Xiyun Guan

Stochastic sensing of TNT with a genetically engineered pore

Engineered versions of the transmembrane protein pore α‐hemolysin (αHL) can be used as stochastic sensing elements for the identification and quantification of a wide variety of analytes at the single‐molecule level. Until now, nitroaromatic analytes have eluded detection by this approach. We now report that binding sites for nitroaromatics can be built within the lumen of the αHL pore from simple rings of seven aromatic amino acid side chains (Phe, Tyr or Trp). By monitoring the ionic current that passes through a single pore at a fixed applied potential, various nitroaromatics can be distinguished from TNT on the basis of the amplitude and duration of individual current‐blocking events. Rings of less than seven aromatics bind the analytes more weakly; this suggests that direct aromatic–aromatic interactions are involved. The engineered pores should be useful for the detection of explosives and, in combination with computational approaches and structural analysis, they could further our understanding of noncovalent interactions between aromatic molecules.

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.

Nanopore Stochastic Detection: Diversity, Sensitivity, and Beyond

Nanopore sensors have emerged as a label-free and amplification-free technique for measuring single molecules. First proposed in the mid-1990s, nanopore detection takes advantage of the ionic current modulations produced by the passage of target analytes through a single nanopore at a fixed applied potential. Over the last 15 years, these nanoscale pores have been used to sequence DNA, to study covalent and non-covalent bonding interactions, to investigate biomolecular folding and unfolding, and for other applications. A major issue in the application of nanopore sensors is the rapid transport of target analyte molecules through the nanopore. Current recording techniques do not always accurately detect these rapid events. Therefore, researchers have looked for methods that slow molecular and ionic transport. Thus far, several strategies can improve the resolution and sensitivity of nanopore sensors including variation of the experimental conditions, use of a host compound, and modification of the analyte molecule and the nanopore sensor. In this Account, we highlight our recent research efforts that have focused on applications of nanopore sensors including the differentiation of chiral molecules, the study of enzyme kinetics, and the determination of sample purity and composition. Then we summarize our efforts to regulate molecular transport. We show that the introduction of various surface functional groups such as hydrophobic, aromatic, positively charged, and negatively charged residues in the nanopore interior, an increase in the ionic strength of the electrolyte solution, and the use of ionic liquid solutions as the electrolyte instead of inorganic salts may improve the resolution and sensitivity of nanopore stochastic sensors. Our experiments also demonstrate that the introduction of multiple functional groups into a single nanopore and the development of a pattern-recognition nanopore sensor array could further enhance sensor resolution. Although we have demonstrated the feasibility of nanopore sensors for various applications, challenges remain before nanopore sensing is deployed for routine use in applications such as medical diagnosis, homeland security, pharmaceutical screening, and environmental monitoring.

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.

Nanopore Detection of Copper Ions using a Polyhistidine Probe

We report a stochastic nanopore sensing method for the detection of Cu(2+) ions. By employing a polyhistidine molecule as a chelating agent, and based on the different signatures of the events produced by the translocation of the chelating agent through an α-hemolysin pore in the absence and presence of target analytes, trace amounts of copper ions could be detected with a detection limit of 40 nM. Importantly, although Co(2+), Ni(2+), and Zn(2+) also interacts with the polyhistidine molecule, since the event residence times and/or blockage amplitudes for these metal chelates are significantly different from those of copper chelates, these metal ions do not interfere with Cu(2+) detection. This chelating reaction approach should find useful application in the development of nanopore sensors for other metal ions.

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.