Brian J. Nablo

Theory for polymer analysis using nanopore-based single-molecule mass spectrometry

Nanometer-scale pores have demonstrated potential for the electrical detection, quantification, and characterization of molecules for biomedical applications and the chemical analysis of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theoretical models that incorporate the interactions between chemicals (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) molecules in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a reduction in the mobile cation concentration inside the pore and an increase in the affinity between the polymer and the pore. The model was used to estimate the free energy of formation for K+-PEG inside the nanopore (≈-49.7 meV) and the free energy of PEG partitioning into the nanopore (≈0.76 meV per ethylene glycol monomer). The results suggest that rational, physical models for the analysis of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chemical and biological applications.

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.

Sizing the Bacillus anthracis PA63 Channel with Nonelectrolyte Poly(Ethylene Glycols)

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pores PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is <2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Uncovering the Contribution of Microchannel Deformation to Impedance-Based Flow Rate Measurements

Changes in electrical impedance have previously been used to measure fluid flow rate in microfluidic channels. Ionic redistribution within the electrical double layer by fluid flow has been considered to be the primary mechanism underlying such impedance based microflow sensors. Here we describe a previously unappreciated contribution of microchannel deformation to such measurements. We found that flow-induced microchannel deformation contributes significantly to the change in electrical impedance of solutions, in particular to those solutions producing an electrical double layer in the order of a few tens of nanometers (i.e., containing relatively high ionic strength). Since the flow velocity at the measurement surface is near zero, due to the laminar nature of the flow, the contribution of the double layer under the conditions mentioned above should be negligible. In contrast, an increase in the fluid flow rate results in an increase in the microchannel cross-sectional area (because of higher local pressure), therefore, producing a decrease in solution resistance between the two electrodes. Our results suggest that microflow sensors based on the concept of elastic deformation could be designed for in situ monitoring and fine control of fluid flow in flexible microfluidics. Finally, we show that purposefully engineering a larger deformability of the microchannel, by changing the geometry and the Youngs modulus of the microchannel, enhances the sensitivity of this flow rate measurement.

Theory of high-resolution single molecule size determination using a solitary nanopore

Fluorescence correlation spectroscopy, fluorescence resonance energy transfer, optical tweezers, and atomic force microscopy are several modern tools used to probe the physical properties of polymers (e.g., intramolecular forces, structural changes and dynamics). Nanopore-based techniques were developed to detect, characterize, and quantify a wide-range of polymer types (e.g., single-stranded RNA/DNA, proteins, biowarfare agents, therapeutic agents against anthrax toxins, and chemically synthesized molecules). For example, it was recently shown that a single nanometer-scale pore permits the discrimination of polymers whose lengths differ by approximately 0.15 nm5. We will discuss the physical basis of this method and its high resolving power.

Electronic Detection of Biomolecules

We are developing metrologies that could enable the rapid screening of therapeutic agents against anthrax infection, determine the structure-function relationship of anthrax toxins, and the rapid detection of pathogens and cellular biomarkers. Our goals are to better understand the threats posed by biowarfare agents and thereby provide the metrology needed to effectively detect and combat these toxins. These techniques might also prove useful for genomic and proteomic applications.