Here we report a rapid, label-free method for monitoring peptide cleavage. Monitoring peptide translocation through an engineered ion channel in the absence and the presence of an enzyme allowed quantitative chemical kinetics information on enzymatic processes to be obtained. In addition to its potential application in disease diagnostics and drug discovery, this peptide/protein cleavage approach is envisioned for further development as a novel rapid, label-free protein sequencing technique.

One of the key challenges to nanopore DNA sequencing is to slow down DNA translocation. Here, we report that the translocation velocities of various DNA homo- and copolymers through protein pores could be significantly decreased by using electrolyte solutions containing organic salts. Using a butylmethylimidazolium chloride (BMIM-Cl) solution instead of the commonly used KCl solution, DNA translocation rates on the order of hundreds of microseconds per nucleotide base were achieved. The much enhanced resolution of the nanopore coupled with different event blockage amplitudes produced by different nucleotides permits the convenient differentiation between various DNA molecules.

The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e., deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, while the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to be used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing.

a b s t r a c tWe report a stochastic nanopore sensing method for the detection of organophosphorus nerve agent hydrolysis products. By employing an engineered ␣-hemolysin single pore embedded in a planar lipid bilayer as the stochastic sensing element and ␤-cyclodextrin as a host molecule, trace amounts of soman and cyclosarin hydrolytes could be detected, with detection limits of 53 nM and 102 nM, respectively. Importantly, sarin, tabun, and VX hydrolysis products, as well as other common pesticides, do not interfere with detection of the analytes. The method offers the potential as a rapid and sensitive sensing technique for use in on-site analysis of nerve agents in environmental monitoring applications at the single-molecule level.

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