Daniel Pedone

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.

Organophosphonate-Based PNA-Functionalization of Silicon Nanowires for Label-Free DNA Detection

We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

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.

Fabrication and electrical characterization of a pore–cavity–pore device

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.