Proteins are responsible for the occurrence and treatment of many diseases, and therefore protein sequencing will revolutionize proteomics and clinical diagnostics. Biological nanopore approach has proved successful for single‐molecule DNA sequencing, which resolves the identities of 4 natural deoxyribonucleotides based on the current blockages and duration times of their translocations across the nanopore confinement. However, open challenges still remain for biological nanopores to sequentially identify each amino acid (AA) of single proteins due to the inherent complexity of 20 proteinogenic AAs in charges, volumes, hydrophobicity and structures. Herein, we focus on recent exciting advances in biological nanopores for single‐molecule protein sequencing (SMPS) from native protein unfolding, control of peptide translocation, AA identification to applications in disease detection.

Proteins are responsible for the occurrence and treatment of many diseases, and therefore protein sequencing will revolutionize proteomics and clinical diagnostics. Biological nanopore approach has proved successful for single‐molecule DNA sequencing, which resolves the identities of 4 natural deoxyribonucleotides based on the current blockages and duration times of their translocations across the nanopore confinement. However, open challenges still remain for biological nanopores to sequentially identify each amino acid (AA) of single proteins due to the inherent complexity of 20 proteinogenic AAs in charges, volumes, hydrophobicity and structures. Herein, we focus on recent exciting advances in biological nanopores for single‐molecule protein sequencing (SMPS) from native protein unfolding, control of peptide translocation, AA identification to applications in disease detection.

Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep‐P was decelerated by up to 46 folds compared to the wild‐type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep‐Ac, monophosphorylated Pep‐P, di‐modified Pep‐P‐Ac and non‐modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non‐covalent interactions between nanopores and single analytes.

The nanopore approach holds the possibility for achieving single-molecule protein sequencing. However, ongoing challenges still remain in the biological nanopore technology, which aims to identify 20 natural amino acids by reading the ionic current difference with the traditional current-sensing model. In this paper, taking aerolysin nanopores as an example, we calculate and compare the current blockage of each of 20 natural amino acids, which are all far from producing a detectable current blockage difference. Then, we propose a modified solution conductivity of σ′ in the traditional volume exclusion model for nanopore sensing of a peptide. The σ′ value describes the comprehensive result of ion mobility inside a nanopore, which is related to but not limited to nanopore–peptide interactions, and the positions, orientations, and conformations of peptides inside the nanopore. The nanopore experiments of a short peptide (VQIVYK) in wild type and mutant nanopores further demonstrate that the traditional volume exclusion model is not enough to fully explain the current blockage contribution and that many other factors such as enhanced nanopore–peptide interactions could contribute to a dominant part of the current change. This modified sensing model provides insights into the further development of nanopore protein sequencing methods.

The prompt introduction of single-molecule science into undergraduates' curricula is an urgent but challenging task. However, the research experiments are often too complicated and the instruments are too expensive to be widely transferred into a compulsory course of undergraduate students. Herein, we introduce a recently developed nanopore technique into an accessible laboratory course for students with the advantages of simple operation, high repeatability, and low cost. The nanopore course serves to help undergraduates in practice to understand single-molecule behaviors, think at a single molecule level, and gradually develop single-molecule solutions for scientific issues. The learning activities for undergraduate students include setup of the instrument, fabrication of a biological nanopore on an artificial lipid bilayer membrane, detection of individual oligonucleotides, and data analysis. An experiment example is single-molecule detection of the model DNA sample poly(dA) n (n = 4, 5) using the aerolysin nanopore. As our developed nanopore sensing system is simple, portable, and easy to handle, the experiment course could be implemented and popularized in undergraduates' laboratory education. For the undergraduate students who have taken general chemistry and/or biochemistry courses, this hands-on experience of nanopore experiments would benefit them with a better understanding of the chemical world at a single-molecule level and further using single-molecule tools in various applications including DNA and/or protein sequencing.