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.

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 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.

Discrete tomography of demanding samples based on a modified SIRT algorithm.

The 3D structure of three particularly challenging samples was reconstructed by electron tomography. Due to sample limitations resulting in a large missing wedge and large tilt increments respectively the 3D structure could not be reconstructed by standard iterative algorithms; even a recently developed discrete algorithm failed until the input parameters for discrete reconstruction were improved. These challenges were addressed by adding a mask in each step of the preceding standard iterative reconstruction, setting all voxels known to be vacuum as zero, thus improving the segmentation and the 3D starting model. The position of these vacuum voxels is obtained from TEM images or other measurement data.

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.