Gregory P. Lafyatis

Nanochannel electroporation delivers precise amounts of biomolecules into living cells

Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.

Electroporation dependence on cell size: optical tweezers study.

Electropermeabilization or electroporation is the electrical disruption of a cells membrane to introduce drugs, DNA/RNA, proteins, or other therapies into the cell. Despite four decades of study, the fundamental science of the process remains poorly understood and controversial. We measured the minimum applied electric field required for permeabilization of suspended spherical cells as a function of the cell radius for three cell lines. Key to this work is our use of optical tweezers to precisely position individual cells and enable well-defined, repeatable measurements on cells in suspension. Our findings call into question fundamental assumptions common to all theoretical treatments that we know of. It is generally expected that, for individual cells from a particular cell line, large cells should be easier to electroporate than small ones: the minimum electric field to cause electropermeabilization should scale inversely with the cell diameter. We found instead that each cell line has its own characteristic field that will, on average, cause permeabilization in cells of that line. Electropermeabilization is a stochastic process: two cells which appear identical may have different permeabilization thresholds. However, for all three cell lines, we found that the minimum permeabilization field for any given cell does not depend on its size.

Photon Counting Using a Large Area Avalanche Photodiode Cooled to 100 K

We have detected single 650 nm photons with quantum efficiencies greater than 60% using a large area silicon avalanche photodiode. We cool a 5 mm diam commercially available device to 100 K and operate in the gain‐mode. For most applications—from the near‐infrared to the ultraviolet—this device is the most sensitive photon counting detector that has ever been demonstrated. In typical photon counting applications, this detector should prove to be between two and one hundred times more sensitive than the best currently available devices.

On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance

Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ∼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.

Stiffness-Independent Highly Efficient On-Chip Extraction of Cell-Laden Hydrogel Microcapsules from Oil Emulsion into Aqueous Solution by Dielectrophoresis

A dielectrophoresis (DEP)-based method achieves highly efficient on-chip extraction of cell-laden microcapsules of any stiffness from oil into aqueous solution. The hydrogel microcapsules can be extracted into the aqueous solution by DEP and interfacial tension forces with no trapped oil, while the encapsulated cells are free from electrical damage due to the Faraday cage effect.

Atomic Carbide Bonding Leading to Superior Graphene Networks

A versatile method for achieving atomic carbide-bonded graphene networks on both metallic and non-metallic substrates is described. This consists of vacuum-assisted thermal exfoliation and floatation of functional graphenes at elevated temperatures, followed by deposition on substrates and in situ formation of carbide bonds. The cross-linked graphene networks with an interlayer distance of angstroms exhibits a unique combination of unprecedented properties.

Characterization of cooled large-area silicon avalanche photodiodes

We characterize the operation of large-area high-gain silicon avalanche photodiodes (APDs) at near liquid-nitrogen temperatures. The APDs that we studied have active areas of 64 mm2 and have gains of up to 20 000 at 85 K. We characterized the devices for both the usual, analog mode of operation and for doing single-photon pulse counting. The experimental results were found to be reasonably well described by the McIntyre theory. We independently measured k, the hole/electron ionization ratio—a key parameter in the McIntyre theory—and found it to be ∼6×10−4. Cooled, large-area, high-gain APDs compare favorably to photomultiplier tubes in applications that require high sensitivity at near-infrared wavelengths.

One- and two-dimensional optical lattices on a chip for quantum computing

We propose a way to make arrays of optical frequency dipole-force microtraps for cold atoms above a dielectric substrate. Traps are nodes in the evanescent wave fields above an optical waveguide resulting from interference of different waveguide modes. The traps have features sought in developing neutral atom based architectures for quantum computing:

Fabrication and characterization of extremely smooth large area gold surfaces

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Cell-cell proximity effects in multi-cell electroporation

of laser power yields very tight traps