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ABSTRACT Detection of ultra‐short peptides is one of the critical steps toward deeper understanding of proteins and the sequencing of amino acids using solid‐state nanopores. The ability of solid‐state nanopores to detect these ultra‐short peptides can help us reveal their hydrodynamic state under different conditions like the concentrations and the external voltage, which may further guide the future development in this field for deeper investigation and possible improvement. In this study, we fabricate Si_xN_ynanopores by CDB with various pore sizes and use them to detect ultra‐short peptides comprised of five different amino acids. The peptide translocation events are extracted under various external voltages. Optimal experimental conditions such as the concentration of electrolytes and analytes, and the range of external voltage are investigated and compared. The statistical results based on volume exclusion analysis indicate that a significant portion of peptides exist in aggregation form. Due to the limitations of Si_xN_ynanopores such as the thickness and the noise, most of the single peptide signals are masked under the baseline noise. In addition, the results show that peptide–pore interactions are dependent upon the diameter of the nanopore. Higher voltage may also influence the degree of peptide aggregations. This study serves to further comprehend the physical and chemical properties of peptides, find possible ways to improve the performance of solid‐state nanopores in the area of protein and peptide detections, and indicate the potential improvements in solid‐state nanopore‐based peptide sequencing. 

Abstract Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid‐state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid‐state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B–the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h⁻¹. The current noise is exceptionally stable with traces typically showing <10 pA h⁻¹increase in noise. Furthermore, a real‐time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid‐state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite. 

Abstract A nanopore device is capable of providing single‐molecule level information of an analyte as they translocate through the sensing aperture—a nanometer‐sized through‐hole—under the influence of an applied electric field. In this study, a silicon nitride (Si_xN_y)‐based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid‐state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture. 

Abstract Electrolyte chemistry plays an important role in the transport properties of analytes through nanopores. Here, we report the translocation properties of the protein human serum transferrin (hSTf) in asymmetric LiCl salt concentrations with either positive (C_trans/C_cis< 1) or negative chemical gradients (C_trans/C_cis> 1). Thecisside concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while thetransside concentration varied between 0.5 to 4 M which resulted in six different configurations, respectively, for both positive and negative gradient types. For positive chemical gradient conditions, translocations were observed in all six configurations for at least one voltage polarity whereas with negative gradient conditions, dead concentrations where no events at either polarity were observed. The flux of Li⁺and Cl⁻ions and their resultant cation or anion enrichment zones, as well as the interplay of electrophoretic and electroosmotic transport directions, would determine whether hSTf can traverse across the pore. 

Abstract Recently, we developed a fabrication method—chemically‐tuned controlled dielectric breakdown (CT‐CDB)—that produces nanopores (through thin silicon nitride membranes) surpassing legacy drawbacks associated with solid‐state nanopores (SSNs). However, the noise characteristics of CT‐CDB nanopores are largely unexplored. In this work, we investigated the 1/fnoise of CT‐CDB nanopores of varying solution pH, electrolyte type, electrolyte concentration, applied voltage, and pore diameter. Our findings indicate that the bulk Hooge parameter (α_b) is about an order of magnitude greater than SSNs fabricated by transmission electron microscopy (TEM) while the surface Hooge parameter (α_s) is ∼3 order magnitude greater. Theα_sof CT‐CDB nanopores was ∼5 orders of magnitude greater than theirα_b, which suggests that the surface contribution plays a dominant role in 1/fnoise. Experiments with DNA exhibited increasing capture rates with pH up to pH ∼8 followed by a drop at pH ∼9 perhaps due to the onset of electroosmotic force acting against the electrophoretic force. The1/fnoise was also measured for several electrolytes and LiCl was found to outperform NaCl, KCl, RbCl, and CsCl. The 1/fnoise was found to increase with the increasing electrolyte concentration and pore diameter. Taken together, the findings of this work suggest the pH approximate 7–8 range to be optimal for DNA sensing with CT‐CDB nanopores. 

We present a successful discrimination of heparin, FGF-1, and heparin–FGF-1 complexes at a single-molecule level through solid-state nanopores. 

Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin’s enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal–protein interactions at the molecular level.