Nanobiotechnology Center

Breckinridge Center, KY, United States

Nanobiotechnology Center

Breckinridge Center, KY, United States
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Wang S.,Nanobiotechnology Center | Haque F.,Nanobiotechnology Center | Rychahou P.G.,University of Kentucky | Evers B.M.,University of Kentucky | And 2 more authors.
ACS Nano | Year: 2013

The ingenious design of the bacterial virus phi29 DNA packaging nanomotor with an elegant and elaborate channel has inspired its application for single molecule detection of antigen/antibody interactions. The hub of this bacterial virus nanomotor is a truncated cone-shaped connector consisting of 12 protein subunits. These subunits form a ring with a central 3.6-nm channel acting as a path for dsDNA to enter during packaging and to exit during infection. The connector has been inserted into a lipid bilayer. Herein, we reengineered an Epithelial Cell Adhesion Molecule (EpCAM) peptide into the C-terminal of nanopore as a probe to specifically detect EpCAM antibody (Ab) in nanomolar concentration at the single molecule level. The binding of Abs sequentially to each peptide probe induced stepwise blocks in current. The distinctive current signatures enabled us to analyze the docking and undocking kinetics of Ab-probe interactions and determine the Kd. The signal of EpCAM antibody can be discriminated from the background events in the presence of nonspecific antibody or serum. Our results demonstrate the feasibility of generating a highly sensitive platform for detecting antibodies at extremely low concentrations in the presence of contaminants. © 2013 American Chemical Society.


Shu Y.,Nanobiotechnology Center | Shu Y.,University of Kentucky | Shu D.,Nanobiotechnology Center | Shu D.,University of Kentucky | And 4 more authors.
Nature Protocols | Year: 2013

RNA nanotechnology is a term that refers to the design, fabrication and use of nanoparticles that are mainly composed of RNAs via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nanodelivery platform. This protocol describes the synthesis, assembly and functionalization of pRNA nanoparticles on the basis of three 'toolkits' derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions and an RNA three-way junction for branch extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and other functionalities can also be fused to the pRNA before the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultralow concentrations. Gene-silencing effects are progressively enhanced with increasing numbers of siRNAs in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nanodelivery scaffold paves a new way for nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3-4 weeks when including in vitro functional assays, or 2-3 months when including in vivo studies. © 2013 Nature America, Inc. All rights reserved.


PubMed | Nanobiotechnology Center
Type: Journal Article | Journal: Nature protocols | Year: 2013

RNA nanotechnology is a term that refers to the design, fabrication and use of nanoparticles that are mainly composed of RNAs via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nanodelivery platform. This protocol describes the synthesis, assembly and functionalization of pRNA nanoparticles on the basis of three toolkits derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions and an RNA three-way junction for branch extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and other functionalities can also be fused to the pRNA before the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultralow concentrations. Gene-silencing effects are progressively enhanced with increasing numbers of siRNAs in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nanodelivery scaffold paves a new way for nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3-4 weeks when including in vitro functional assays, or 2-3 months when including in vivo studies.


News Article | November 24, 2016
Site: www.medicalnewstoday.com

Interactions between an animal cell and its environment, a fibrous network called the extracellular matrix, play a critical role in cell function, including growth and migration. But less understood is the mechanical force that governs those interactions. A multidisciplinary team of Cornell engineers and colleagues from the University of Pennsylvania have devised a method for measuring the force a cell - in this case, a breast cancer cell - exerts on its fibrous surroundings. Understanding those forces has implications in many disciplines, including immunology and cancer biology, and could help scientists better design biomaterial scaffolds for tissue engineering. The group, led by Mingming Wu, associate professor in the Department of Biological and Environmental Engineering, developed 3-D traction-force microscopy to measure the displacement of fluorescent marker beads distributed in a collagen matrix. The beads are displaced by the pulling of migrating breast cancer cells embedded in the matrix. An important part of the puzzle was to calculate the force exerted by the cells using the displacement of the beads. That calculation was carried out by the team led by Vivek Shenoy, professor of materials science and engineering at the University of Pennsylvania. The group's paper, "Fibrous nonlinear elasticity enables positive mechanical feedback between cells and extracellular matrices," published online in Proceedings of the National Academy of Sciences. Matthew Hall, Ph.D. '16, now a postdoctoral researcher at the University of Michigan, is lead author and engineered the collagen matrices used in the study. Wu - who also was affiliated with the Cornell Center on the Microenvironment and Metastasis at Weill Cornell Medicine, which existed from 2009 through 2015 - said her group's work centered on a basic question: How much force do cells exert on their extracellular matrix when they migrate? "The matrix is like a rope, and in order for the cell to move, they have to exert force on this rope," she said. "The question arose from cancer metastasis, because if the cells don't move around, it's a benign tumor and generally not life-threatening." It's when the cancerous cell migrates that serious problems can arise. That migration occurs through "cross-talk" between the cell and the matrix, the group found. As the cell pulls on the matrix, the fibrous matrix stiffens; in turn, the stiffening of the matrix causes the cell to pull harder, which stiffens the matrix even more. This increased stiffening also increases cell force transmission distance, which can potentially promote metastasis of cancer cells. "We've shown that the cells are able to align the fibers in their vicinity by exerting force," Hall said. "We've also shown that when the matrix is more fibrous - less like a continuous material and more like a mesh of fibers - they're able to align the fibers through the production of force. And once the fiber is aligned and taut, it's easier for cells to pull on them and migrate." "I'm a strong believer that every new science discovery goes hand-in-hand with new technology development," she said. "And with every new tool, you discover something new." This research was supported by grants from the National Institutes of Health, the National Cancer Institute and the National Science Foundation, and made use of the Cornell NanoScale Science and Technology Facility, the Cornell Biotechnology Resource Center Imaging Facility, the Cornell Center for Materials Research and the Cornell Nanobiotechnology Center. Article: Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs, Matthew S. Hall, Farid Alisafaei, Ehsan Ban, Xinzeng Feng, Chung-Yuen Hui, Vivek B. Shenoy, and Mingming Wu, PNAS, doi: 10.1073/pnas.1613058113, published online 21 November 2016.


News Article | November 22, 2016
Site: www.eurekalert.org

Interactions between an animal cell and its environment, a fibrous network called the extracellular matrix, play a critical role in cell function, including growth and migration. But less understood is the mechanical force that governs those interactions. A multidisciplinary team of Cornell engineers and colleagues from the University of Pennsylvania have devised a method for measuring the force a cell -- in this case, a breast cancer cell -- exerts on its fibrous surroundings. Understanding those forces has implications in many disciplines, including immunology and cancer biology, and could help scientists better design biomaterial scaffolds for tissue engineering. The group, led by Mingming Wu, associate professor in the Department of Biological and Environmental Engineering, developed 3-D traction-force microscopy to measure the displacement of fluorescent marker beads distributed in a collagen matrix. The beads are displaced by the pulling of migrating breast cancer cells embedded in the matrix. An important part of the puzzle was to calculate the force exerted by the cells using the displacement of the beads. That calculation was carried out by the team led by Vivek Shenoy, professor of materials science and engineering at the University of Pennsylvania. The group's paper, "Fibrous nonlinear elasticity enables positive mechanical feedback between cells and extracellular matrices," published online Nov. 21 in Proceedings of the National Academy of Sciences. Matthew Hall, Ph.D. '16, now a postdoctoral researcher at the University of Michigan, is lead author and engineered the collagen matrices used in the study. Wu -- who also was affiliated with the Cornell Center on the Microenvironment and Metastasis at Weill Cornell Medicine, which existed from 2009 through 2015 -- said her group's work centered on a basic question: How much force do cells exert on their extracellular matrix when they migrate? "The matrix is like a rope, and in order for the cell to move, they have to exert force on this rope," she said. "The question arose from cancer metastasis, because if the cells don't move around, it's a benign tumor and generally not life-threatening." It's when the cancerous cell migrates that serious problems can arise. That migration occurs through "cross-talk" between the cell and the matrix, the group found. As the cell pulls on the matrix, the fibrous matrix stiffens; in turn, the stiffening of the matrix causes the cell to pull harder, which stiffens the matrix even more. This increased stiffening also increases cell force transmission distance, which can potentially promote metastasis of cancer cells. "We've shown that the cells are able to align the fibers in their vicinity by exerting force," Hall said. "We've also shown that when the matrix is more fibrous - less like a continuous material and more like a mesh of fibers - they're able to align the fibers through the production of force. And once the fiber is aligned and taut, it's easier for cells to pull on them and migrate." "I'm a strong believer that every new science discovery goes hand-in-hand with new technology development," she said. "And with every new tool, you discover something new." This research was supported by grants from the National Institutes of Health, the National Cancer Institute and the National Science Foundation, and made use of the Cornell NanoScale Science and Technology Facility, the Cornell Biotechnology Resource Center Imaging Facility, the Cornell Center for Materials Research and the Cornell Nanobiotechnology Center.

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