A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection.

Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing.

A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection.

Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral approach for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing.

Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing.

Viral vectors represent a bottleneck in the manufacturing of cellular therapies. Electroporation has emerged as an approach for non-viral transfection of primary cells, but standard cuvette-based approaches suffer from low throughput, difficult optimization, and incompatibility with large-scale cell manufacturing. Here, we present a novel electroporation platform capable of rapid and reproducible electroporation that can efficiently transfect small volumes of cells for research and process optimization and scale to volumes required for applications in cellular therapy.

High surface-area carbon microcantilevers.

Microscale porous carbon mechanical resonators were formed using carbon nanotube templated microfabrication. These cantilever resonators exhibited nanoscale porosity resulting in a high surface area to volume ratio which could enable sensitive analyte detection in air. These resonators were shown to be mechanically robust and the porosity could be controllably varied resulting in densities from 10 to 10 kg m, with pore diameters on the order of hundreds of nanometers.

Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias.

Single cell whole genome amplification is susceptible to amplification biases that impact the accuracy of single cell sequencing data. To address this, we have developed a microfluidic device for the isolation and purification of genomic DNA from individual cells. The device uses a micropillar array to physically capture single cells and its chromosomal DNA upon extraction.

Microfluidic Device for Aptamer-Based Cancer Cell Capture and Genetic Mutation Detection.

We present a microfluidic device for specifically capturing cancer cells and isolating their genomic DNA (gDNA) for specific amplification and sequence analysis. To capture cancer cells within the device, nucleic acid aptamers that specifically bind to cancer cells were immobilized within a channel containing micropillars designed to increase capture efficiency. The captured cells were lysed in situ, and their gDNA was isolated by physical entanglement within a second smaller-dimensioned micropillar array.

Low-Power Photothermal Self-Oscillation of Bimetallic Nanowires.

We investigate the nonlinear mechanics of a bimetallic, optically absorbing SiN-Nb nanowire in the presence of incident laser light and a reflecting Si mirror. Situated in a standing wave of optical intensity and subject to photothermal forces, the nanowire undergoes self-induced oscillations at low incident light thresholds of <1 μW due to engineered strong temperature-position (T-z) coupling. Along with inducing self-oscillation, laser light causes large changes to the mechanical resonant frequency ω and equilibrium position z that cannot be neglected.

Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles.

Near-field fluorescence cross-correlation spectroscopy on planar membranes.

The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes.

Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device.

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library.

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity.

RAPID-SELEX for RNA aptamers.

Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps.

Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics.

Deoxyribonucleic acid (DNA) is the blueprint on which life is based and transmitted, but the way in which chromatin - a dynamic complex of nucleic acids and proteins - is packaged and behaves in the cellular nucleus has only begun to be investigated. Epigenetic modifications sit 'on top of' the genome and affect how DNA is compacted into chromatin and transcribed into ribonucleic acid (RNA). The packaging and modifications around the genome have been shown to exert significant influence on cellular behaviour and, in turn, human development and disease.

Graphene metallization of high-stress silicon nitride resonators for electrical integration.

High stress stoichiometric silicon nitride resonators, whose quality factors exceed one million, have shown promise for applications in sensing, signal processing, and optomechanics. Yet, electrical integration of the insulating silicon nitride resonators has been challenging, as depositing even a thin layer of metal degrades the quality factor significantly. In this work, we show that graphene used as a conductive coating for Si3N4 membranes reduces the quality factor by less than 30% on average, which is minimal when compared to the effect of conventional metallization layers such as chromium or aluminum.

Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.

Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association.

Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e.

Control of the graphene-protein interface is required to preserve adsorbed protein function.

Graphene's suite of useful properties makes it of interest for use in biosensors. However, graphene interacts strongly with hydrophobic components of biomolecules, potentially altering their conformation and disrupting their biological activity. We have immobilized the protein Concanavalin A onto a self-assembled monolayer of multivalent tripodal molecules on single-layer graphene.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions.

Photothermal self-oscillation and laser cooling of graphene optomechanical systems.

By virtue of their low mass and stiffness, atomically thin mechanical resonators are attractive candidates for use in optomechanics. Here, we demonstrate photothermal back-action in a graphene mechanical resonator comprising one end of a Fabry-Perot cavity. As a demonstration of the utility of this effect, we show that a continuous wave laser can be used to cool a graphene vibrational mode or to power a graphene-based tunable frequency oscillator.

Ordered arrays of native chromatin molecules for high-resolution imaging and analysis.

Individual chromatin molecules contain valuable genetic and epigenetic information. To date, there have not been reliable techniques available for the controlled stretching and manipulation of individual chromatin fragments for high-resolution imaging and analysis of these molecules. We report the controlled stretching of single chromatin fragments extracted from two different cancerous cell types (M091 and HeLa) characterized through fluorescence microscopy and atomic force microscopy (AFM).

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state.

Zero-mode waveguides for single-molecule analysis.

We review the optical properties, fabrication, and applications of zero-mode waveguides (ZMWs) for single-molecule studies. These simple nano-structures allow individual molecules to be isolated for optical analysis at high concentrations. Fluorescent species are observed in a sufficiently small volume that the average number of fluorescent molecules is less than one, even at concentrations high enough for biochemical reactions to proceed at normal rates.

Nanostructures for sensors and analytical systems.

Professor Harold Craighead attended the University of Maryland (MD, USA) where he received his Bachelor of Science Degree in Physics. He moved to Cornell University (NY, USA) for his PhD in Physics, which he completed in 1980. He subsequently worked as a member of technical staff in the Device Physics Research Department at Bell Laboratories (NJ, USA) until 1984 when he joined Bellcore to form and manage the Quantum Structures research group.