CMOS microcavity arrays for single-cell electroporation and lysis.

Transfection is a key function for many single-cell analyses. Reversible electroporation (EP) using high intensity electric fields is a simple means of transfection applicable to most cell types. For reversible EP, precise control over the electric field is critical to regulate the induced pore densities in the membrane and maintain cell viability. Individually accessible microelectrode arrays enabled by semiconductor fabrication methods have emerged as a viable technology for single-cell analyses but do not provide for effective electroporation capabilities due to the planar arrangement of electrodes. Towards the goal of a fully integrated single-cell analysis platform, we utilize a commercial complementary metal-oxide-semiconductor (CMOS) process to realize microcavities which allow for single-cell confinement with integrated three-dimensionally aligned electrodes for effective poration. The structure is formed using the inherent metal stack available within the CMOS process as a hard etch mask for deep-reactive ion etching. Using this structure, to our knowledge, we present the first on-CMOS demonstration of controlled electroporation with the goal of transfection using human embryonic kidney cells (HEK-293) stained with Calcein as a model. We report an increase in calcein leaching from the cells subject to increasing electric field intensities with subsequent reuptake confirming cell viability post electroporation. These results are supported by numerical simulation of theoretical pore density which are in good agreement with numerical simulation. Combined with simple optical or electrical feedback, the structure is suitable for precise electroporation control in single-cells.

A flow through device for simultaneous dielectrophoretic cell trapping and AC electroporation.

Isolation of cells and their transfection in a controlled manner is an integral step in cell biotechnology. Electric field approaches such as dielectrophoresis (DEP) offers a more viable method for targeted immobilization of cells without any labels. For transfection of cells to incorporate exogenous materials, electrical methods such as electroporation, are preferred over chemical and viral delivery methods since they minimally affect cell viability and can target many types. However prior approaches to both methods required multiple excitation sources, an AC source for DEP-based trapping and another DC source for electroporation. In this paper, we present a first of its kind flow through lab-on-chip platform using a single AC excitation source for combined trapping using negative dielectrophoresis (nDEP) and AC electroporation. Use of AC fields for electroporation eliminates the unwanted side effects of electrolysis or joule heating at electrodes compared to DC electroporation. Adjusting the flow rate and the electrical parameters of the incident AC field precisely controls the operation (trap, trap with electroporation and release). The platform has been validated through trapping and simultaneous transfection of HEK-293 embryonic kidney cells with a plasmid vector containing a fluorescent protein tag. Numerical scaling analysis is provided that indicates promise for individual cell trapping and electroporation using low voltage AC fields.

A three-dimensional electrochemical paper-based analytical device for low-cost diagnostics.

Paper-based microfluidic devices with screen-printed electrodes (SPEs) for electrochemical sensing are popular for low-cost point-of-care diagnostics. The electroactive sensing area in these devices is always the irregular, bottom-SPE surface which is in contact with the analyte flowing within the paper substrate. Unfortunately, this results in an electroactive area which varies widely from sensor to sensor. In this paper, we present a three-dimensional paper-based analytical device with a hollow 3D fluid reservoir which allows for use of a more uniform top-SPE surface as the electroactive sensing area. The use of this isolated reservoir eliminates the need for dielectric inks used in conventional SPEs on paper. Our sensors are fabricated using a combination of wax-printing, screen-printing and simple folding via a cleanroom free process without the need for expensive equipment. Additionally, for the first time, we demonstrate an electrochemical paper-based analytical device with a custom designed potentiostat integrated circuit (IC) as a miniaturized reader. The versatility of the sensor is demonstrated through voltammetric, amperometric and potentiometric measurements of important biochemical analytes such as dopamine, glucose and pH. The 3D ePAD together with a custom CMOS potentiostat demonstrates a low-cost, versatile, self-contained system suitable for point-of-care diagnostic devices.

Origami microfluidic paper-analytical-devices (omPAD) for sensing and diagnostics.

Recent research activities in the area of low-cost sensing and diagnostics that are realized on cellulosic paper substrate are presented. First a three-dimensional origami paper-based analytical device (omPAD) with multiple electrochemical sensors, an integrated sample reservoir and tight integration with a custom CMOS potentiostat is presented. Second, an optical sensor array with built-in microfluidic channel for sample delivery is presented. The sensors are fabricated using a combination of wax printing and screen-printing using a solution based approach in ambient conditions without the need for expensive fabrication equipment or a cleanroom. Readout is based on using existing consumer grade electronic devices like flatbed scanner (for optical sensor) or custom designed CMOS potentiostat (for electrochemical sensors). Together the 3D paper-based analytical device with integrated sensor, microfluidics and portable readout instrumentation demonstrates a low-cost, self-contained system suitable for sensing and point-of-care diagnostics.

Transcriptional repression by the proximal exonic region at the human TERT gene.

In humans, the enzyme telomerase (hTERT) is responsible for the synthesis of new repeat sequences at the telomeres of chromosomes. Although active in early embryogenesis, the hTERT gene is transcriptionally silenced in almost all somatic cells in the adult, but is aberrantly re-activated in over 90% of human cancers. The molecular mechanisms responsible for repression of this gene are thought to involve the transcription factor CTCF. In this study, we bioinformatically identify putative CTCF binding sites in the hTERT proximal exonic region (PER) and determine their functional relevance in mediating transcriptional silencing at this gene. Tests using a reporter gene assay in HeLa cancer cells demonstrate that a sub-region of the PER exhibits strong transcriptional repressive activity. This repression is independent of the previously identified CTCF binding site near the transcriptional start site of the hTERT gene. In addition, site directed mutagenesis of three predicted CTCF binding sites, including a previously characterized in vivo site in exon 2, does not result in a loss of the repression mediated by the PER. The results from this study indicate that expression of the hTERT gene in HeLa cells is regulated by sequences in the PER. This transcriptional control is mediated through additional regulatory molecular mechanisms, independent of CTCF binding.