Alfred Kick

Surface plasmon resonance platform technology for multi-parameter analyses on polymer chips

The development of a surface plasmon resonance (SPR) spectrometer comprising angular‐resolved analysis for quasi‐monochromatic illumination is reported. The optical system utilizes disposable, injection‐molded chips combined with a lateral imaging optical system for parallel analysis of one‐dimensional spot arrays. Further parallelization is achieved by introducing a segmented light source. This source sequentially illuminates three neighbored one‐dimensional arrays in order to keep angular‐resolved analysis without introducing any mechanically moving parts. This system is applied to detect genetic variations among different DNA samples obtained from polymerase chain reaction (PCR). For this purpose, 135 spots on the chip surface have been prepared by spotting and analyzed separately.

DNA microarrays for hybridization detection by surface plasmon resonance spectroscopy

We report on the development of a new platform technology for the detection of genetic variations by means of surface plasmon resonance (SPR) spectroscopy. TOPAS chips with integrated optics were exploited in combination with microfluidics. Within minutes, the detection of hybridization kinetics was achieved simultaneously at all spots of the DNA microarray. A nanoliter dispenser is used to deposit thiol-modified single-stranded probe DNA on the gold surface of the chips. We investigated the influence of different parameters on hybridization using model polymerase chain reaction (PCR) products. These PCR products comprised a single-stranded tag sequence being complementary to an anti-tag sequence of probes immobilized on the gold surface. The signals increased with increasing length of PCR products (60, 100 or 300 base pairs) as well as with their concentration. We investigated hybridizations on DNA microarrays comprising 90 spots of probe DNA with three different sequences. Furthermore, we demonstrate that sequences with possible hairpin structures significantly lower the binding rate, and thus, the SPR signals during hybridization.

Electronic Structure of Genomic DNA: A Photoemission and X-ray Absorption Study

The electronic structure of genomic DNA has been comprehensively characterized by synchrotron-based X-ray absorption and X-ray photoelectron spectroscopy. Both unoccupied and occupied states close to the Fermi level have been unveiled and attributed to particular sites within the DNA structure. A semiconductor-like electronic structure with a band gap of approximately 2.6 eV has been found at which the pi and pi* orbitals of the nucleobase stack make major contributions to the highest occupied and lowest unoccupied molecular orbitals, respectively, in agreement with previous theoretical predictions.

A novel platform technology for the detection of genetic variations by surface plasmon resonance

We report on a novel approach to identify genetic variations based on the detection of specific polymerase-chain-reaction products by surface plasmon resonance. We use a recently developed, home-made spectrometer which exploits chips with integrated optics and microfluidics. The gold film at the chip surface is locally functionalized with single-stranded, thiol-modified probe DNA by applying a nanoliter dispenser. We discuss the chemical conditions for the achievement of maximum SPR signal strength and spot array density, and evaluate the SPR detection of selective binding of a set of 17 PCR products.

EGNAS: an exhaustive DNA sequence design algorithm

BackgroundThe molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA) is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences) offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences.ResultsThe algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented.ConclusionsWe developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use athttp://www.chm.tu-dresden.de/pc6/EGNAS.

Rapid detection of DNA hybridization on surface plasmon resonance based microarrays

The detection of DNA hybridization in medical diagnostics ought to be rapid, sensitive and specific. A platform technology based on surface plasmon resonance (SPR) is presented. We use TOPAS R chips with integrated optics and combined with microfluidics. Applying a nanoliter dispenser, thiol-modified single-stranded probe DNA (anti-tag) is deposited on the gold surface of the chips to create a DNA microarray. We fabricate chips with sufficiently high probe density, which is a key factor for DNA chips and can be controlled by adding MgCl2 to the immobilization solution. This technology offers the possibility of detecting PCR products comprising a single-stranded tag sequence being complementary to an anti-tag sequence of immobilized probes on the microarray. Consequently, this universal platform can be applied for detection of DNA hybridization based on the tag/anti-tag system. We demonstrate detection of specific hybridization of different 300 base pairs-long PCR products by SPR within less than five minutes. We checked for expected cross hybridizations with seven base pairs-long sequences at different positions in the tag/anti-tag sequence. Depending on the distance to the sensor surface we could observe crosshybridization if the according complementary sequence part is more distant from the surface. The initial binding rates (response units/min) at different PCR product concentrations were determined. Within five minutes a PCR product concentration of 2.6 nM is sufficient for distinct detection without crosshybridizations.

Tunable Fluorescence of a Semiconducting Polythiophene Positioned on DNA Origami

A novel approach for the integration of π-conjugated polymers (CPs) into DNA-based nanostructures is presented. Using the controlled Kumada catalyst-transfer polycondensation, well-defined thiophene-based polymers with controllable molecular weight, specific end groups, and water-soluble oligoethylene glycol-based side chains were synthesized. The end groups were used for the easy but highly efficient click chemistry-based attachment of end-functionalized oligodeoxynucleotides (ODNs) with predesigned sequences. As demonstrated by surface plasmon resonance spectroscopy, the prepared block copolymers (BCPs), P3(EO)3T-b-ODN, comprising different ODN lengths and specific or repetitive sequences, undergo specific hybridization with complementary, thiol-functionalized ODNs immobilized on a gold surface. Furthermore, the site-specific attachment of the BCPs to DNA origami structures is studied. We demonstrate that a nanoscale object, that is, a single BCP with a single ODN handle, can be directed and bound to the DNA origami with reasonable yield, site-specificity, and high spatial density. On the basis of these results, we are able to demonstrate for the first time that optical properties of CP molecules densely immobilized on DNA origami can be locally fine-tuned by controlling the attractive π-π-stacking interactions between the CPs. In particular, we show that the fluorescence of the immobilized CP molecules can be significantly enhanced by surfactant-induced breakup of π-π-stacking interactions between the CPs backbones. Such molecular control over the emission intensity of the CPs can be valuable for the construction of sophisticated switchable nanophotonic devices and nanoscale biosensors.

Polymer microarrays for surface plasmon resonance based sensors

Surface plasmon resonance spectroscopy (SPR) is used to detect pH changes in acidic solutions. SPR is an optical, label-free method for highly sensitive detection of refractive index changes. A microarray of pH-sensitive polymers, using poly(2-vinylpyridine) (P2VP) and poly(4-vinylpyridine) (P4VP), as recognition elements, is investigated on a gold surface. Swelling and protonation of the polymers lead to refractive index changes at the sensor surface. For the first time, a polymer microarray in a microfluidic channel is used to detect pH values smaller than 5 by SPR in aqueous hydrochloric acid and buffered solutions of different ionic strength. Every polymer has a distinct pH range where it shows its highest pH sensitivity. pH measurements are performed in a range between pH 0.75 and 5. The ionic strength influences the swelling behavior and the pH sensitivity of the polymers. The applied polymers are pH-selective in the used buffers containing sodium, phosphate, citrate, potassium and chloride ions.

DNA-Sequenzauswahl und ihr Einfluss auf die Hybridisierung auf SPR-Mikroarrays

In dieser Arbeit wird auf Grundlage experimenteller Ergebnisse an einem computergenerierten Modellsystem gezeigt, wie DNA-Sequenzen die Detektion von Hybridisierungen auf einem DNAMikroarray beeinflussen. Die Ergebnisse zeigen, dass neben Kreuzhybridisierungen auch Sekundarstrukturen (z. B. Haarnadelstrukturen) vermieden werden. Anderenfalls verringert sich die Empfindlichkeit und Nachweisgrenze des Sensors. Die vorgestellten Untersuchungen beruhen auf dem Messprinzip der Oberflachenplasmonenresonanzspektroskopie (SPR). Es ist eine markierungsfreie Methode. Die Sensorschicht besteht aus einem 50 nm dicken Goldfilm, auf dem einzelstrangige und thiolmodifizierte SondenDNA immobilisiert ist. Die Adresssequenzen dieser Sonden werden als Anti-TAGs bezeichnet. Detektiert wird die Hybridisierung von einem Produkt der Polymerase-Kettenreaktion (PCR). Das PCR-Produkt besteht aus einer 300 Basenpaar-langen Doppelhelix mit einem 25 Basen-langen einzelstrangigen Uberhang (TAG), der komplementar zu einer Anti-TAG-Sequenz ist (TAG/Anti-TAG-System).

Entwicklung polymerbeschichteter DNA-Chip-Oberflächen für enzymatisch unterstützte Genotypisierungen

Die DNA-Chiptechnologie bildet einen innovativen und effizienten Ansatz in der genetischen Diagnostik. Fur diese Technologie stellen wir die Entwicklung von Polymerbeschichtungen auf Glasoberflachen vor, welche die Analyse geringer DNA-Mengen durch die Methode der ALR (Arrayed Ligation Reaction) ermoglicht. Dieses Verfahren wurde fur den spezifischen Nachweis von Schimmelund Hausfaulepilzen bei der Biotype Diagnostic GmbH etabliert (Tafel 1). Neben der Hybridisierung komplementarer DNA-Strange findet dabei eine spezifische Enzymreaktion statt, bei der ein fluoreszenzmarkiertes DNA-Oligonukleotid kovalent an immobilisierte DNA-Sonden des Mikroarrays und damit an den DNA-Chip gebunden wird (Bild 1). Diese Ligationsreaktion findet jedoch nur statt, wenn die DNA-Zielsequenz zur Sonde und zum Ligationsoligonukleotid komplementar ist. [1-3]