4 research outputs found
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 precisionno other method offers such capabilities
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
In Vitro Characterization of Surface Properties Through Living Cells
The ability to probe an interface beneath a layer of living cells in situ without the need for labeling and fixation has the potential to unlock some of the key questions in cell biology and biointerfacial phenomena. Here, we show that vibrational sum frequency generation (SFG) spectroscopy can be used to detect alkanethiol self-assembled monolayers (SAMs) buried underneath a layer of living erythrocytes (ECs). SFG spectra with and without ECs showed the spectral signatures typical of these SAMs, indicating that the signal was being generated solely by the SAM and was not influenced by the presence of cells. Direct comparison of infrared spectroscopy to SFG measurements of cells adhered on a fibronectin layer showed that the SFG signal emanated solely from this layer. These results have important implications for the characterization of surfaces in biomedical, environmental, and industrial applications
Biological Noise Abatement: Coordinating the Responses of Autonomous Bacteria in a Synthetic Biofilm to a Fluctuating Environment Using a Stochastic Bistable Switch
Noise is inherent to single cell
behavior. Its origins can be traced
to the stochasticity associated with a few copies of genes and low
concentrations of protein and ligands. We have studied the mechanisms
by which the response of noisy elements can be entrained for biological
signal processing. To elicit predictable biological function, we have
engineered a gene environment that incorporates a gene regulatory
network with the stringently controlled microenvironment found in
a synthetic biofilm. The regulatory network leverages the positive
feedback found in quorum-sensing regulatory components of the <i>lux</i> operon, which is used to coordinate cellular responses
to environmental fluctuations. Accumulation of the Lux receptor in
cells, resulting from autoregulation, confers a rapid response and
enhanced sensitivity to the quorum-sensing molecule that is retained
after cell division as epigenetic memory. The memory of the system
channels stochastic noise into a coordinated response among quorum-sensing
signal receivers in a synthetic biofilm in which the noise diminishes
with repeated exposure to noisy transmitters on the input of a signaling
cascade integrated into the same biofilm. Thus, gene expression in
the receivers, which are autonomous and do not communicate with each
other, is synchronized to fluctuations in the environment