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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 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.