Single Cell Transfection with Single Molecule Resolution Using a Synthetic Nanopore

We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient but also transfects with single molecule resolution at low voltage (1 V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precisionno other method offers such capabilities

Direct, Concurrent Measurements of the Forces and Currents Affecting DNA in a Nanopore with Comparable Topography

We report direct, concurrent measurements of the forces and currents associated with the translocation of a single-stranded DNA molecule tethered to the tip of an atomic force microscope (AFM) cantilever through synthetic pores with topagraphies comparable to the DNA. These measurements were performed to gauge the signal available for sequencing and the electric force required to impel a single molecule through synthetic nanopores ranging from 1.0 to 3.5 nm in diameter in silicon nitride membranes 6–10 nm thick. The measurements revealed that a molecule can slide relatively frictionlessly through a pore, but regular fluctuations are observed intermittently in the force (and the current) every 0.35–0.72 nm, which are attributed to individual nucleotides translating through the nanopore in a turnstile-like motion

Wiring Together Synthetic Bacterial Consortia to Create a Biological Integrated Circuit

The promise of adapting biology to information processing will not be realized until engineered gene circuits, operating in different cell populations, can be wired together to express a predictable function. Here, elementary biological integrated circuits (BICs), consisting of two sets of transmitter and receiver gene circuit modules with embedded memory placed in separate cell populations, were meticulously assembled using live cell lithography and wired together by the mass transport of quorum-sensing (QS) signal molecules to form two isolated communication links (comlinks). The comlink dynamics were tested by broadcasting “clock” pulses of inducers into the networks and measuring the responses of functionally linked fluorescent reporters, and then modeled through simulations that realistically captured the protein production and molecular transport. These results show that the comlinks were isolated and each mimicked aspects of the synchronous, sequential networks used in digital computing. The observations about the flow conditions, derived from numerical simulations, and the biofilm architectures that foster or silence cell-to-cell communications have implications for everything from decontamination of drinking water to bacterial virulence

Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (<i>i.e.</i>, a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of <i>a single protein molecule</i> by measuring volumes of 0.1 nm³ per read

Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (<i>i.e.</i>, a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of <i>a single protein molecule</i> by measuring volumes of 0.1 nm³ per read

Direct Visualization of Single-Molecule Translocations through Synthetic Nanopores Comparable in Size to a Molecule

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time

Single-molecule protein identification by sub-nanopore sensors - Fig 2

<p>(a) An example of a pore current trace acquired from a denatured H3.3 histone translocating through sub-nanopore with a nominal diameter of 0.5-nm. (b) The bottom trace is a magnified view of a 600 ms region of a top trace, showing a current blockade associated with the translocation of a single protein molecule. In the figure, higher values correspond to larger blockade currents. Blockades, associated with the translocation of single proteins were identified as regions with fluctuations five standard deviations above the noise level and with duration > 1 ms.); and then the raw current <i>I</i> was converted into <i>fractional blockade current</i>.</p

Datasets summary.

<p>Datasets summary.</p

Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, <i>Escherichia coli</i> and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD₅₀ = 30 e^–/nm², which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the <i>E. coli</i> membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD₅₀. On the other hand, with a cumulative dose below LD₅₀ (and lower SNR), it was still possible to visualize the infection of <i>E. coli</i> by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution

TEM micrograph of sub-nanopore is shown with a nominal diameter of 0.5 nm sputtered through silicon nitride membrane about 10-nm thick.

<p>The shot noise is associated with electron transmission through the pore. (center) Multi-slice simulations of the TEM image are consistent with the experimental imaging conditions. The simulations correspond to a bi-conical pore with a 0.5 x 0.4 <i>nm</i>² cross-section and a 15 cone angle at defocus of -40 nm. (right) Space-filled model of the same pore is shown where the <i>Si</i> atoms are represented by spheres with a 0.235 nm diameter and <i>N</i> atoms by spheres with a 0.13 nm diameter. The scale bars are 1 nm.</p