Nanopore sensing has shown great potential; however, one of its major challenges remains the fast and uneven translocation of analytes in free translocation nanopore measurements. We recently introduced Scanning Ion Conductance Spectroscopy (SICS), a technique that enables precise control of translocation speed by immobilizing the analyte on the surface and regulating the speed through controlled capillary movement. This methodology requires a unique preparation of the analyte over conventional free-translocation experiments, as the analyte is captured by and immobilized along a DNA carrier that is attached to a glass surface. This chapter describes the preparation of the essential components required for experiments, including the capillary, buffer, measurement system, and, most importantly, the preparation of DNA carriers that can be functionalized with aptamers.

Abstract Biomolecular condensates are important regulators of cellular compartmentalization and biochemical processes. Understanding their material properties is critical to elucidate how they control molecular organization and dynamics within cells. However, quantitatively probing these properties remains challenging due to the wide range of length scales, concentrations, and timescales over which condensates operate, as well as the limited force ranges accessible to current nanoscale mechanical mapping methods. We explored the use of a non-contact 3D imaging tool Scanning Ion Conductance Microscopy (SICM) for stiffness measurements of liquid–liquid phase-separated biomolecular condensates. We focus on the Dhh1 protein, which is a regulator of cytoplasmic processing bodies (PBs) membrane-less cytoplasmic condensates that control the storage and degradation of untranslated mRNA. In our study, we investigate the properties of mCherry2- or His-mCherry2-tagged full-length Dhh1 and N- or C-terminus tail-deletion constructs, as well as the catalytically inactive mutant DQAD, under different pH and incubation times. We mapped both spatial and temporal changes in the material properties of the condensates, highlighting the capabilities of the instrument. We found that the removal of either of the two tails led to an increase in condensate stiffness upon shifting the pH from a stress-associated cellular environment (pH 6.5) to physiological conditions (pH 7.5). Additionally, the choice of protein tags led to vastly different results depending on the pH where mCherry2-Dhh1 exhibited a stiffening going from pH 6.0 to 6.5 while the double-tagged His-mCherry2 did not. Our measurements are verified and corroborated by established techniques such as optical tweezer-based fusion assays and fluorescence recovery after photobleaching (FRAP). Furthermore, we were able to track the same biomolecular condensate sample for up to 20 days getting insights on the ‘ageing’ and evolution of the condensates. Overall, our study demonstrates the applicability of SICM for direct measurement of the material properties of biomolecular condensate.

Solid-state nanopores offer the possibility of detecting disease biomarkers in early diagnostic applications. Standard approaches harness fingerprinting, where protein targets are bound to DNA carriers and detected in free translocation with a solid-state nanopore. However, they suffer from several drawbacks, including uncontrolled fast translocations, which lead to low detection accuracy and a low signal-to-noise ratio (SNR). This has hampered their application in clinical settings. Here, we propose a nanopore-based system capable of sensing selected molecules of interest from biological fluids by harnessing programmable aptamer sequences attached to DNA carrier systems that are tethered to glass surfaces. This allows for spatial and velocity control over translocation in the x, y, and z directions and enables the repeated scanning of the same analyte. The scanning ion conductance spectroscopy (SICS) based approach distinguishes itself from standard nanopore-based approaches with its ability to repeatedly scan the same aptamer molecule target site more than 5 times. We designed a DNA carrier with multiple binding sites for different aptamers to increase the yield of the experiment. Our approach achieves a detection rate of up to 74%, significantly higher than the 14% achieved with standard solid-state nanopore measurements. The strong spatial control also allows for significantly increased densities of aptamer target sites along the same DNA carrier, thereby paving the way for multiplexed sensing. The system offers user-defined programmability with different aptamer sequences, potentially expanding the use of our system to sense other disease biomarkers.

Two-dimensional (2D) materials offer potential as substrates for biosensing devices, as their properties can be engineered to tune interactions between the surface and biomolecules. Yet, not many methods can measure these interactions in a liquid environment without introducing labeling agents such as fluorophores. In this work, we harness interferometric scattering (iSCAT) microscopy, a label-free imaging technique, to investigate the interactions of single molecules of long dsDNA with 2D materials. The millisecond temporal resolution of iSCAT allows us to capture the transient interactions and to observe the dynamics of unlabeled DNA binding to a hexagonal boron nitride (hBN) surface in solution for extended periods (including a fraction of 10%, of trajectories lasting longer than 110 ms). Using a focused ion beam technique to engineer defects, we find that DNA binding affinity is enhanced at defects; when exposed to long lanes, DNA binds preferentially at the lane edges. Overall, we demonstrate that iSCAT imaging is a useful tool to study how biomolecules interact with 2D materials, a key component in engineering future biosensors.

Interferometric scattering microscopy (iSCAT) is a label-free imaging technique that achieves single-molecule spatial and milli-second temporal resolution. In this technique, we use the combination of reflected and scattered light of the nanoparticle itself as our signal obviating the need of a label or tags as commonly used in fluorescence microscopy. With iSCAT, photobleaching is not an issue as the signal donor (the particle of interest) provides an infinite budget of scattered photons and hence indefinite imaging times.

2D materials offer huge potential as substrates to build devices for biosensing applications but are plagued by unwanted interactions such as binding/sticking. Controlling such interactions will be critical for the continued exploration of 2D materials in biosensing. In this work, we engineer and tune the surface interactions of hexagonal boron nitride (hBN) to direct the motion and diffusion of DNA. Using ISCAT and fluorescence microscopy techniques, we explore the nanoscopic interactions of DNA with different 2D materials. We show that pristine hBN flakes exhibit the lowest surface interactions and DNA bind preferentially to the edges and regions of high defect density of the hBN flake. We tap into a recently reported Xenon Focused Ion Beam (FIB) technique to engineer edges and defects on hBN flakes. Our technique harnesses a Xenon-FIB to lightly irradiate the desired regions of the hBN flake followed by subsequent etching in water which allows for a much cleaner hBN surface. We are able to enhance DNA binding and affinity at defined locations by inducing defects using FIB. By creating long tracks of defects, we induce diffusion along our created tracks, thereby allowing us to direct motion of the DNA molecules. We envision future devices where such engineered interactions are able to direct biomolecules to sensing regions (such as a nanopore) on 2D material based devices thereby increasing the rate of analyte capture and sensitivity of single molecule sensing devices.

Solid-state nanoapertures (nanometer sized holes within a membrane) allow for the interrogation of single molecules by probing them within their nanoscale volume through which molecules pass in a single-file manner. Molecules are probed using two main techniques: ionic sensing where a salt solution and applied voltage is used to produce an ionic current through the nanopore, and optical sensing through a shift in the resonance wavelength of the plasmonic nanoaperture. Here, we briefly review the basic principles, applications, and challenges in sensing with solid-state nanoapertures, as well as some strategies for further improvements. We compare the complimentary features of the two approaches and highlight recent attempts to combine them into new sensing platforms.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanopar-ticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extru-sion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring -form-ing subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

Nucleoid-associated proteins (NAPs) are a class of highly abundant DNA-binding proteins in bacteria and archaea. While both the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding and stabilizing higher-order nucleoprotein complexes in the bacterial chromosome. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding site arrangements. We find that spatial organization of the protein binding sites can govern the higher-order architecture of the nucleoprotein complexes. Based on sequence arrangement the complexes differed in their global shape and compaction as well as the extent of FIS and H-NS binding. Our observations highlight the important role the DNA sequence plays in driving structural differentiation within the bacterial chromosome.

2D nanoslit devices, where two crystals with atomically flat surfaces are separated by only a few nanometers, have attracted considerable attention because their tunable control over the confinement allows for the discovery of unusual transport behavior of gas, water, and ions. Here, the passage of double-stranded DNA molecules is studied through nanoslits fabricated from exfoliated 2D materials, such as graphene or hexagonal boron nitride, and the DNA polymer behavior is examined in this tight confinement. Two types of events are observed in the ionic current: long current blockades that signal DNA translocation and short spikes where DNA enters the slits but withdraws. DNA translocation events exhibit three distinct phases in their current-blockade traces-loading, translation, and exit. Coarse-grained molecular dynamics simulation allows the different polymer configurations of these phases to be identified. DNA molecules, including folds and knots in their polymer structure, are observed to slide through the slits with near-uniform velocity without noticeable frictional interactions of DNA with the confining graphene surfaces. It is anticipated that this new class of 2D-nanoslit devices will provide unique ways to study polymer physics and enable lab-on-a-chip biotechnology.

We have developed a fabrication methodology for label-free optical trapping of individual nanobeads and proteins in inverted-bowtie-shaped plasmonic gold nanopores. Arrays of these nanoapertures can be reliably produced using focused ion beam (FIB) milling with gap sizes of 10-20 nm, single-nanometer variation, and with a remarkeie stability that allows for repeated use. We employ an optical readout where the presence of the protein entering the trap is marked by an increase in the transmission of light through the nanoaperture from the shift of the plasmonic resonance. In addition, the optical trapping force of the plasmonic nanopores allows 20-nm polystyrene beads and proteins, such as beta-amylase and Heat Shock Protein (HSP90), to be trapped for very long times (approximately minutes). On demand, we can release the trapped molecule for another protein to be interrogated. Our work opens up new routes to acquire information on the conformation and dynamics of individual proteins.

Small MethodsVolume 3, Issue 7 1970018 Cover PictureFree Access Solid State Nanopores: Nanopore Formation via Tip-Controlled Local Breakdown Using an Atomic Force Microscope (Small Methods 7/2019) Yuning Zhang, Yuning Zhang Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorYoichi Miyahara, Yoichi Miyahara Department of Physics, Texas State University, San Marcos, TX, 78666 USASearch for more papers by this authorNassim Derriche, Nassim Derriche Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorWayne Yang, Wayne Yang Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, 2629 HZ Delft, The NetherlandsSearch for more papers by this authorKhadija Yazda, Khadija Yazda Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorXavier Capaldi, Xavier Capaldi Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorZezhou Liu, Zezhou Liu Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorPeter Grutter, Peter Grutter Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorWalter Reisner, Walter Reisner Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this author Yuning Zhang, Yuning Zhang Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorYoichi Miyahara, Yoichi Miyahara Department of Physics, Texas State University, San Marcos, TX, 78666 USASearch for more papers by this authorNassim Derriche, Nassim Derriche Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorWayne Yang, Wayne Yang Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, 2629 HZ Delft, The NetherlandsSearch for more papers by this authorKhadija Yazda, Khadija Yazda Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorXavier Capaldi, Xavier Capaldi Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorZezhou Liu, Zezhou Liu Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorPeter Grutter, Peter Grutter Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this authorWalter Reisner, Walter Reisner Department of Physics, McGill University, Montreal, QC, H3A 2T8 CanadaSearch for more papers by this author First published: 08 July 2019 https://doi.org/10.1002/smtd.201970018Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Developing a scalable method for creating highly localized single nanopore and nanopore arrays in ultra-thin membranes has long been the Achilles' Heel of the vastly growing field of nanopore bio-sensing. In article number 1900147, Yuning Zhang, Yoichi Miyahara, Peter Grutter, Walter Reisner and co-workers report a new method for creating nanoscale pores and nanopore arrays in thin SiNx membranes. Tip-controlled local breakdown (TCLB) uses a conductive tip in an atomic force microscope to create pores at precise positions on a membrane by inducing highly localized dielectric breakdown. TCLB is able to fabricate sub 5 nm diameter nanopores within tens of milliseconds, with high scalability, and with nanometer positioning precision. The cover depicts the process of a single nanopore forming at the tip, with pore arrays already fabricated in the background. Citing Literature Volume3, Issue7July 10, 20191970018 RelatedInformation

The dielectric breakdown approach for forming nanopores has greatly accelerated the pace of research in solid-state nanopore sensing, enabling inexpensive formation of nanopores via a bench top setup. Here we demonstrate the potential of tip controlled dielectric breakdown (TCLB) to fabricate pores 100$\times$ faster, with high scalability and nanometre positioning precision. A conductive atomic force microscope (AFM) tip is brought into contact with a nitride membrane positioned above an electrolyte reservoir. Application of a voltage pulse at the tip leads to the formation of a single nanoscale pore. Pores are formed precisely at the tip position with a complete suppression of multiple pore formation. In addition, our approach greatly accelerates the electric breakdown process, leading to an average pore fabrication time on the order of 10 ms, at least 2 orders of magnitude shorter than achieved by classic dielectric breakdown approaches. With this fast pore writing speed we can fabricate over 300 pores in half an hour on the same membrane.

We report a simple and scalable technique for the fabrication of nanopore arrays on freestanding SiN and graphene membranes based on electron-beam lithography and reactive ion etching. By controlling the dose of the single-shot electron-beam exposure, circular nanopores of any size down to 16 nm in diameter can be fabricated in both materials at high accuracy and precision. We demonstrate the sensing capabilities of these nanopores by translocating dsDNA through pores fabricated using this method, and find signal-to-noise characteristics on par with transmission-electron-microscope-drilled nanopores. This versatile lithography-based approach allows for the high-throughput manufacturing of nanopores and can in principle be used on any substrate, in particular membranes made out of transferable two-dimensional materials.

The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (i) pore-to-pore transfer, which can be controlled at near 100% efficiency, and (ii) DNA molecules bridging between the two nanopores, which enables detection with an enhanced temporal resolution (e.g., an increase of more than 2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resolution readout of biological molecules.