Kontrolle und Manipulation von DNA auf Au und deren Anwendungen für die Biosensorik

DNA molecules tethered to gold surfaces are of great importance for bio-sensor applications. The present work reports on methods to control and manipulate oligonucleotides on gold substrates with electric fields. Extremely smooth and stable gold surfaces are prepared and characterized. Novel methods to precisely adjust the DNA surface coverage are developed and subsequently applied in high-sensitivity bio-sensing experiments. Finally, the functional DNA layers are combined with micro-fluidic cells and it is demonstrated that the integrated setup allows for the label-free detection of proteins of biological interest.Auf Goldoberflächen verankerte DNA Moleküle sind von großer Bedeutung für Anwendungen in der Biosensorik. Die vorliegende Arbeit berichtet über Methoden Oligonukleotide auf Goldsubstraten mittels elektrischer Felder zu kontrollieren und zu manipulieren. Extrem glatte und stabile Goldoberflächen wurden hergestellt und charakterisiert. Es wurden neue Methoden entwickelt, um die DNA Oberflächenbedeckung präzise einzustellen; weiters werden diese Methoden in hoch-sensitiven Biosensorik Experimenten angewendet. Die entwickelten funktionellen DNA Schichten wurden mit mikro-fluidischen Zellen kombiniert und es wurde gezeigt, dass der integrierte Aufbau die label-freie Detektion von Proteinen von biologischer Bedeutung erlaubt

Electrical Manipulation of DNA on Metal Surfaces

Summary We review recent work on the active manipulation of DNA on metal substrates by electric fields. This includes the controlled positioning, alignment, or release of DNA on or into dedicated locations and the control of hybridization. In this context, we discuss techniques for immobilizing DNA on metal surfaces and methods of characterizing such hybrid systems. In particular, we focus on electrically induced, conformational changes of monolayers of short oligonucleotides on gold substrates. Such switchable layers allow for molecular dynamics studies at interfaces and have demonstrated large potential in label-free biosensing applications

Excessive Counterion Condensation on Immobilized ssDNA in Solutions of High Ionic Strength

We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (η(rel)) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, η(rel) decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations

Microfluidic biosensor for the detection of DNA by fluorescence enhancement and the following streptavidin detection by fluorescence quenching

We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA-biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA-biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions

Dissimilar Kinetic Behavior of Electrically Manipulated Single- and Double-Stranded DNA Tethered to a Gold Surface

We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment