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SKOKIE, IL, United States

Bernal R.A.,Northwestern University | Ramachandramoorthy R.,Northwestern University | Espinosa H.D.,Northwestern University | Espinosa H.D.,Infinitesimal, Llc
Ultramicroscopy | Year: 2015

MEMS and other lab-on-a-chip systems are emerging as attractive alternatives to carry out experiments in situ the electron microscope. However, several electrical connections are usually required for operating these setups. Such connectivity is challenging inside the limited space of the TEM side-entry holder. Here, we design, implement and demonstrate a double-tilt TEM holder with capabilities for up to 9 electrical connections, operating in a high-resolution TEM. We describe the operating principle of the tilting and connection mechanisms and the physical implementation of the holder. To demonstrate the holder capabilities, we calibrate the tilting action, which has limits of ±15°, and establish the insulation resistance of the electronics to be 36. GΩ, appropriate for measurements of currents down to the nano-amp (nA) regime. Furthermore, we demonstrate tensile testing of silver nanowires using a previously developed MEMS device for mechanical testing, using the implemented holder as the platform for electronic operation and sensing. The implemented holder can potentially have broad application to other areas where MEMS or electrically-actuated setups are used to carry out in situ TEM experiments. © 2015 Elsevier B.V. Source


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 149.14K | Year: 2012

This Small Business Innovation Research Phase I project aims at developing and commercializing a cost-effective cell injection system based on nanofountain probe (NFP) technology. The NFP has been recently used to investigate drug delivery and direct cell injection. Benefits of this method include easy integration with existing infrastructure, force control of injection, and the ability to control injection volumes at the picoliter level. Injection of large number of cells is necessary for drug discovery and associated large scale genomic and proteomic studies. Such studies require delivery of drugs, conjugated nanoparticles, DNA, siRNA, and proteins into living cells to study spatial and temporal molecular regulatory mechanisms within the cell. Considering the delicate nature of living cells, such a task is nontrivial. For direct drug delivery, micropipette based injection has been used for many years. However, its viability (cell survival) and lack of automation limit its broad use. In this project, a single cell injection system will be developed based on leveraging probe-cell force control and fluidic handling capabilities of NFPs. The system has the potential for parallel cell injection and ultimately automated operation, which would greatly enhance viability as a workhorse tool for cell injection in research labs and pharmaceutical companies.

The broader impact/commercial potential of this project is the development of a new platform for high throughput single cell drug delivery, allowing for efficient evaluation of drug efficacy and the development of diagnostics and next generation therapeutics. The application of nanotechnology to medicine (termed nanomedicine) has provided numerous emerging opportunities in healthcare, particularly given the increasing demand for in vitro toxicology and diagnostics as a pathway towards personalized medicine. Drug delivery to individual cells and monitoring of associated pathways is at the core of most research toward in vitro diagnostics and toxicology. Personalized medicine will require the systematic incorporation of genetic information from individuals in optimizing preventive and therapeutic care, and much more efficient biomedical tools. Commercialization of this platform would allow research centers and pharmaceutical companies to have access to state-of-the-art nanotechnology tools in their endeavor toward a patient-centered health care system. Furthermore, the new single cell injection system will find utility in laboratories in universities across the U.S., exposing the next generation of scientists to nanotechnology and its impact on medicine.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2014

DESCRIPTION: Single-cell studies are essential for biochemical characterization of heterogeneous cell behaviors. However, studying direct correlation between cell input (transfection) and output (biochemical analysis) is still challenging due to lack of high throughpu methodologies with cell selectivity and high enough sensitivity. The tool proposed here is a microwell device that will allow temporal sampling and analysis of internal biomolecules, with the additional functionality of activating cells by delivering genes, drugs, or other molecules when appropriate for the biological problem. This tool would enable fundamental studies of biological variability, input- output relationships that control mechanistic pathways, and with multiplexed automation, could serve as a drug screening platform. The major innovations of the proposed device include (1) isolation of 1,000 individual single cells in a microwell array, (2) long-term cell culture on a micro device, (3) temporal transfection and extraction of b


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1000.00K | Year: 2015

DESCRIPTION provided by applicant This proposed Phase II project will lead to a commercial product capable of transfecting individual target cells This new biotool uses a patented microfabricated chip called a nanofountain probe NFP to deliver molecules into live individual target cells by single cell electroporation which induces temporary nanopores in the cell membrane via the application of an electrical field that is localized at the tip of a microfludic cantilever on the NFP chip This novel transfection technique is called nanofountain probe electroporation NFP E and it has shown incredible promise for translation into a novel commercial biotool Existing techniques for single cell transfection such as microinjection and electroporation by micropipette require extensive operator training are highly user dependent and labor intensive and routinely damage cells from excessive mechanical force on the cell membrane The NFP cantilever reduces the mechanical stress on the cell membrane and can be tailored during fabrication The long term objective of this project is to develop the first sinle cell transfection system that is easy to use gentle on cells and automated to offer relatively high throughput and eliminate user dependent variability The specific aims for this proposal are the following to optimize the design of product components for plug and play assembly and easy solution loading and recovery to develop software for image processing and analysis to simplify NFP Electroporation by automating the process of locating target cells detecting contact of the NPF tip with a cell membrane and applying the electric field and to perform three key transfection experiments to demonstrate the potential of the NFP E in Randamp D to establish experimental protocols collect statistics of efficiency viability verify specifications efficiency viability throughput and refine the image recognition software for a number of cell lines and primary cells Accomplishing these aims will produce a product that could enable new capabilities for single cell research and therapeutics including cell reprogramming differentiation cell cell signaling gene expression and protein interaction cell to cell variability drug discovery personalized drug response diagnostics and personalized medicine PUBLIC HEALTH RELEVANCE This project will lead to the development of the nanofountain probe electroporation system NFP E System the first commercial product capable of delivering molecules into individual target cells in an automated easy to use and gentle method This new biotool for single cell research and development will offer high throughput and eliminate user dependent variability to enable new capabilities for biological research and therapeutics particularly for drug discovery personalized drug response diagnostics and personalized medicine based on stem cells


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: STTR PHASE II | Award Amount: 485.09K | Year: 2013

This Small Business Innovation Research (SBIR) Phase II project is aimed at increasing the throughput of a novel single-cell technology based on nanofountain probe electroporation (NFP-E). Progress in biotechnology research in recent years has shown promise for cell reprogramming and extremely sensitive medical diagnostics, yet this research requires effective, precise, and gentle transfection of cells - for which a robust tool is currently lacking. The NFP-E technology is capable of filling this need by enabling single-cell electroporation for dosage-controlled transport of biomolecules, proteins, or drugs into a cell. By applying a low electric potential to a target cell, small pores in the cell membrane are generated, which allows for delivery of molecules into cells in a way that is more efficient and less invasive than any other method of transfection used today. This proposed project has three primary goals: to establish protocols for various cell types and transfection agents, to fabricate a microwell plate that couples with the NFP-E, and to develop software algorithms to automate alignment and electroporation control. These developments will create a robust system with exceptional process control and cell viability that is suitable for relatively high throughput single-cell transfection applications.

The broader impact/commercial potential of this project stems from the unprecedented capabilities that the novel tool will provide to researchers and biotechnology companies for manipulation and interrogation of cellular processes, benefiting fundamental biology research and the development of personalized medicine applications. This project will enhance the scientific and technological understanding of fundamental electroporation mechanisms and single cell analysis techniques by combining nanofabrication, microfluidics, biophysics, and molecular biology aspects. The NFP-E tool will allow scientists to use primary cells for research applications, providing a more meaningful link between laboratory research and human disease pathophysiology. In addition, this new tool could make the promise of personalized medicine feasible and practical by enabling new capabilities in biotechnology and providing a robust commercial instrument for single-cell studies toward discoveries that elucidate disease mechanisms, focus drug discovery efforts, and personalize disease diagnosis and therapies.

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