SKOKIE, IL, United States
SKOKIE, IL, United States

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Kang W.,Northwestern University | Kang W.,Infinitesimal, Llc | Yavari F.,Northwestern University | Minary-Jolandan M.,Northwestern University | And 6 more authors.
Nano Letters | Year: 2013

The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the past decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells. © 2013 American Chemical Society.


Kang W.,Infinitesimal, Llc | McNaughton R.L.,Infinitesimal, Llc | Espinosa H.D.,Infinitesimal, Llc
Trends in Biotechnology | Year: 2016

Several recent micro- and nanotechnologies have provided novel methods for biological studies of adherent cells because the small features of these new biotools provide unique capabilities for accessing cells without the need for suspension or lysis. These novel approaches have enabled gentle but effective delivery of molecules into specific adhered target cells, with unprecedented spatial resolution. We review here recent progress in the development of these technologies with an emphasis on in vitro delivery into adherent cells utilizing mechanical penetration or electroporation. We discuss the major advantages and limitations of these approaches and propose possible strategies for improvements. Finally, we discuss the impact of these technologies on biological research concerning cell-specific temporal studies, for example non-destructive sampling and analysis of intracellular molecules. Miniaturization of biotools: micro-/nanoscale biotools for cell transfection and analysis are being developed to achieve cell-specific experimental capabilities and localized cell-tool interfaces. This allows minimal perturbation to cells and unprecedented spatial resolution, which is essential for fundamental cell studies.Biotools for adherent cells: despite the risk for altering phenotype or stressing cells, conventional biotechnologies often require suspending and replating cells during in vitro studies. Novel micro/nanotechnologies are being developed to transfect and analyze adhered cells, which is particularly advantageous for longitudinal studies of individual cells or for investigating cell mechanisms.Combination of micro-/nanotechnologies with conventional biotechnologies: various strategies use micro-/nanotechnologies with conventional analytical tools such as fluorescence array readers and atomic force microscopes. This assembly approach promises revolutionary advances in biology and medicine. © 2016 Elsevier Ltd.


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.


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: STTR | Phase: Phase I | Award Amount: 100.16K | Year: 2012

DESCRIPTION (provided by applicant): Application of nanotechnology in medicine, nanomedicine, has provided numerous emerging opportunities in healthcare, particularly given ever increasing demand for in vitro toxicology and diagnostics towards personalized medicine. With increasing demand for personalized medicine, pharmaceutical companies strive in taking advantage of the potential of nanotechnology tools in search of next generation therapeutics. Such efforts require systematic incorporation of genetic information from each individual in optimizing preventive and therapeutic care, which evidently would require much more efficient biomedical tools. The so-called nanofountain probe (NFP), which was developed at Northwestern University and is currently commercialized by iNfinitesimal LLC, is a nanotechnology tool that enables such endeavors. These probes have been used to investigate drug delivery through substrate nanopatterning and direct cell injection. In this regard, unique attributes of the NFP technology are: Easy integration with bio-AFMs and inverted fluorescent microscopes. Ultra-low mechanical stiffness of the probe allowing force-controlled injection to ensure minimal cell damage, i.e., high cell viability. Special probe morphology with a sharp tip that creates a small rupture in the cell membrane while the outer shell guides biomolecules to the point of injection. Picoliter control in fluid injection Large scale genomic and proteomic 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 in response to a particular treatment. For direct drug delivery, micropipette based injection has been used for many years. However, its viability (cell survival) and limited automation limit its extensive application. This STTR Phase I project aims at developing and commercializing a cost effective and high-throughput cell injection system based on the nanofountain probe technologythat can potentially be automated towards much faster drug delivery to individual cell with high viability. Leveraging probe-cell force control and fluidic handling capabilities of nanofountain probes are the core of the new proposed tool. These attributeswould greatly enhance its potential to be the instrument of choice for viable cell injection in research labs and pharmaceutical companies, allowing them to have access to state-of-the-art nanotechnology tools in their endeavor toward patient-centered healthcare system. Key words: Single cell Injection, Drug delivery, Nanofountain Probes, Personalized medicine, Atomic force microscopy, Fluorescent microscopy, Cell viability, Force control. PUBLIC HEALTH RELEVANCE: This STTR Phase I project aims atdeveloping and commercializing a cost effective, high- throughput, and potentially automated single cell injection system towards much faster drug delivery to individual cells. The system will be applicable to large scale genomic and proteomic studies required for the ever increasing demand for personalized medicine .


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: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 285.00K | 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.


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.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | 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

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