can be classified as biological, [8] solidstate, [9] or a combination of the two. [10] Biological nanopores include channel proteins such as the product of Curli specific gene G (CsgG) from Escherichia coli, [11] α-hemolysin (αHL) from Staphylococcus aureus, [12] Mycobacterium smegmatis porin A (MspA) [13] and Aeromonas hydrophila Aerolysin (AeL), [14] which spontaneously embed themselves in a lipid bilayer. These types of nanopores can be produced in large numbers by protein expression and purification, have well-characterized crystal structures, and have played a critical role in advancing the resistive pulse sensing technique. [15-17] It is, however, difficult to generate protein pores with diameters that exceed 4 nm. Another limitation is the need to reconstitute these protein pores into lipid or block copolymer membranes, which can be mechanically and chemically fragile. [18] Conversely, solid-state nanopores can be fabricated in various materials such as silicon nitride, silicon oxide, aluminum oxide, hafnium oxide, or graphene. [9,19] Their diameters and geometries can be tuned depending on user requirements, and they can be used repeatedly for experiments with considerable experimental flexibility (e.g., temperature, buffer conditions, presence of detergents or solvents, extreme pH values, applied potential differences, etc.). [20-22] Despite these attractive characteristics, solid-state nanopores suffer from at least three drawbacks. First, they usually interact nonspecifically with biomolecules, leading to resistive pulse artifacts such as attenuated rotation, translation and Nanopore-based resistive-pulse recordings represent a promising approach for single-molecule biophysics with applications ranging from rapid DNA and RNA sequencing to "fingerprinting" proteins. Based on advances in fabrication methods, solid-state nanopores are increasingly providing an alternative to proteinaceous nanopores from living organisms; their widespread adoption is, however, slowed by nonspecific interactions between biomolecules and pore walls, which can cause artifacts and pore clogging. Although efforts to minimize these interactions by tailoring surface chemistry using various physisorbed or chemisorbed coatings have made progress, a straightforward, robust, and effective coating method is needed to improve the robustness of nanopore recordings. Here, covalently attached nanopore surface coatings are prepared from three different polymers using a straightforward "dip and rinse" approach and compared to each other regarding their ability to minimize nonspecific interactions with proteins. It is demonstrated that polymer coatings approach the performance of fluid lipid coatings with respect to minimizing these interactions. Moreover, these polymer coatings enable accurate estimates of the volumes and spheroidal shapes of freely translocating proteins; uncoated or inadequately coated solid-state pores do not have this capability. In addition, these polymer coatings impart physical and chemical stability and enable effic...

Through the use of optical tweezers we performed controlled translocations of DNA–protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on λ-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the nonequilibrium work done during the translocation of a DNA–protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA–protein complexes simultaneous to the localization of their binding sites.

By combining hydraulic pressure with ultrathin and 2D nanopores we demonstrate how pressure can support wetting of nanopores, as well as how pressure induced fluid flow can be used to identify and eliminate wetting artifacts.