Brandon R. Bruhn

Real-time shape approximation and fingerprinting of single proteins using a nanopore

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

Osmosis-Based Pressure Generation: Dynamics and Application

This paper describes osmotically-driven pressure generation in a membrane-bound compartment while taking into account volume expansion, solute dilution, surface area to volume ratio, membrane hydraulic permeability, and changes in osmotic gradient, bulk modulus, and degree of membrane fouling. The emphasis lies on the dynamics of pressure generation; these dynamics have not previously been described in detail. Experimental results are compared to and supported by numerical simulations, which we make accessible as an open source tool. This approach reveals unintuitive results about the quantitative dependence of the speed of pressure generation on the relevant and interdependent parameters that will be encountered in most osmotically-driven pressure generators. For instance, restricting the volume expansion of a compartment allows it to generate its first 5 kPa of pressure seven times faster than without a restraint. In addition, this dynamics study shows that plants are near-ideal osmotic pressure generators, as they are composed of many small compartments with large surface area to volume ratios and strong cell wall reinforcements. Finally, we demonstrate two applications of an osmosis-based pressure generator: actuation of a soft robot and continuous volume delivery over long periods of time. Both applications do not need an external power source but rather take advantage of the energy released upon watering the pressure generators.

Dual-pore glass chips for cell-attached single-channel recordings

While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance. Here, femtosecond laser ablation was used to fabricate dual-pore glass chips optimized for use in cell-attached single-channel recordings that circumvent this problem by using different pores to position a cell and to establish a seal. This dual-pore design also permitted the use of a relatively small patch aperture (D ~ 150 to 300 nm) that is better-suited for establishing high-resistance seals than the micropores used typically in planar patch-clamp setups (D ~ 1 to 2 μm) without compromising the ability of the device to position a cell. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (rate of gigaseal formation = 61%, mean seal resistance = 53 GΩ), maintained gigaseals for prolonged durations (up to 6 hours), achieved RMS noise values as low as 0.46 pA at 5 kHz bandwidth, and enabled single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp.

Dual-Pore Glass Chips for Single-Channel Recording

Despite the widespread use of high-throughput planar patch-clamp instruments, the conventional pipette-based technique remains the method of choice for recording single-channel activity. Generally, planar platforms are not well suited for single-channel studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms typically use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, thereby reducing the seal resistance. Here, femtosecond laser ablation is used to fabricate dual-pore glass chips for use in low-noise, single-channel recordings that circumvent this problem. One pore positions a cell by suction while another nearby pore, the recording pore, avoids contamination by maintaining positive pressure until a cell is positioned and then establishes a seal. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (>10 GΩ), maintained gigaseals for prolonged durations (up to 6 hrs), and enabled single-channel recordings in cell-attached mode that are comparable to those obtained by conventional patch-clamp.View Large Image | View Hi-Res Image | Download PowerPoint Slide