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1. 发明授权

US06335201B1 Method and apparatus for detecting enzymatic activity using molecules that change electrophoretic mobility 失效
公开(公告)号：US06335201B1
公开(公告)日：2002-01-01
申请号：US09358504
申请日：1999-07-21
申请人： Nancy L. Allbritton , Christopher E. Sims , Michael W. Berns , Gavin D. Meredith , Tatiana B. Krasieva , Bruce J. Tromberg
发明人： Nancy L. Allbritton , Christopher E. Sims , Michael W. Berns , Gavin D. Meredith , Tatiana B. Krasieva , Bruce J. Tromberg
IPC分类号： G01N3348
CPC分类号： C12M41/46 , C12M35/02 , C12M47/06 , C12Q1/00 , C12Q1/485 , G01N27/44721 , G01N27/44743 , G01N33/50 , G01N33/5017
摘要： The activity of intracellular chemical reactions of molecules is measured by the use of fluorescently labeled substrate molecules that undergo a change in electrophoretic mobility upon chemical reaction such as that catalyzed by an enzyme. Specificity is achieved by using labeled substrate molecules that can be acted upon only by specific enzymes. Thus the activity of a specific enzyme or class of enzymes can be determined. Measurements are made with the intracellular presence of such substrate molecules, at some time of interest, typically after exposure of the cell to a stimulus that activates a particular enzymatic pathway. To ensure accuracy, measurements must be made in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. Fast controllable laser lysis is used to obtain the contents of a single cell into which reporter substrate molecules have been introduced. The cell contents are then subjected to capillary electrophoresis and enzymatic activity is determined by comparing amounts of substrate molecules to amounts of enzymatically altered substrate molecules which are separated by the electrophoresis and identified by the presence of a fluorescent label.

2. 发明授权

US6156576A Fast controllable laser lysis of cells for analysis 失效
标题翻译：快速可控激光裂解细胞进行分析
公开(公告)号：US6156576A
公开(公告)日：2000-12-05
申请号：US36706
申请日：1998-03-06
申请人： Nancy L. Allbritton , Christopher E. Sims , Michael W. Berns , Gavin D. Meredith , Tatiana B. Krasieva , Bruce J. Tromberg
发明人： Nancy L. Allbritton , Christopher E. Sims , Michael W. Berns , Gavin D. Meredith , Tatiana B. Krasieva , Bruce J. Tromberg
IPC分类号： G01N33/48 , C12M1/33 , C12N13/00 , G01N27/447 , G01N33/483
CPC分类号： G01N27/44743 , C12M47/06 , G01N27/44721
摘要： Fast lysis of a single cell or cellular component thereof is performed by generating a shock wave in a medium in which the cell or cellular component thereof is positioned. The cell or cellular component thereof is either positioned by laser tweezers or cultured as an adhered cell or cellular component thereof to minimize manipulation trauma. The disclosed method completely lyses a single cell or cellular component thereof in a controllable manner in milliseconds or less followed immediately by the loading of the cellular contents into a capillary for analyte separation and detection. The cell or cellular component thereof is adjacent the inlet of an electrophoretic column through which a gravity siphon flow of the medium is maintained. The lysed contents of the cell or cellular component thereof enter the electrophoretic column in less than 33 msec, are separated and analyzed by laser induced fluorescence. The method takes advantage of the shock wave produced by a highly focused laser pulse which is created in a medium adjacent to the cell or cellular component thereof. In the illustrated embodiment the laser pulse is focused in the glass substrate at or near a glass-to-buffer interface of a cell chamber in which the cell or cellular component thereof to be lysed has been cultured.
摘要翻译：单细胞或其细胞成分的快速裂解是通过在细胞或细胞成分定位的培养基中产生冲击波来进行的。其细胞或细胞组分由激光镊子定位或作为粘附细胞或其细胞组分培养，以最小化操作创伤。所公开的方法以几毫秒或更短的可控方式完全溶解单细胞或细胞组分，随后将细胞内容物加载到用于分析物分离和检测的毛细管中。其电池或细胞组分邻近电泳柱的入口，通过该入口保持介质的重力虹吸流。细胞或细胞成分的裂解内容物在小于33毫秒内进入电泳柱，通过激光诱导荧光分离和分析。该方法利用了在与其细胞或细胞组分相邻的介质中产生的高度聚焦的激光脉冲产生的冲击波。在所示实施例中，激光脉冲聚焦在玻璃基板中或其附近，其中待裂解细胞或其细胞组分的细胞室的玻璃 - 缓冲液界面已被培养。

3. 发明授权

US5773227A Bifunctional chelating polysaccharides 失效
标题翻译：双功能螯合多糖
公开(公告)号：US5773227A
公开(公告)日：1998-06-30
申请号：US82269
申请日：1993-06-23
申请人： Michael A. Kuhn , Tobias Meyer , Nancy L. Allbritton
发明人： Michael A. Kuhn , Tobias Meyer , Nancy L. Allbritton
IPC分类号： C08B37/02 , G01N33/84 , G01N33/566 , C07K17/10
CPC分类号： G01N33/84 , C08B37/0021 , G01N2400/22
摘要： This invention describes bifunctional polysaccharides conjugated to both a chelating group suitable for the selective complexation of metal cations, and a targeting peptide specific for a cellular substructure. These bifunctional polysaccharides are primarily useful for the regulation, detection and quantification of metal ion levels, such as Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, or Li.sup.+, in specific cellular structures. Localization within the cellular structure is accomplished by the targeting peptide, whereupon the large, water-soluble polysaccharide prevents diffusion of the chelating group from the targeted site. When the target cell structure is the nucleus of a fertilized egg cell, the polysaccharide-chelator conjugate remains sequestered within the nucleus until the breakdown of the nuclear envelope, whereupon the reagent becomes sequestered into both daughter nuclei. This means of tracking daughter cells is practical even through several cell divisions.
摘要翻译：本发明描述了与适用于金属阳离子的选择性络合的螯合基团结合的双功能多糖，以及针对细胞亚结构的靶向肽。这些双功能多糖主要用于在特定细胞结构中调节，检测和定量金属离子水平，如Ca2 +，Mg2 +，Na +，K +或Li +。细胞结构内的定位是通过靶向肽完成的，因此大的水溶性多糖可防止螯合基团从目标位点的扩散。当靶细胞结构是受精卵细胞的核时，多糖 - 螯合剂缀合物保留在细胞核内，直到核包膜破裂，于是该试剂被隔离到两个子核中。跟踪子细胞的这种手段甚至通过几个细胞分裂是切实可行的。

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