Ulrich Rant

Stochastic sensing of proteins with receptor-modified solid-state nanopores

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.

Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis

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.

DNA origami gatekeepers for solid-state nanopores.

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.

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

Photonic crystal nanostructures for optical biosensing applications.

We present the design, fabrication and optical investigation of photonic crystal (PhC) nanocavity drop filters for use as optical biosensors. The resonant cavity mode wavelength and Q-factor are studied as a function of the ambient refractive index and as a function of adsorbed proteins (bovine serum albumin) on the sensor surface. Experiments were performed by evanescent excitation of the cavity mode via a PhC waveguide. This in turn is coupled to a ridge waveguide that allows the introduction of a fluid flow cell on a chip. A response of partial delta lambda/delta c=(4.54+/-0.66)x10(5)nm/M is measured leading to a measured detection limit as good as Delta m=4.0+/-0.6 fg or Delta m/Delta A=(4.9+/-0.7)x10(2)pg/mm(2)in the sensitive area.

Conformations of End-Tethered DNA Molecules on Gold Surfaces: Influences of Applied Electric Potential, Electrolyte Screening, and Temperature

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.

Data Analysis of Translocation Events in Nanopore Experiments

Nanopores have become important tools for single molecule experiments, where information about the properties of DNA/RNA or proteins is inferred from current pulses elicited by individual molecules as they traverse a single pore. However, because of necessary electronic filters employed in the measurement technique, the extraction of meaningful information from short pulses is limited. This restricts the use of nanopores for the investigation of small molecules which cross the pore rapidly. Here we present a method which significantly improves the accuracy of the analysis of noise-filtered current pulses. We introduce improved criteria to measure the pulse width and propose a method to evaluate the pulse height from the falling edge of the pulse, which renders the identification of a pulse plateau unnecessary. The new methods are compared to conventional routines and validated by analyzing representative current pulses as well as experimental protein translocation data. It is demonstrated that the pulse properties can be recovered with satisfying accuracy beyond the usual limitations of Bessel filters, i.e., from pulses featuring a width of merely 0.3f(c)(-1) (f(c) being the filter cutoff frequency).

Fabrication of Metallized Nanopores in Silicon Nitride Membranes for Single‐Molecule Sensing

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.

Multiplexed parallel single transport recordings on nanopore arrays.

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.

Electrical manipulation of oligonucleotides grafted to charged surfaces

The electrical manipulation of short DNA molecules on surfaces offers novel functionalities with fascinating possibilities in the field of bio-interfaces. Here we present systematic investigations of the electrical interactions which govern the structure of oligonucleotides on charged gold surfaces. Successively, we address influences of the applied field strength, the role of DC electrode potentials, in particular for polycrystalline surfaces, as well as screening effects of the surrounding electrolyte solution. Data obtained for single and double stranded DNA exhibit differences which can be attributed to the dissimilar flexibility of the different molecular conformations. A comparison of the experimental results with a basic model shows how the alignment of the molecules adjusts according to a balance between electrically induced ordering and stochastic thermal motions. The presented conclusions are expected to be of general relevance for the behaviour of polyelectrolytes exposed to localized electric fields at interfaces.