Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites, bacteria, blood cells and circulating tumour cells in blood. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of 'liquid biopsies', are secreted by cells and contain nucleic acid and protein information about their originating tissue. One challenge in the study of exosome biology is to sort exosomes by size and surface markers. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235 nm. We show that at low Péclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110 nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.

No abstract

Understanding as well as rapidly screening the interaction of nanoparticles with cell membranes is of central importance for biological applications such as drug and gene delivery. Recently, we have shown that "striped" mixed-monolayer-coated gold nanoparticles spontaneously penetrate a variety of cell membranes through a passive pathway. Here, we report an electrical approach to screen and readily quantify the interaction between nanoparticles and bilayer lipid membranes. Membrane adsorption is monitored through the capacitive increase of suspended planar lipid membranes upon fusion with nanoparticles. We adopt a Langmuir isotherm model to characterize the adsorption of nanoparticles by bilayer lipid membranes and extract the partition coefficient, K, and the standard free energy gain by this spontaneous process, for a variety of sizes of cell-membrane-penetrating nanoparticles. We believe that the method presented here will be a useful qualitative and quantitative tool to determine nanoparticle interaction with lipid bilayers and consequently with cell membranes.

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.

No abstract

No abstract

We introduce a method to fabricate solid-state nanopores with sub-20 nm diameter in membranes with embedded metal electrodes across a 200 mm wafer using CMOS compatible semiconductor processes. Multi-layer (metal-dielectric) structures embedded in membranes were demonstrated to have high uniformity (± 0.5 nm) across the wafer. Arrays of nanopores were fabricated with an average size of 18 ± 2 nm in diameter using a Reactive Ion Etching (RIE) method in lieu of TEM drilling. Shorts between the membrane-embedded metals were occasionally created after pore formation, but the RIE based pores had a much better yield (99%) of unshorted electrodes compared to TEM drilled pores (<10%). A double-stranded DNA of length 1 kbp was translocated through the multi-layer structure RIE-based nanopore demonstrating that the pores were open. The ionic current through the pore can be modulated with a gain of 3 using embedded electrodes functioning as a gate in 0.1 mM KCl aqueous solution. This fabrication approach can potentially pave the way to manufacturable nanopore arrays with the ability to electrically control the movement of single or double-stranded DNA inside the pore with embedded electrodes.