Qitao Zhao

Real-Time Monitoring of Peptide Cleavage Using a Nanopore Probe

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.

Slowing DNA translocation through nanopores using a solution containing organic salts.

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.

Study of Peptide Transport through Engineered Protein Channels

Peptides play important roles in a variety of biological processes. Here, we studied the transport of peptides containing mainly aromatic amino acids in protein pores engineered with aromatic binding sites. With an increase in the length of the peptide, both the event mean dwell time and the current blockage amplitude increased. The dissociation rate constants k(off) decreased significantly, while the association rate constants k(on) decreased slowly as the peptide length increased. Thus, the overall reaction formation constants K(f), and hence the binding affinities of various peptides to the protein pore, are largely dependent upon the dissociation rate constants rather than the association rate constants. Furthermore, in a protein channel modified with aromatic binding sites, aromatic amino acid components contributed more to the dwell time and current blockage of the events than other types of amino acids, although the van der Waals volumes of amino acids also affected the event signatures. The effect of protein structure on peptide translocation was also investigated. With more aromatic binding sites engineered inside the lumen of the protein pore, a stronger binding affinity between peptides and the pore was observed. With the much enhanced resolution of the engineered protein pore, a series of short peptides, including those differing by a single amino acid, was successfully differentiated and simultaneously quantified. In addition to providing a rapid and cost-effective method for the peptide detection, the engineered protein pore approach offers the potential for peptide and protein sequencing.

Nanopore Stochastic Detection of a Liquid Explosive Component and Sensitizers Using Boromycin and an Ionic Liquid Supporting Electrolyte

We report a rapid and sensitive stochastic nanopore sensing method for the detection of monovalent cations and liquid explosive components and their sensitizers. The sensing element is a wild-type alpha-hemolysin protein pore with boromycin as a molecular adaptor, while a solution containing an ionic liquid was used as the background electrolyte. The analyte-boromycin complexes showed significantly different signatures. Specifically, their event mean dwell times and amplitudes were sufficiently distinct to permit the convenient differentiation and even simultaneous detection of liquid explosive components in aqueous environments. In addition, the results also demonstrate that the usage of specific ionic liquid salt solutions instead of NaCl or KCl solution as supporting electrolyte provides a useful means to greatly enhance the sensitivity of the nanopore for some analytes in stochastic sensing.

Stochastic study of the effect of ionic strength on noncovalent interactions in protein pores.

Salt plays a critical role in the physiological activities of cells. We show that ionic strength significantly affects the kinetics of noncovalent interactions in protein channels, as observed in stochastic studies of the transfer of various analytes through pores of wild-type and mutant α-hemolysin proteins. As the ionic strength increased, the association rate constant of electrostatic interactions was accelerated, whereas those of both hydrophobic and aromatic interactions were retarded. Dramatic decreases in the dissociation rate constants, and thus increases in the overall reaction formation constants, were observed for all noncovalent interactions studied. The results suggest that with the increase of salt concentration, the streaming potentials for all the protein pores decrease, whereas the preferential selectivities of the pores for either cations or anions drop. Furthermore, results also show that the salt effect on the rate of association of analytes to a pore differs significantly depending on the nature of the noncovalent interactions occurring in the protein channel. In addition to providing new insights into the nature of analyte-protein pore interactions, the salt-dependence of noncovalent interactions in protein pores observed provides a useful means to greatly enhance the sensitivity of the nanopore, which may find useful application in stochastic sensing.

Probing mercury(II)-DNA interactions by nanopore stochastic sensing.

In this work, DNA-Hg(II) interactions were investigated by monitoring the translocation of DNA hairpins in a protein ion channel in the absence and presence of metal ions. Our experiments demonstrate that target-specific hairpin structures could be stabilized much more significantly by mercuric ions than by the stem length and the loop size of the hairpin due to the formation of Thymine-Hg(II)-Thymine complexes. In addition, the designed DNA probe allows the development of a highly sensitive nanopore sensor for Hg(2+) with a detection limit of 25 nM. Further, the sensor is specific, and other tested metal ions including Pb(2+), Cu(2+), Cd(2+), and so on with concentrations of up to 2 orders of magnitude greater than that of Hg(2+) would not interfere with the mercury detection.

Translocation of single-stranded DNA through the α-hemolysin protein nanopore in acidic solutions

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, whereas 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 being 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.

Stochastic sensing of biomolecules in a nanopore sensor array

In this study, we demonstrate that a pattern-recognition stochastic sensor can be constructed by employing an array of protein pores modified with a variety of non-covalent bonding sites as effective sensing elements. The collective responses of each of the individual component nanopores to a compound produce diagnostic patterns characterized by event dwell time, amplitude, and voltage dependence, which can independently or collectively serve as (an) analyte signature(s). With an increase in the dimensionality of the signal, the nanopore sensor array provides enhanced resolution for the differentiation of analytes compared to a single-pore configuration. This allows identification of a target analyte from a mixture or the potential for simultaneous detection. The pattern-recognition nanopore method is envisaged for further development as a miniaturized and automated sensing technique, which could find potential use as a laboratory or clinical tool for routine sensor applications, including environmental monitoring, drug discovery, medical diagnosis, and homeland security.

Stochastic Detection of Terrorist Agents and Biomolecules in a Biological Channel

Stochastic sensing can detect analytes at the single-molecule level, in which a biological ion channel embedded in a lipid bilayer or a nano-scale sized pore fabricated in a solid-state membrane is used as the sensing element. By monitoring the ionic current modulations induced by the passage of the target analyte through the single pore, both the concentration and the identity of the analyte can be revealed. In this chapter, we highlight recent advances in the stochastic detection of terrorist agents and biomolecules, and in real-world sample analysis using alpha-hemolysin protein ion channels.