Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric Point.

Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concentrations can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irrespective of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelectric point (pI) of Cas9 was found to be correlated and a critical factor in our experiments. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be critical for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.

DNA Coil Dynamics and Hydrodynamic Gating of Pressure-Biased Nanopores.

Nanopores are ideally suited for the analysis of long DNA fragments including chromosomal DNA and synthetic DNA with applications in genome sequencing and DNA data storage, respectively. Hydrodynamic fluid flow has been shown to slow down DNA transit time within the pore, however other influences of hydrodynamic forces have yet to be explored. In this report, a broad analysis of pressure-biased nanopores and the impact of hydrodynamics on DNA transit time, capture rate, current blockade depth, and DNA folding are conducted. Using a 10 nm pore, it is shown that hydrodynamic flow inhibits the early stages of linearization of DNA and produces predominately folded events which are initiated by folded DNA (2-strands) entering the pore. Furthermore, utilizing larger pores (30 nm) leads to unique DNA gating behavior in which DNA events can be switched on and off with the application of pressure. A computational model, based on combining electrophoretic drift velocities with fluid velocities, accurately predicts the pore size required to observe DNA gating. Hydrodynamic fluid flow generated by a pressure bias, or potentially more generally by other mechanisms like electroosmotic flow, is shown to have significant effects on DNA sensing and can be useful for DNA sensing technologies.

Nanodiagnostics: A review of the medical capabilities of nanopores.

Modern diagnostics strive to be accurate, fast, and inexpensive in addition to properly identifying the presence of a disease, infection, or illness. Early diagnosis is key; catching a disease in its early stages can be the difference between fatality and treatment. The challenge with many diseases is that detectability of the disease scales with disease progression. Since single molecule sensors, e.g., nanopores, can sense biomolecules at low concentrations, they have the potential to become clinically relevant in many of today's medical settings. With nanopore-based sensing, lower volumes and concentrations are required for detection, enabling it to be clinically beneficial. Other advantages to using nanopores include that they are tunable to an enormous variety of molecules and boast low costs, and fabrication is scalable for manufacturing. We discuss previous reports and the potential for incorporating nanopores into the medical field for early diagnostics, therapeutic monitoring, and identifying relapse/recurrence.

On the origins of conductive pulse sensing inside a nanopore.

Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concentrations. Contrarily, conductive pulses are observed under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).

Measuring trapped DNA at the liquid-air interface for enhanced single molecule sensing.

Nanopore sensing is a promising tool with widespread application in single-molecule detection. Borosilicate glass nanopores are a viable alternative to other solid-state nanopores due to low noise and cost-efficient fabrication. For dielectric materials, including borosilicate glass, the capacitive noise is one of the major contributors to noise, which depends on the wall thickness and the surface area submerged in an ionic solution. Here, we investigated the root mean square (IRMS) noise and ionic conductance for borosilicate nanopores in different depths (i.e., tip submersion depth) ranging from the solution surface (assumed to be zero) to 5000 µm. Our findings demonstrate a decrease in IRMS noise as the pipette moves toward the surface. We further demonstrate that borosilicate nanopores can detect single lambda DNA (λ-DNA) molecules with a high signal-to noise ratio close to the liquid-air interface. Specifically, our results indicate a higher signal to noise ratio as the submersion depth is reduced owing to the reduced surface area and thus capacitive noise. Further, our experimental results show higher DNA capture frequency at the air-water interface due to a combined effect of evaporation and an evaporation-induced thermal gradient at the surface. Therefore, our findings demonstrate that borosilicate glass nanopores are suitable for studying interfacial concentration gradients of molecules, specifically DNA, with a higher signal to noise.

Optimization of the Mechanical Properties and the Cytocompatibility for the PMMA Nanocomposites Reinforced with the Hydroxyapatite Nanofibers and the Magnesium Phosphate Nanosheets.

Commercial poly methyl methacrylate (PMMA)-based cement is currently used in the field of orthopedics. However, it suffers from lack of bioactivity, mechanical weakness, and monomer toxicity. In this study, a PMMA-based cement nanocomposite reinforced with hydroxyapatite (HA) nanofibers and two-dimensional (2D) magnesium phosphate MgP nanosheets was synthesized and optimized in terms of mechanical property and cytocompatibility. The HA nanofibers and the MgP nanosheets were synthesized using a hydrothermal homogeneous precipitation method and tuning the crystallization of the sodium-magnesium-phosphate ternary system, respectively. Compressive strength and MTT assay tests were conducted to evaluate the mechanical property and the cytocompatibility of the PMMA-HA-MgP nanocomposites prepared at different ratios of HA and MgP. To optimize the developed nanocomposites, the standard response surface methodology (RSM) design known as the central composite design (CCD) was employed. Two regression models generated by CCD were analyzed and compared with the experimental results, and good agreement was observed. Statistical analysis revealed the significance of both factors, namely, the HA nanofibers and the MgP nanosheets, in improving the compressive strength and cell viability of the PMMA-MgP-HA nanocomposite. Finally, it was demonstrated that the HA nanofibers of 7.5% wt and the MgP nanosheets of 6.12% wt result in the PMMA-HA-MgP nanocomposite with the optimum compressive strength and cell viability.

On the structure and chemistry of iron oxide cores in human heart and human spleen ferritins using graphene liquid cell electron microscopy.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.