In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Recently it has been proposed to use sensors based on genetically engineered reporter cells to perform continuous online water monitoring. Here we describe the design, assembly and performance of a novel flow-through device with immobilized genetically modified yeast cells that produce a fluorescent protein upon stimulation with diclofenac whose intensity is then detected by fluorescence microscopy. Although other devices employing immobilized cells for the detection of various analytes have already been described before, as novelty our system allows safe enclosure of the sensor cells, and thus, to obtain fluorescent signals that are not falsified by a loss of cells. Furthermore, the yeast cells are prevented from being released into the environment. Despite the safe containment, the immobilized reporter cells are accessible to nutrients and analytes. They thus have both the ability to grow and respond to the analyte. Both in cell culture medium and standardized synthetic wastewater, we are able to differentiate between diclofenac concentrations in a range from 10 to 100 μM. As particularly interesting feature, we show that only the biologically active fraction of diclofenac is detected. Nowadays, contamination of wastewater with diclofenac and other pharmaceutical residues is becoming a severe problem. Our investigations may pave the way for an easy-to-use and cost-efficient wastewater monitoring method.

ZusammenfassungDie Impedanzspektroskopie ermöglicht in situ eine schnelle sowie zerstörungsfreie Charakterisierung lebender Zellen, was beispielsweise für die Referenzierung von Messwerten von Bedeutung ist.Zur Detektion lebender Hefezellen (Saccharomyces cerevisiae) mittels Impedanzspektroskopie wurden hier interdigitale Platindickschichtelektroden verwendet.Dabei wurde die komplexe Impedanz in einem Bereich zwischen 0,01 Hz und 1 MHz sowohl für Elektroden mit lebenden als auch Hitzeinaktivierten Hefezellen, suspendiert in Nährmedium bzw.immobilisiert in Agar über einen Zeitraum von 300 min bestimmt.Für ein besseres Verständnis der Vorgänge an den Elektroden wurde ein Ersatzschaltbild aufgestellt und analysiert.Dieses enthält ein constant phase element (zur Beschreibung der Doppelschichtkapazität an den Elektroden),

We present a novel concept for the synthesis of a self-thermophoretic nanoswimmer, a construct consisting of a gold nanoparticle as a heating element and an artificial DNA structure as a thermophoretic active part. For the latter, DNA origami technique was applied to design and synthesize rod-like bundle structures as well as a more complex 2-leg construct. This novel concept is based on the versatile and easy-to-apply DNA origami toolbox to realize Janus particle-like structures with various geometries. We synthesized a variety of different nanoswimmers and characterized them in terms of structure, yield, and thermal stability.[GRAPHICS].Schematic representation of the construction principle of a self-thermophoretic nanoswimmer consisting of a DNA origami structure and a gold nanoparticle. Inset: TEM image of such a nanoswimmer structure.(C) 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Interdigitated, screen-printed platinum microelectrodes are used for non-Faradaic impedance spectroscopy detection of viable Saccharomyces cerevisiae. The time dependence of the complex impedance of both living and heat-inactivated yeast cells immersed in plain medium or immobilized in agar is measured between 0.01 Hz and 1 MHz for 300 min. To understand the processes probed by the impedance measurement, an equivalent electrical circuit, containing a constant phase element, a charge transfer resistance and a suspension resistance, is established and analyzed. For both heat-inactivated and living yeast cells in plain medium, the suspension resistance increases over time caused by sedimentation of the yeast cells into the stray field of the electrodes. The measured increase is found to be larger for living cells than for heat-inactivated ones. The time dependence of the double-layer capacitance is rather dominated by the metabolic activity of living cells, but a possible impact of cell sedimentation cannot be completely neglected. In experiments carried out with cells immobilized in agar, cell sedimentation is suppressed, and thus, the time evolution of the double-layer capacitance is solely caused by metabolic active cells. Therefore, the time-dependent change of the double-layer capacitance can be used to directly monitor the viability of Saccharomyces cerevisiae in situ, since inactivated cells do not contribute to the signal generation in this case. (C) 2017 Elsevier B.V. All rights reserved.

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 CP's 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.

The determination of the double-layer capacitance of planar interdigitated gold electrodes in a microfluidic channel by electrical impedance spectroscopy is presented. A simple and miniaturized two-electrode setup is used to investigate electrodes coated with pH-sensitive self-assembled thiol layers of 4-mercaptopyridine (4-MP) or 4-mercaptobenzoic acid (4-MBA). The total sensing area is about 0.25mm(2). Surface modifications and pH have a pronounced influence on the double-layer capacitance of the electrodes. The interfacial capacitance rises significantly at pH<3 on the 4-MP and at pH>10 on the 4-MBA layer. This effect is related to surface protonation or deprotonation of the pyridine or carboxylic moieties, respectively. It is shown that this double-layer capacitance is widely independent from the electrolyte concentration. Such electrodes can be used as pH indicators in microfluidic channels, additionally allowing the determination of the electrolyte conductivity. This kind of sensor is supposed to be used in lab-on-a-chip systems for fast readout of local chemical and physical process parameters.

Layers of pH‐sensitive poly(2‐vinylpyridine) are immobilised on gold surfaces of surface plasmon reson­ance (SPR) or quartz crystal microbalance (QCM) sen­sors. Two different polymers are used, one (P2VP‐COOH) with and another (P2VP) without a terminal carboxylic group. Swelling and stiffening are preferably observable in the low protonation state of the polymer layers by QCM. In solutions of hydrochloric acid QCM measurements reveal that P2VP‐COOH forms denser and stiffer layers than P2VP. Both polymers clearly show viscoelastic properties. The SPR sensor detects dilution as well as the incorporation of hydrochloric acid into the sensing layers at lower pH, and therefore, higher protona­tion state of the polymers. SPR and QCM are complementarily used to characterise changes of the mechanical and optical properties of pH‐sensitive poly­mer layers.

In dieser Arbeit wird die Herstellung eines Polymer-Mikroarray vorgestellt. Die dazu verwendeten Losungen der pH-sensitiven Polyvinylpyridine werden auf einer reaktiv beschichteten Goldoberflache durch ein Mikropipettiersystem tropfenweise in Abstanden von 290 μm auf 20 verschiedenen Messorten abgesetzt. Mittels der Methode der Oberflachenplasmonenresonanz (SPR) werden pH-WertAnderungen in sauren, wassrigen Losungen auf dem Mikroarray detektiert. Die SPR-Methode erlaubt eine hochempfindliche und markierungsfrei optische Bestimmung der Brechungsindexanderung. Das Quellen und die Protonierung der Polymere fuhren zu messbaren Brechungsindexanderungen an der Sensoroberflache. Das Mikroarray wird in einem mikrofluidischen Kanal eingesetzt, um pH-Werte kleiner als 5 in salzsauren Losungen mittels SPR schnell zu detektieren. Jedes der Polymere zeigt einen bestimmten pH-Bereich, in dem es die hochste Sensitivitat aufweist.

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.

We present the use of thiol‐modified and phosphorothioate (PT)‐modified oligonucleotides for building DNA microarrays on the gold surface of surface plasmon resonance (SPR) chips. PT‐modified oligonucleotides (PTOs) have several advantages in comparison to thiol‐modified ones. They do not form disulfides and the PT groups can be introduced in any desired position of the molecular backbone of the oligonucleotide. Additionally, modifications with PT groups are not as cost‐intensive as thiol groups. The direct immobilization of the oligonucleotides via thiol and PT groups is compared. The affinity of the PT groups to gold is lower than that of thiol groups. Nevertheless, the hybridization kinetics of a model polymerase chain reaction (PCR) product could be studied in real time on a DNA microarray for both types of modified oligonucleotides. This contributes to clarification of previous, contradictory reports on the use of PTOs for their attachment to gold surfaces. The immobilization of PTOs could be improved by the introduction of iodoacetylated surfaces, which are reactive to bind PTs as well as thiols with high efficiency. Furthermore, the influence of the probe structure on the probe density and the hybridization was investigated.

Background The 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. Results The 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. Conclusions We 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 at http://www.chm.tu-dresden.de/pc6/EGNAS .

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.

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]