Synthetic protein-conductive membrane nanopores built with DNA

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells

The decay pattern of the Pygmy Dipole Resonance of ¹⁴⁰Ce

The decay properties of the Pygmy Dipole Resonance (PDR) have been investigated in the semi-magic N=82 nucleus ¹⁴⁰Ce using a novel combination of nuclear resonance fluorescence and γ–γcoincidence techniques. Branching ratios for transitions to low-lying excited states are determined in a direct and model-independent way both for individual excited states and for excitation energy intervals. Comparison of the experimental results to microscopic calculations in the quasi-particle phonon model exhibits an excellent agreement, supporting the observation that the Pygmy Dipole Resonance couples to the ground state as well as to low-lying excited states. A 10% mixing of the PDR and the [2+1×PDR]is extracted

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

Nanometre spaced electrodes on a cleaved AlGaAs surface

We present a novel technique for fabricating nanometre spaced metal electrodes on a smooth crystal cleavage plane with precisely predetermined spacing. Our method does not require any high-resolution nanolithography tools, all lateral patterning being based on conventional optical lithography. Using molecular beam epitaxy we embedded a thin gallium arsenide (GaAs) layer in between two aluminium gallium arsenide (AlGaAs) layers with monolayer precision. By cleaving the substrate an atomically flat surface is obtained exposing the AlGaAs–GaAs sandwich structure. After selectively etching the GaAs layer, the remaining AlGaAs layers are used as a support for deposited thin film metal electrodes. We characterized these coplanar electrodes by atomic force microscopy and scanning electron microscopy; this revealed clean, symmetric and macroscopically flat surfaces with a maximum corrugation of less than 1.2 nm. In the case of a device with a 20 nm thick GaAs layer the measured electrode distance was 22.5 nm with a maximum deviation of less than 2.1 nm. To demonstrate the electrical functionality of our device we positioned single colloidal gold nanoparticles between the electrodes by an alternating voltage trapping method; this resulted in a drop of electrical resistance from ~11 G Ω to ~1.5 k Ω at 4.2 K. The device structure has large potential for the manipulation of nanosized objects like molecules or more complex aggregates on flat surfaces and the investigation of their electrical properties in a freely suspended configuration

Morphological and electrochemical properties of different PNA-based sensing platforms – Impact of the receptor-surface binding modes

By using self-assembled monolayers of phosphonic acids (SAMPs) on silicon native oxide surfaces as anchor platforms, two distinct organic interfaces with a high density of PNA bioreceptors are prepared. The impact of the PNA-bioreceptor orientation on the surface properties of the sensing platform is characterized in detail by water contact angle (CA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our results suggest that multidentate binding of PNA bioreceptor via attachment groups at the γ-points along the PNA backbone produces an extended, protruding and netlike 3D-metastructure with no preferential spatial direction. Contrary, a spatially more localized and cylindrical metastructure is realized by the monodentate binding. Furthermore, cyclic voltammetry measurements performed in a redox buffer solution, which is containing a small and highly mobile Ru-based redox active complex, reveal strikingly different insulating properties (diffusion kinetics) of these two PNA layers. Finally, investigation by electrochemical impedance spectroscopy confirms that the binding mode has a significant impact on the electrochemical properties of the functional PNA sensing surface. Here, we could observe changes of the conductance and capacitance of the underlying silicon-based semiconducting substrate in the range of 30-50 % which are strongly depending on the surface organization of the bioreceptors at different bias potential regimes. Consequently, a well-chosen modification of the PNA backbone is a valid approach to influence the sensing properties of surface-immobilized PNA bioreceptors, which might provide an additional parameter to further tune and tailor the sensing capabilities of PNA-based biosensing devices

Role of Different Receptor-Surface Binding Modes in the Morphological and Electrochemical Properties of Peptide-Nucleic-Acid-Based Sensing Platforms

Label-free detection of charged biomolecules, such as DNA, has experienced an increase in research activity in recent years, mainly to obviate the need for elaborate and expensive pretreatments for labeling target biomolecules. A promising label-free approach is based on the detection of changes in the electrical surface potential on biofunctionalized silicon field-effect devices. These devices require a reliable and selective immobilization of charged biomolecules on the device surface. In this work, self-assembled monolayers of phosphonic acids are used to prepare organic interfaces with a high density of peptide nucleic acid (PNA) bioreceptors, which are a synthetic analogue to DNA, covalently bound either in a multidentate ( - PNA) or monodentate ( - PNA) fashion to the underlying silicon native oxide surface. The impact of the PNA bioreceptor orientation on the sensing platform's surface properties is characterized in detail by water contact angle measurements, atomic force microscopy, X-ray photoelectron spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Our results suggest that the multidentate binding of the bioreceptor via attachment groups at the ?-points along the PNA backbone leads to the formation of an extended, protruding, and netlike three-dimensional metastructure. Typical "mesh" sizes are on the order of 8 \ub1 2.5 nm in diameter, with no preferential spatial orientation relative to the underlying surface. Contrarily, the monodentate binding provides a spatially more oriented metastructure comprising cylindrical features, of a typical size of 62 \ub1 23 7 12 \ub1 2 nm 2 . Additional cyclic voltammetry measurements in a redox buffer solution containing a small and highly mobile Ru-based complex reveal strikingly different insulating properties (ion diffusion kinetics) of these two PNA systems. Investigation by electrochemical impedance spectroscopy confirms that the binding mode has a significant impact on the electrochemical properties of the functional PNA layers represented by detectable changes of the conductance and capacitance of the underlying silicon substrate in the range of 30-50% depending on the surface organization of the bioreceptors in different bias potential regimes

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10^–3 s^–1. Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution