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1. Nanopores map the acid-base properties of a single site in a single DNA molecule

   https://doi.org/10.1093/nar/gkae518  

   Smith, Drew C.; Thomas, Christopher A.; Craig, Jonathan M.; Brinkerhoff, Henry; Abell, Sarah J.; Franzi, Michaela C.; Carrasco, Jessica D.; Hoshika, Shuichi; Benner, Steven A.; Gundlach, Jens H.; et al (June 2024, Nucleic Acids Research)

   Abstract Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas. 

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2. Ensuring fair assessment of solid-state nanopore sensors with reporting baseline current

   https://doi.org/10.1063/5.0167402  

   Dong, Ming; Tang, Zifan; Guan, Weihua (October 2023, Applied Physics Letters)

   In developing solid-state nanopore sensors for single molecule detection, comprehensive evaluation of the nanopore quality is important. Existing studies typically rely on comparing the noise root mean square or power spectrum density values. Nanopores exhibiting lower noise values are generally considered superior. This evaluation is valid when the single molecule signal remains consistent. However, the signal can vary, as it is strongly related to the solid-state nanopore size, which is hard to control during fabrication consistently. This work emphasized the need to report the baseline current for evaluating solid-state nanopore sensors. The baseline current offers insight into several experimental conditions, particularly the nanopore size. Our experiments show that a nanopore sensor with more noise is not necessarily worse when considering the signal-to-noise ratio (SNR), particularly when the pore size is smaller. Our findings suggest that relying only on noise comparisons can lead to inaccurate evaluations of solid-state nanopore sensors, considering the inherent variability in fabrication and testing setups among labs and measurements. We propose that future studies should include reporting baseline current and sensing conditions. Additionally, using SNR as a primary evaluation tool for nanopore sensors could provide a more comprehensive understanding of their performance. 

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3. Threading single proteins through pores to compare their energy landscapes

   https://doi.org/10.1073/pnas.2202779119  

   Tripathi, Prabhat; Firouzbakht, Arash; Gruebele, Martin; Wanunu, Meni (September 2022, Proceedings of the National Academy of Sciences)

   Translocation of proteins is correlated with structural fluctuations that access conformational states higher in free energy than the folded state. We use electric fields at the solid-state nanopore to control the relative free energy and occupancy of different protein conformational states at the single-molecule level. The change in occupancy of different protein conformations as a function of electric field gives rise to shifts in the measured distributions of ionic current blockades and residence times. We probe the statistics of the ionic current blockades and residence times for three mutants of the $\lambda$-repressor family in order to determine the number of accessible conformational states of each mutant and evaluate the ruggedness of their free energy landscapes. Translocation becomes faster at higher electric fields when additional flexible conformations are available for threading through the pore. At the same time, folding rates are not correlated with ease of translocation; a slow-folding mutant with a low-lying intermediate state translocates faster than a faster-folding two-state mutant. Such behavior allows us to distinguish among protein mutants by selecting for the degree of current blockade and residence time at the pore. Based on these findings, we present a simple free energy model that explains the complementary relationship between folding equilibrium constants and translocation rates. 

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4. Noise in nanopore sensors: Sources, models, reduction, and benchmarking

   https://doi.org/10.1016/j.npe.2019.12.008  

   Liang, Shengfa; Xiang, Feibin; Tang, Zifan; Nouri, Reza; He, Xiaodong; Dong, Ming; Guan, Weihua (December 2019, Nanotechnology and Precision Engineering)

   Label-free nanopore sensors have emerged as a new generation technology of DNA sequencing and have been widely used for single molecule analysis. Since the first α-hemolysin biological nanopore, various types of nanopores made of different materials have been under extensive development. Noise represents a common challenge among all types of nanopore sensors. The nanopore noise can be decomposed into four components in the frequency domain (1/f noise, white noise, dielectric noise, and amplifier noise). In this work, we reviewed and summarized the physical models, origins, and reduction methods for each of these noise components. For the first time, we quantitatively benchmarked the root mean square (RMS) noise levels for different types of nanopores, demonstrating a clear material-dependent RMS noise. We anticipate this review article will enhance the understanding of nanopore sensor noises and provide an informative tutorial for developing future nanopore sensors with a high signal-to-noise ratio. 

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5. Mechanism of molecular interaction of acrylate-polyethylene glycol acrylate copolymers with calcium silicate hydrate surfaces

   https://doi.org/10.1039/C9GC03287H  

   Jamil, Tariq; Javadi, Ali; Heinz, Hendrik (March 2020, Green Chemistry)

   Obtaining insights into the adsorption and assembly of polyelectrolytes on chemically variable calcium silicate hydrate (C-S-H) surfaces at the atomic scale has been a longstanding challenge in the chemistry of sustainable building materials and mineral–polymer interactions. Specifically, polycarboxylate ethers (PCEs) based on acrylate and poly(ethylene glycol) acrylate co-monomers are widely used to engineer the fluidity and hydration of cement and play an important role in the search for building materials with a lower carbon footprint. We report the first systematic study of PCE interactions with C-S-H surfaces at the molecular level using simulations at single molecule coverage and comparisons to experimental data. The mechanism of adsorption of the ionic polymers is a two-step process with initial cation adsorption that reverses the mineral surface charge, followed by adsorption of the polymer backbone through ion pairing. Free energies of binding are tunable in a wide range of 0 to −5 kcal mol −1 acrylate monomer. Polymer attraction increases for higher calcium-to-silicate ratio of the mineral and higher pH value in solution, and varies significantly with PCE composition. Thereby, successive negatively charged carboxylate groups along the backbone induce conformation strain and local detachment from the surface. Polyethylene glycol (PEG) side chains in the copolymers avoid contact with the C-S-H surfaces. The results guide in the rational design of adsorption strength and conformations of the comb copolymers, and lay the groundwork to explore the vast phase space of C-S-H compositions, surface morphologies, electrolyte conditions, and PCE films of variable surface coverage. Chemically similar minerals and copolymers also find applications in other structural and biomimetic materials. 

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