Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.

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The information that can be extracted from DNA base sequences, is important for our understanding of biology, for insights in disease, and the way we practice medicine. The technology that one uses to determine DNA-base sequences is called ‘DNA sequencing’. A portable device that enables fast and accurate sequencing, can potentially greatly improve the quality of healthcare. Nanopores in graphene have potential for a new DNA sequencing technology that is superior to the current solutions.@en

Many theoretical studies predict that DNA sequencing should be feasible by monitoring the transverse current through a graphene nanoribbon while a DNA molecule translocates through a nanopore in that ribbon. Such a readout would benefit from the special transport properties of graphene, provide ultimate spatial resolution because of the single-atom layer thickness of graphene, and facilitate high-bandwidth measurements. Previous experimental attempts to measure such transverse inplane signals were however dominated by a trivial capacitive response. Here, we explore the feasibility of the approach using a custom-made differential current amplifier that discriminates between the capacitive current signal and the resistive response in the graphene. We fabricate well-defined short and narrow (30 nm × 30 nm) nanoribbons with a 5 nm nanopore in graphene with a high-temperature scanning transmission electron microscope to retain the crystallinity and sensitivity of the graphene. We show that, indeed, resistive modulations can be observed in the graphene current due to DNA translocation through the nanopore, thus demonstrating that DNA sensing with inplane currents in graphene nanostructures is possible. The approach is however exceedingly challenging due to low yields in device fabrication connected to the complex multistep device layout.@en

We present a technique to fabricate ultrathin (down to 20 nm) uniform electron transparent windows at dedicated locations in a SiN membrane for in situ transmission electron microscopy experiments. An electron-beam (e-beam) resist is spray-coated on the backside of the membrane in a KOH-etched cavity in silicon which is patterned using through-membrane electron-beam lithography. This is a controlled way to make transparent windows in membranes, whilst the topside of the membrane remains undamaged and retains its flatness. Our approach was optimized for MEMS-based heating chips but can be applied to any chip design. We show two different applications of this technique for (1) fabrication of a nanogap electrode by means of electromigration in thin free-standing metal films and (2) making low-noise graphene nanopore devices.@en

Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.@en

Graphene nanopores are receiving great attention due to their atomically thin membranes and intrinsic electrical properties that appear greatly beneficial for biosensing and DNA sequencing. Here, we present an extensive study of the low-frequency 1/f noise in the ionic current through graphene nanopores and compare it to noise levels in silicon nitride pore currents. We find that the 1/f noise magnitude is very high for graphene nanopores: typically two orders of magnitude higher than for silicon nitride pores. This is a drawback as it significantly lowers the signal-to-noise ratio in DNA translocation experiments. We evaluate possible explanations for these exceptionally high noise levels in graphene pores. From examining the noise for pores of different diameters and at various salt concentrations, we find that in contrast to silicon nitride pores, the 1/f noise in graphene pores does not follow Hooge's relation. In addition, from studying the dependence on the buffer pH, we show that the increased noise cannot be explained by charge fluctuations of chemical groups on the pore rim. Finally, we compare single and bilayer graphene to few-layer and multi-layer graphene and boron nitride (h-BN), and we find that the noise reduces with layer thickness for both materials, which suggests that mechanical fluctuations may be the underlying cause of the high 1/f noise levels in monolayer graphene nanopore devices.@en

Structural defects strongly impact the electrical transport properties of graphene nanostructures. In this Perspective, we give a brief overview of different types of defects in graphene and their effect on transport properties. We discuss recent experimental progress on graphene self-repair of defects, with a focus on in situ transmission electron microscopy studies. Finally, we present the outlook for graphene self-repair and in situ experiments.@en