Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel one-step method based on a wild-type aerolysin nanopore, which enables kinase activity detection without labeling/modification, immobilization, cooperative enzymes and complicated procedures. By virtual of the positively charged confinement of the aerolysin nanopore, the kinase-induced phosphopeptides are specially captured while the positively charged substrate peptides might move away from the pore by the electric field. Combining with internal standard method, the event frequency of the phosphopeptides exhibited a dose-dependent response with kinases. The detection limit of 0.005 U/μL has been achieved with protein kinase A as a model target. This method also allowed kinase inhibitor screening, kinase activity sensing in cell lysates and the real-time monitoring of kinase-catalyzed phosphorylation at singe molecule level, which could further benefit fundamental biochemical research, clinical diagnosis and kinase-targeted drug discovery. Moreover, this nanopore sensor shows strong capacity for the other enzymes that altered substrate charge (e.g., sulfonation, carboxylation, or amidation).

The open-close states of the ion channels in a living system are regulated by multiple stimuli such as ligand, pH, potential and light. Functionalizing natural channels by using synthetic chemistry would provide biological nanopores with novel properties and applications. Here we use para-sulfonato-calix[4]arene-based host-guest supramolecular system to develop artificial gating mechanisms aiming at regulating wild-type α-HL commanded by both ligand and light stimuli. Using the gating property of α-hemolysin, we studied the host-guest interactions between para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. Subsequently, we have extended the application of this gating system to the real-time study of light-induced molecular shuttle based on para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. These experiments provide a more efficient method to develop a general tool to analyze the individual motions of supramolecular systems by using commercially available α-HL nanopores.

Nanopore-based sensing is an emerging analytical technique with a number of important applications, including single-molecule detection and DNA sequencing. In this paper, we developed a Modified Hidden Markov Model (MHMM) to analyze directly the raw (unfiltered) nanopore current blockade data, which significantly reduced the filtering-induced distortion of the nanopore events. Traditionally, prior to further analysis, the measured nanopore data need to be pre-filtered to supress the strong noises. Nonetheless, this would result in the distortion of the shape of the blockade current especially for rapid translocations and bumping blockades. The HMM has been proved to be robust with respect to highly noisy data and thus ideally suitable for processing raw nanopore data directly. Unfortunately, its performance is somehow sensitive to the initial parameters usually preset arbitrarily. To overcome this problem, we use the Fuzzy c-Means (FCM) algorithm to initialize the HMM parameters automatically. Then we use the Viterbi training algorithm to optimize the HMM. Finally, the application results on both the simulated and experimental data are presented to demonstrate the practicability of the developed method for accurate detection of the nanopore current blockade events. The proposed method enables detection of the nanopore events at the highest bandwidth of the commercial instruments to extract the true useful information about the single molecules under analysis.

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore. Furthermore, the accurate isomerization efficiency between the trans and cis form was obtained with single molecule resolution.

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

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The self-assembly process from a 1 : 1 to a 1 : 2 complex, facilitated by para-sulfonatocalix[6]arenes (SC6) as host and methyl viologen (MV(2+)) as guest, was analyzed at the single-molecule level through an α-hemolysin nanopore. Especially, the assembled complex structures were discriminated in real time in the mixture of SC6 and MV(2+).

Inspired by the critical role of ion channel proteins in the regulation of cellular activities, here we developed a new type of synthetic ion channel by simple benzocrown ether-based derivatives M1and M2, where M1 had a dodecyl tail and M2 had a diethylene glycol-conjugated dodecyl tail.Being amphiphilic in nature, the two small molecules were assumed to form crown ether channels through supramolecular interactions in bilayer lipid membranes (BLMs). The efficient ion transport was investigated by both a fluorescence-based vesicle assay and a planar bilayer conductance measurement, and M2 with diethylene glycol substitution exhibited more efficient activity comparable to amphotericin B. Moreover, the presence of a photosensitive o-nitrobenzyl group provided the light-regulation to deactivate ion transport by destroying the channel assembly of the molecules in BLMs, which provides new opportunities for developing intelligent light-regulated systems for biomedical applications based on synthetic small molecules. † Electronic supplementary information (ESI) available: The detailed synthesis and characterizations of compounds, self-assembled properties, some HPTS assays, doping and solubility measurements. See