pathogens very important. With a variety of biosensors being developed for extremely sensitive and rapid nucleic acid diagnostics, it has become even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample preparation. The application of ultrasound has the potential to create DNA fragments from genomic material with lengths that are suitable for determination using biosensors and microarrays. For example, application of 85 W power at a frequency of 20 kHz can produce a preponderance of fragments of 100-400 base pairs (bp) within several seconds, and sample processing can lead to over 75% conversion from genomic material to fragments in times of 20-30 s. A proportion of these fragments are in a single-stranded state and are suitable for hydridization with immobilized single-stranded DNA probe oligonucleotides using a fiber optic biosensor. Control of factors such as salt concentration, exposure time, ultrasound power, and the initial temperature of the solution, can affect the length and form (single- or double-stranded) of DNA fragments that are generated by ultrasound, and average fragment length can be adjusted by selection of these operating parameters.

Biosensing Techniques↗

PubMed2004

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1-1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10-100-fold), permitting for the completion of measurements in under 1 min.

Biosensing Techniques↗

PubMed2003

Modeling of DNA hybridization kinetics for spatially resolved biochips.

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.

Biosensing Techniques↗

PubMed2003

Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones.

The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, alpha-crystallin and reticulocyte lysate (RL) in bulk solution were found to re-activate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors.

Animals↗

PubMed2003

Fluorescence polarization spectroscopy in protein analysis.

Information about concentration, molecular structure, binding events, and motion can be obtained using fluorescence spectroscopy methods. Fluorescence polarization spectroscopy is one such method, which studies the relationship between the polarization of light that is used for excitation and light that is subsequently detected from fluorescence. The extent of change of polarization between excitation and emission can be used to study physical processes such as rotational diffusion, extent of denaturation, and orientation at surfaces. In this article Mann and Krull describe the underlying principles to the technique and show how fluorescence polarization spectroscopy has contributed to protein analysis.

Animals↗

PubMed2003

Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage.

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Animals↗

PubMed2002

An AFM determination of the effects on surface roughness caused by cleaning of fused silica and glass substrates in the process of optical biosensor preparation.

The covalent attachment of organic films and of biological molecules to fused silica and glass substrates is important for many applications. For applications such as biosensor development, it is desired that the immobilised molecules be assembled in a uniform layer on the surface so as to provide for reproducibility and speed of surface interactions. For optimal derivatisation the surface must be appropriately cleaned to remove contamination, to create surface attachment sites such as hydroxyl groups, and to control surface roughness. The irregularity of the surface can be significant in defining the integrity and density of immobilised films. Numerous cleaning methods exist for fused silica and glass substrates and these include gas plasmas, and combinations of acids, bases and organic solvents that are allowed to react at varying temperatures. For many years, we have used a well established method based on a combination of washing with basic peroxide followed by acidic peroxide to clean and hydroxylate the surface of fused silica and glass substrates before oligonucleotide immobilisation. Atomic force microscopy (AFM) has been used to evaluate the effect of cleaning on surface roughness for various fused silica and glass samples. The results indicate that surface roughness remains substantial after use of this common cleaning routine, and can provide a surface area that is more than 10% but less than 30% larger than anticipated from geometric considerations of a planar surface.

Biosensing Techniques↗