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Patent Issued for Method for Live-Cell Activity Assay

September 10, 2014



By a News Reporter-Staff News Editor at Journal of Engineering -- From Alexandria, Virginia, VerticalNews journalists report that a patent by the inventors Yang, Eun-Gyeong (Seoul, KR); Na, Yu-Ran (Seoul, KR); Park, Hong-Kun (Lexington, MA); Marsela, Jorgolli (Arlington, MA), filed on October 8, 2010, was published online on August 26, 2014.

The patent's assignee for patent number 8815523 is Korea Institute of Science and Technology (Seoul, KR).

News editors obtained the following quote from the background information supplied by the inventors: "(a) Field of the Invention

"This disclosure relates to technologies for direct measurement of activity of a bioactive substance in cell using nanowires. In particular, a method of measuring intracellular activity of a bioactive substance using a support having nanowires to which a cell is immobilized and another support having nanowires to which a target substance for a bioactive substance to be detected is immobilized, and a chip for measuring intracellular activity of a bioactive substance including a support having nanowires to which a cell is immobilized and another support having nanowires to which a targeting substance for a bioactive substance to be detected are immobilized, are provided.

"(b) Description of the Related Art

"In general, enzyme activity has been studied using purified enzymes and substrates, and the methods include spectrophotometric assays measuring absorbance changes according to changes of substrates due to enzyme activities to identify enzyme activities, fluorometric assays measuring enzyme activities using differences in fluorescence between a product produced by enzyme activity and an initial substrate, calorimetry assays measuring heat absorbed or emitted by chemical reactions to measure changes of substrates due to enzyme reactions using microcalorimeter, chemiluminescent assays measuring light emission due to chemical reactions to measure enzyme activities, chromatographic assays measuring a product generated by enzyme activity using chromatography, and radiometric assays identifying enzyme activities using radioisotopes as substrates, and the like.

"Since most of the methods for measuring enzyme activity are conducted in vitro using purified enzymes and substrates, various external conditions (ionic strength, salt concentration, temperature, pH, enzyme concentration, and etc.) may be controlled to conduct assays under best conditions for enzyme activity, which differs from endogenous enzyme conditions in cells. Since the existence of large quantities of macromolecules in cells may influence functional and structural stability of protein, and enzyme reaction including rate of reaction, the result of activity analysis using purified enzyme cannot always be regarded as representing enzyme action in cells.

"Moreover, since in many cases, another enzyme reversing the reaction of one enzyme exists (for example, protein kinase and phosphatase which respectively cause phosphorylation and dephosphorylation exist simultaneously in cells), enzyme activity in the cell context may not be predicted simply by in vitro cell activity analysis. Recently, for measurement of endogenous enzyme activity in cells, various methods involving instantaneous cell lysis followed by assays have been extensively employed. However, the possibility that enzyme environment and activity may be changed during the cell lysis and lysate preparation cannot be excluded, and thus a method for analysis of enzyme activity with maintaining cells alive is required.

"Nanotechnology enables characterization and control of material in atomic and molecular unit, and it has recently been combined with biotechnology and developed as new technology for future. Many study results have been reported on low dimensional nanostructures, and particularly one-dimensional nanomaterials such as nanorods, nanowires, nanotubes, nanobelts and the like with excellent optical, electrical, chemical and physical properties have been applied in various fields.

"So far, biomedical applications of nanotechnology have mainly been focused on elucidating characteristics of nanoparticles for bio-imaging technology, biosensor, and local drug delivery. Since early 2000's, an intracellular drug delivery technique named nanotube spearing using nanomaterial supernatants has been developed. In 2007, Peidong Yang et al. of UC Berkeley, U.S.A. reported a study result of incubation of mice embryonic stem cells using vertically grown silicon nanowires and DNA transfection upon penetrating cells with the nanowires (Kim W, Ng J K, Kunitake M E, Conklin B R, Yang P, J. (2007) Am. Chem. Soc. 128:8990-8991), and suggested a possibility of application of vertically grown nanowires to live cells for the first time, although the transfection efficiency was very low as less than 1%."

As a supplement to the background information on this patent, VerticalNews correspondents also obtained the inventors' summary information for this patent: "Accordingly, the present inventors successfully established a method to immobilize live cells and specific enzyme substrates to vertically grown nanowires, cultured the cells immobilized to a piece of nanowires with the enzyme substrates immobilized to another piece of nanowires in a sandwich format, and succeeded in measuring the enzyme activity in live cells, to complete the present invention.

"Therefore, one embodiment of the present invention provides a method of measuring intracellular activity of a bioactive substance comprising immobilizing cells to nanowires, immobilizing target substance for a bioactive substance to be detected covalently to separate nanowires, culturing the cells together with the target substance in a sandwich format, and detecting reaction of the target substance and/or degree of the reaction inside the cells.

"Another embodiment provides a chip for measuring intracellular activity of a bioactive substance including nanowires to which cells are immobilized and nanowires to which target substances for bioactive substance to be detected are immobilized, the cell-immobilized nanowires and the target substance-immobilized nanowires being in contact with each other in a sandwich manner.

"Another embodiment provides a kit for measuring intracellular activity of a bioactive substance including said chip for measuring intracellular activity of the bioactive substance and a mean for detecting reaction of the target substance and/or degree of the reaction inside the cells."

For additional information on this patent, see: Yang, Eun-Gyeong; Na, Yu-Ran; Park, Hong-Kun; Marsela, Jorgolli. Method for Live-Cell Activity Assay. U.S. Patent Number 8815523, filed October 8, 2010, and published online on August 26, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8815523.PN.&OS=PN/8815523RS=PN/8815523

Keywords for this news article include: Emerging Technologies, Enzymes and Coenzymes, Korea Institute of Science and Technology, Nanotechnology, Science And Technology.

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Source: Journal of Engineering


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