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The binding properties of bispecific antibodies (bsAb) are crucial for their function, especially when two antigens are targeted on the same cell surface. Dynamic interactions between each of the antibody's arms and its cognate target cause the formation and decay of a biologically functional ternary complex. How association and dissociation processes work cooperatively, and how they influence the avidity of the ternary complex, is still poorly understood. Here, we present a biosensor assay for the simultaneous measurement of the binding kinetics of the therapeutic bsAb emicizumab (Hemlibra®) and its two targets, the blood coagulation factors IX and X (FIX, FX). We describe an automated workflow to characterize binary and ternary-binding modes, utilizing a Y-shaped DNA nanostructure to immobilize the antigens on a sensor and to emulate conditions on a cell or platelet surface by presenting the antigens with optimal accessibility for the bsAb flown over the sensor as analyte. We find that emicizumab binds FX much stronger than FIX (K

The characterization of novel therapeutic antibodies with multivalent or multispecific binding sites requires new measurement modalities for biosensors, to discriminate the engagement of antigens via one, two, or even more binding moieties. The presentation of antigens on a sensor surface in a well-controlled spatial arrangement is a prerequisite for the successful interpretation of binding kinetics measurements of multivalent analytes, but the adjustment of defined distances between immobilized ligands is difficult to achieve in state-of-the-art biosensor systems. Here, we introduce a simple DNA nanostructure resembling a slingshot, which can be configured with two identical or two different antigens (bivalent or bispecific), which are spaced at a defined distance. We characterize the slingshot structure with a chip-based biosensor using electrically switchable DNA nanolevers and demonstrate that bivalent and monovalent antibodies selectively interact with slingshots that have been functionalized with two identical or two different antigens, respectively. The dissociation kinetics are quantified in real-time measurements and we show that the slingshot structure enables a clear differentiation between affinity and avidity effects.

Electrically controllable deoxyribonuclic acid (DNA) nanolevers are used to investigate the binding interaction between Influenza A/Aichi/2/1968 and the peptide called “PeB”, which specifically binds the viral surface protein hemagglutinin. PeB is immobilized on gold electrodes of a “switchSENSE” biochip by conjugation to DNA-strands that are hybridized to complementary anchors. The surface-tethered DNA strand carries a fluorophore while the complementary strand is a multivalent arrangement carrying up to three PeB peptides. The nanolevers are kept upright (static) by applying a negative potential. Signal read-out for this static measurement mode is the change in fluorescence intensity due to changes in the local environment of the dye upon binding. Measurements of virus-peptide interaction show that the virus material specifically binds to the immobilized peptides and remains bound throughout the measurement time. Immobilized viruses are subsequently used as ligands to characterize oligovalent peptide binding to hemagglutinin, revealing rate constants of the interaction. Moreover, three Influenza A subtypes are compared in their binding behavior. Overall, this paper shows the ability to immobilize virus material on a sensor surface, which allows to target virus-proteins in their native environment. The “switchSENSE” method is therefore applicable to characterize virus-receptor interactions.

PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (15–16 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11. Electronic supplementary material The online version of this article (doi:10.1007/s10577-017-9552-1) contains supplementary material, which is available to authorized users.

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10−3 s−1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.

Dynamic methods of biosensing based on electrical actuation of surface-tethered nanolevers require the use of levers whose movement in ionic liquids is well controllable and stable. In particular, mechanical integrity of the nanolevers in a wide range of ionic strengths will enable to meet the chemical conditions of a large variety of applications where the specific binding of biomolecular analytes is analyzed. Herein, we study the electrically induced switching behavior of different rodlike DNA origami nanolevers and compare to the actuation of simply double-stranded DNA nanolevers. Our measurements reveal a significantly stronger response of the DNA origami to switching of electrode potential, leading to a smaller potential change necessary to actuate the origami and subsequently to a long-term stable movement. Dynamic measurements in buffer solutions with different Mg2+ contents show that the levers do not disintegrate even at very low ion concentrations and constant switching stress and thus provide s...

Development of electrically powered DNA origami nanomachines requires effective means to actuate moving origami parts by externally applied electric fields. We demonstrate how origami nanolevers on an electrode can be manipulated (switched) at high frequency by alternating voltages. Orientation switching is long-time stable and can be induced by applying low voltages of 200 mV. The mechanical response time of a 100 nm long origami lever to an applied voltage step is less than 100 μs, allowing dynamic control of the induced motion. Moreover, through voltage assisted capture, origamis can be immobilized from folding solution without purification, even in the presence of excess staple strands. The results establish a way for interfacing and controlling DNA origamis with standard electronics, and enable their use as moving parts in electro-mechanical nanodevices.

Self-assembled monolayers of charged polymers are an integral component of many state-of-the-art nanobiosensors. Electrical interactions between charged surfaces and charged biomolecules, adopting the roles of linkers or capture molecules, are not only crucial to the sensor performance but may also be exploited for novel sensing concepts based on electrically actuated interfaces. Here we introduce an analytical model describing the behavior of double-stranded DNA and proteins tethered to externally biased microelectrodes. Continuum electrostatic Poisson-Boltzmann models and the drift-diffusion (Smoluchowski) equation are used to calculate the steady state as well as the dynamic behavior of oligonucleotide rods in DC and AC electric fields. The model predicts the oligonucleotide orientation on the surface and calculates how the increased hydrodynamic drag caused by a protein bound to the DNA's distal end affects the molecular dynamics of the DNA-protein complex. The results of the model are compared to experiments with electrically switchable DNA layers, and very good agreement between theory and experiment is found. The hydrodynamic diameter of the bound protein can be analyzed from experimental data of the slowed motion of the DNA-protein conjugate with angstrom precision.

The conformation of charged molecules tethered to conducting substrates can be controlled efficiently through the application of external voltages. Biomolecules like DNA or oligopeptides can be forced to stretch away from—or fold onto—surfaces biased at moderate potentials of merely hundreds of millivolts. These externally controlled conformation changes can be used to switch the biological function of molecular monolayers on and off, by revealing or concealing molecular recognition sites at will. Moreover, the electrical actuation of biomolecular surface probes bears great potential as a novel, label-free, yet highly sensitive measurement modality for the analysis of molecular interactions. The binding of target molecules to an oscillating probe layer significantly alters the layer’s switching behavior in terms of the conformation switching amplitude and, most remarkably, with respect to the molecular switching dynamics. Analyzing the switching response of target–probe complexes from the low- to the high-frequency regime reveals a wealth of previously inaccessible information. Besides “classical” interaction parameters like binding affinities and kinetic rate constants, information on the size, shape, bending flexibility, and elasticity of the target molecule may be obtained in a single assay. This review describes the advent of electrically switchable biosurfaces, focusing on DNA monolayers. The preparation of self-assembled switchable oligonucleotide monolayers and their electrical interactions with charged substrates are highlighted. Special attention is paid to the merits of evaluating the dynamic response of charged biolayers which are operated at high driving frequencies. Several applications of biosensors based on electrically manipulated molecules are exemplified. It is emphasized that the electrical actuation of biomolecules bears many advantages over passive sensor surfaces.

A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K(D) in the pM regime), and the influence of competitive binders (EC(50) values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

The first example of a programmable DNA photonic wire is reported utilizing fluorophore-tethered pyrrole-imidazole polyamides for site-directed fluorophore assembly along a pre-formed DNA duplex (see scheme; PB=Pacific Blue, Cy3=Cyanine 3; orange rectangles=fluorophore). The importance of such control is revealed by efficient energy transport over distances in excess of 27 nm.

We introduce a nanofabricated silicon chip for massively multiplexed analysis of membrane channels and transporters in suspended lipid membranes that does not require any surface modification or organic solvent. Transport processes through single membrane complexes are monitored by fluorescence. The chip consists of an array of well-defined nanopores, addressing an individual pyramidal back-reflecting 30-fL compartment. The setup allows simultaneous analyses of ∼1,000 single transmembrane events in one field of view, observing translocation kinetics of transmembrane complexes.

The fabrication and characterization of a metallized nanopore structure for the sensing of single molecules is described. Pores of varying diameters (>10 nm) are patterned into free-standing silicon nitride membranes by electron-beam lithography and reactive ion etching. Structural characterization by transmission electron microscopy (TEM) and tomography reveals a conical pore shape with a 40 degrees aperture. Metal films of Ti/Au are vapor deposited and the pore shape and shrinking are studied as a function of evaporated film thickness. TEM tomography analysis confirms metalization of the inner pore walls as well as conservation of the conical pore shape. In electrical measurements of the transpore current in aqueous electrolyte solution, the pores feature very low noise. The applicability of the metallized pores for stochastic sensing is demonstrated in real-time translocation experiments of single lambda-DNA molecules. We observe exceptionally long-lasting current blockades with a fine structure of distinct current levels, suggesting an attractive interaction between the DNA and the PEGylated metallic pore walls.

Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was found to be complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was observed. Here we study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential zeta(pore) and the protein zeta potential zeta(protein) are measured independently as a function of solution pH. Our results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, respectively; the resulting translocation direction can be predicted according to the difference in zeta potentials, zeta(protein) - zeta(pore). When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

The toxic nature of polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), neccessitates the monitoring of PAH contamination levels in food and the environment. Here we introduce an indirect immunoassay format using electro-switchable biosurfaces (ESB) for the detection of B[a]P in water. The association of anti-B[a]P antibodies to microelectrodes is analyzed in real-time by measuring changes in the oscillation dynamics of DNA nanolever probes, which are driven to switch their orientations by high-frequency electrical actuation. From the association kinetics, the active concentration of anti-B[a]P, and hence the B[a]P contamination of the sample, can be determined with picomolar sensitivity. The detection limit of the assay improves with measurement time because increasingly accurate analyses of the binding kinetics become possible. It is demonstrated that an exceedance of the permissible 10 ng/L (40 pM) limit for B[a]P is detectable in an unprecedented short assay time (1 h), using a simple three-step workflow involving minimal sample preparation. The reproducibility was satisfying with standard deviations below 5%. Further, the utility of the assay for practical applications is exemplified by analyzing a river water sample.

Polymers that are terminally attached to solid supports, so called brushes, form the basis for a wide variety of different applications in colloidal and biophysical sciences. For grafted charged chains a conducting surface allows to manipulate the brush structure by applying electric fields across the brush. The dynamics of oligomeric DNA molecules under the action of repulsive and attractive surface electric fields is studied by Brownian dynamics simulations including hydrodynamic effects and compared to experimental results. The difference in flexibility between double and single stranded DNA molecules leads to a change in the switching dynamics when repeatedly reversing the surface charge. This effect allows to detect hybridization of surface anchored DNA. Similar kinetic changes occur when other molecules bind terminally to DNA, opening the possibility to use end grafted polymers for general biosensing applications. We in particular discuss the influence of the adsorbate size and change on the switching dynamics. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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.

A novel concept for the integration of liquid phase charge sensors into microfluidic devices based on silicon-on-insulator (SOI) technology is reported. Utilizing standard silicon processing we fabricated basic microfluidic cross geometries comprising of 5-10-mm-long and 55-/spl mu/m-wide channels of 3 /spl mu/m depth by wet sacrificial etching of the buried oxide of an SOI substrate. To demonstrate the feasibility of fluid manipulation along the channel we performed electroosmotic pumping of a dye-labeled buffer solution. At selected positions along the channel we patterned the 205-nm thin top silicon layer into freely suspended, 10-/spl mu/m wide bars bridging the channel. We demonstrate how these monolithically integrated bars work as thin-film resistors that sensitively probe changes of the surface potential via the field effect. In this way, a combination of electrokinetic manipulation and separation of charged analytes together with an on-chip electronic detection can provide a new basis for the label-free analysis of, for example, biomolecular species as envisaged in the concept of micrototal analysis systems (/spl mu/TAS) or Lab-on-Chip (LOC).

We report on the dynamic control over the orientation of short oligonucleotide strands which are tethered to gold surfaces in electrolyte solution. By applying alternating electrical bias potentials to the supporting electrodes we are able to induce a switching of the layer conformation between a “lying” and a “standing” state, simultaneously monitored in a contactless mode by fluorescence techniques. We demonstrate that our electrooptical experiments allow for an in-depth investigation of the intriguing molecular dynamics of DNA at surfaces and, moreover, how the dynamic response of these switchable biomolecular layers opens new prospects in label-free biosensing.

A novel label-free biosensing technology is introduced which employs a dynamically switchable biosurface. Contrary to conventional biosensors, switchSENSE allows to determine affinity, reaction rates, and molecular size of the target in one analytical run. It constitutes a high-content analytical platform for the discovery of biological drugs and molecular interactions. The determination of KD values and the detection of conformational changes of a Fab-fragment are described here.

We report the synthesis of novel phenylenevinylene derivatives that allow for the introduction of meta versus para connections on the phenylene rings, as well as the incorporation of nitrogen atoms within the conjugated backbone and the attachment of electroactive substituents. We assess the impact of the various derivatization schemes on the electronic and optical properties by means of gas-phase ultraviolet photoelectron spectroscopy (UPS) and optical absorption and emission measurements; the evolution of the experimental data is further supported by the results of quantum-chemical calculations. We demonstrate that the electronic and optical properties of phenylenevinylene chains, and by extension those of many conjugated materials, can be tuned over a large energy range by a tailored design of the molecular structures.

Phenylene-based materials are of particular interest for applications in organic luminescent devices. To gain deeper insight into the emissive properties of this class of materials, we have systematically studied the relation between the degree of conjugation and singlet exciton lifetimes. Two classes of phenylenes are investigated by time resolved frequency domain spectroscopy: oligo-para-phenylenes (OPPs) with n-alkyl substitutions at the central phenylene ring of the molecule and ladder-type-oligo-para-phenylenes (LOPPs) featuring rigid backbones with fixed interring twist angles. Additional quantum yield (QY) measurements allow to assess the intrinsic lifetime of the radiative decay. The experimental data are compared to the results of quantum-chemical calculations yielding an excellent agreement for both the classes of materials.

Over the last years, translocation experiments with proteins through solid-state nanopores have consistently produced anomalously long residence times that are far beyond a ballistic passage process. It has been suggested that attractive interactions to the pore wall might cause a retardation of the protein, but the origin of these interactions has remained elusive up to now.Here we present experimental evidence that this interaction is of hydrophobic nature: We quantitatively compared the translocation times of a variety of different proteins from our own experiments as well as from other authors and analyzed the hydrophobicity of the respective protein surfaces from available atomic structure data. We found that the occurrence of very long translocation times correlates with the existence of large hydrophobic patches on the protein surface. This strongly suggests that hydrophobic interactions are a dominant factor in determining the passage time of proteins through artificial nanopores, and that it is necessary to engineer the hydrophobicity of artificial nanopores in order to control the passage of proteins through the pore.

Eine neue chipbasierte Messmethode detektiert Proteine und bestimmt gleichzeitig deren Grose — labelfrei und in Echtzeit.

We study the coadsorption of mercaptohexanol onto preimmobilized oligonucleotide layers on gold. Monitoring the position of the DNA relative to the surface by optical means directly shows the mercaptohexanol-induced desorption of DNA and the reorientation of surface-tethered strands in situ and in real time. By simultaneously recording the electrochemical electrode potential, we are able to demonstrate that changes in the layer conformation are predominantly of electrostatic origin and can be reversed by applying external bias to the substrate.

There is great interest in establishing efficient means of organizing nanoparticles into complex structures, especially in fields like nano-optical devices. One of the demonstrated routes uses biomolecular scaffolds, like the streptavidin–biotin system, to deterministically separate and structure particle complexes. However, controlled formation of streptavidin-linked nanoparticle dimers or trimers is challenging, and large aggregates are often formed under conditions that are difficult to regulate. Here, we studied the aggregates and interlinking kinetics of biotin-functionalized 20 nm gold nanoparticles in the presence of the interlinking protein, streptavidin. We found two different protein-linker concentration regions where small stable particle aggregates are formed: when the protein and nanoparticle concentrations are similar and when the protein to nanoparticle concentration ratio exceeds intermediate concentrations (10:1–100:1) that promote precipitation of large aggregates. We attribute this behavior to the limited availability of free-linker molecules and the limited availability of free ligand (biotin) on the particle surface for low and high protein concentrations, respectively. Furthermore, we show that the product can be additionally enriched up to 25 % through either centrifugation in sucrose or size-exclusion chromatography. These results provide additional understanding into the assembly of ligand-functionalized nanoparticles with water-soluble linkers and provide a facile way to produce well-defined small aggregates for potential use in, for instance, surface-enhanced spectroscopy.

Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements., The comprehensive bioanalysis of proteins usually requires multi-step surface and mobile phase measurements. Here, the authors use chips functionalized with dynamically actuated nanolevers—DNA strands that can be switched in an electric field—to obtain motional dynamic measurements of proteins on a chip.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of β2-glycoprotein I (β2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of β2-GPI to the cardiolipin surface. Using the switchSENSE sensor, β2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between β2-GPI-dependent autoimmune- and β2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

We report the photo-induced nucleation and growth of silver nanoparticles in aqueous solution in the presence of DNA oligomers. An organic dye (Cy5) was used as a photosensitizer to initiate the nanoparticle growth upon illumination with 647 nm light. The formation of nanoparticles and growth kinetics were observed by extinction spectroscopy, dynamic light scattering, and transmission electron microscopy. Irradiation of the precursor solutions with light at the Cy5 absorption maximum triggered the instantaneous formation of spherical particles with a metallic core ~15 nm in diameter. Remarkably, the particles feature significantly larger effective hydrodynamic diameters (35 nm) in solution, indicative of a DNA ad-layer on the nanoparticle surface. Centrifugation experiments confirmed that DNA was inseparably associated with the nanoparticles and indicated that DNA oligomers adsorb onto the nanoparticle surface during growth, playing the role of a capping agent. The introduced method is a fast and facile way to prepare DNA-capped silver nanoparticles in a single growth step.

DNA has it covered: DNA origami gatekeeper nanoplates convert nanopores in solid-state membranes into versatile devices for label-free macromolecular sensing applications. The custom apertures in the nanoplates can be chemically addressed for sequence-specific detection of DNA.

Electrophoretic transport through a solid-state nanodevice comprised of two stacked nanopore sensors is used to determine the free-solution mobility of DNA molecules based on their “time-of-flight” between the two pores. Mobility measurements are possible at very low (100 pM) DNA concentration and for low as well as high salt concentrations (here 30 mM and 1 M KCl). The mechanism of DNA transport through the device is elucidated by statistical analysis, showing the free-draining nature of the translocating DNA polymers and a barrier-dominated escape through the second pore. Furthermore, consecutive threading of single molecules through the two pores can be used to gain more detailed information on the dynamics of the molecules by correlation analysis, which also provides a direct electrical proof for translocation.

We present the fabrication and characterization of nanopore microcavities for the investigation of transport processes in suspended lipid membranes. The cavities are situated below the surface of silicon-on-insulator (SOI) substrates. Single cavities and large area arrays were prepared using high resolution electron-beam lithography in combination with reactive ion etching (RIE) and wet chemical sacrificial underetching. The locally separated compartments have a circular shape and allow the enclosure of picoliter volume aqueous solutions. They are sealed at their top by a 250 nm thin Si membrane featuring pores with diameters from 2 µm down to 220 nm. The Si surface exhibits excellent smoothness and homogeneity as verified by AFM analysis. As biophysical test system we deposited lipid membranes by vesicle fusion, and demonstrated their fluid-like properties by fluorescence recovery after photobleaching. As clearly indicated by AFM measurements in aqueous buffer solution, intact lipid membranes successfully spanned the pores. The nanopore cavity arrays have potential applications in diagnostics and pharmaceutical research on transmembrane proteins.

Spatial confinement from the nano- to the microscale is ubiquitous in nature. Striving to understand the behavior of nanoscale objects in confined domains we present a nanofluidic silicon device which consists of two stacked nanopores forming the in/outlets to a pyramidal cavity of micrometer dimensions (10 fL volume). Being electrically addressable, charged objects can be actively loaded into, trapped inside, and unloaded from the "pore-cavity-pore" (PCP) device. When operated passively, confined Brownian motion and the entropy barriers of the nanopores govern the behavior of nano-objects within the PCP device. We present measurements with single fluorescent nanoparticles as well as particle-ensembles and analyze their trajectories and residence times. Experimental data are compared to random walk simulations and analytical theories on confined diffusion and the Brownian escape of nano-objects across entropy barriers. Single particle data corroborate analytical solutions of the narrow escape problem, but ensemble measurements indicate crowding effects even at low particle concentrations. The utilization of the device to trap biomolecules is demonstrated for single λ-DNA molecules.

We present a solid state nanopore device structure comprising two nanopores which are stacked above each other and connected via a pyramidal cavity of 10 fl volume. The process of fabrication of the pore-cavity-pore device (PCP) relies on the formation of one pore in a Si(3)N(4) membrane by electron beam lithography, while the other pore is chemically etched into the Si carrier by a feedback controlled process. The dimensions of the two nanopores as well as the cavity can be adjusted independently, which is confirmed by transmission electron microscopy. The PCP device is characterized with respect to its electrical properties, including noise analysis and impedance spectroscopy. An equivalent circuit model is identified and resistance, capacitance, and dielectric loss factors are obtained. Potential and electric field distributions inside the electrically biased device are simulated by finite element methods. The low noise characteristics of the PCP device (comparable to a single solid state nanopore) make it suitable for the stochastic sensing of single molecules; moreover, the pore-cavity-pore architecture allows for novel kinds of experiments including the trapping of single nano-objects and single molecule time-of-flight measurements.

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation-switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies, antibody-fragments, and small proteins like cytochrome c, which can be detected in quantities of less than 1 amol on the sensor surface. We show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response as well as by time-resolved fluorescence measurements. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments and small proteins by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

We describe the behavior of 72mer oligonucleotides that are end-tethered to gold surfaces under the influence of applied electric fields. The DNA extension is measured by fluorescence energy transfer as a function of the DNA hybridization state (single- and double-stranded), the concentration of monovalent salt in solution (100 microM to 1 M NaCl), the applied electrode potential (-0.6 to +0.1 V vs Pt), and the temperature (1 to 50 degrees C). At high ionic strength, the DNA conformations are very robust and independent of the applied electrode potential and temperature variations. In solutions of medium ionic strength, the DNA conformation can be manipulated efficiently by applying bias potentials to the Au electrodes. The molecules are repelled at negative potentials and attracted to the surface at positive potentials. The conformation transition occurs abruptly when the electrode bias is swept by merely 0.1 V across the transition potential, which shifts negatively when the salinity is decreased. The behavior can be understood by electrostatic screening arguments and, in the case of single-stranded DNA, when secondary structures are taken into account. At low ionic strength, the experiments reveal an intriguing temperature-dependent stiffening of single-stranded DNA, which can be rationalized by combining counterion condensation theory with the Odjik-Skolnick-Fixman description of the electrostatic persistence length and the unstacking of bases at elevated temperatures.

We have utilized basic principles of optical interference and resonance and applied our experience in resonant optoelectronic devices [1] to biological applications demonstrating nanometer scale measurement capability in fluorescence microscopy and label-free sensing of protein binding in a high-throughput micro-array format. A novel application of resonance to fluorescence microscopy promises nanometer resolution in biological imaging. We have developed a technique - spectral self-interference fluorescent microscopy (SSFM) - that transforms the variation in the intensity for different wavelengths in emission of fluorophores on a layered surface to nanoscale height information. Using SSFM, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization [2]. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields. Recently, we have also applied SSFM to study the conformational changes of polymers [3] and DNA-protein complexes f41.