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  1. Article ; Online: Combining Active Carbonic Anhydrase with Nanogels: Enzyme Protection and Zinc Sensing.

    Si, Di / Nie, Guochao / Hurst, Tamiika K / Fierke, Carol A / Kopelman, Raoul

    International journal of nanomedicine

    2021  Volume 16, Page(s) 6645–6660

    Abstract: Background: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization ... ...

    Abstract Background: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo.
    Methods and results: Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al
    Conclusion: This work demonstrates universal methods for immobilizing highly fragile bio-macromolecules inside nanoparticle carriers, while preserving their structural integrity and biological activity. The advantages and limitations are discussed, as well as the potential biomedical applications.
    MeSH term(s) Carbonic Anhydrases ; Enzymes, Immobilized ; Nanogels ; Nanoparticles ; Zinc
    Chemical Substances Enzymes, Immobilized ; Nanogels ; Carbonic Anhydrases (EC 4.2.1.1) ; Zinc (J41CSQ7QDS)
    Language English
    Publishing date 2021-09-27
    Publishing country New Zealand
    Document type Journal Article
    ZDB-ID 2364941-0
    ISSN 1178-2013 ; 1176-9114
    ISSN (online) 1178-2013
    ISSN 1176-9114
    DOI 10.2147/IJN.S321099
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 6606: Show issues Location:
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  2. Article ; Online: Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli.

    Wang, Da / Hurst, Tamiika K / Thompson, Richard B / Fierke, Carol A

    Journal of biomedical optics

    2011  Volume 16, Issue 8, Page(s) 87011

    Abstract: Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. ...

    Abstract Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ∼0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.
    MeSH term(s) Biosensing Techniques/instrumentation ; Biosensing Techniques/methods ; Carbonic Anhydrases/genetics ; Carbonic Anhydrases/metabolism ; Escherichia coli/chemistry ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Fluorescence Resonance Energy Transfer/methods ; Luminescent Proteins/chemistry ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Models, Biological ; Molecular Biology/methods ; Zinc/analysis ; Zinc/metabolism ; Red Fluorescent Protein
    Chemical Substances Luminescent Proteins ; Carbonic Anhydrases (EC 4.2.1.1) ; Zinc (J41CSQ7QDS)
    Language English
    Publishing date 2011-09-04
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 1309154-2
    ISSN 1560-2281 ; 1083-3668
    ISSN (online) 1560-2281
    ISSN 1083-3668
    DOI 10.1117/1.3613926
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 4699: Show issues Location:
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  3. Article: Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives.

    Hurst, Tamiika K / Wang, Da / Thompson, Richard B / Fierke, Carol A

    Biochimica et biophysica acta

    2009  Volume 1804, Issue 2, Page(s) 393–403

    Abstract: Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn(2+) and Cu(2+). Subtle modification of the ... ...

    Abstract Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn(2+) and Cu(2+). Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu(2+) in seawater.
    MeSH term(s) Carbonic Anhydrase II/chemistry ; Carbonic Anhydrase II/metabolism ; Humans ; Zinc/chemistry ; Zinc/metabolism
    Chemical Substances Carbonic Anhydrase II (EC 4.2.1.-) ; Zinc (J41CSQ7QDS)
    Language English
    Publishing date 2009-10-08
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 60-7
    ISSN 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    ISSN (online) 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
    ISSN 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    DOI 10.1016/j.bbapap.2009.09.031
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Fluorescence lifetime imaging of physiological free Cu(II) levels in live cells with a Cu(II)-selective carbonic anhydrase-based biosensor.

    McCranor, Bryan J / Szmacinski, Henryk / Zeng, Hui Hui / Stoddard, Andrea K / Hurst, Tamiika / Fierke, Carol A / Lakowicz, J R / Thompson, Richard B

    Metallomics : integrated biometal science

    2014  Volume 6, Issue 5, Page(s) 1034–1042

    Abstract: Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in ... ...

    Abstract Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed.
    MeSH term(s) Animals ; Biosensing Techniques ; Calibration ; Carbonic Anhydrase II/metabolism ; Copper/metabolism ; Humans ; Microscopy, Fluorescence ; PC12 Cells ; Rats
    Chemical Substances Copper (789U1901C5) ; Carbonic Anhydrase II (EC 4.2.1.-)
    Language English
    Publishing date 2014-03-26
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2474317-3
    ISSN 1756-591X ; 1756-5901
    ISSN (online) 1756-591X
    ISSN 1756-5901
    DOI 10.1039/c3mt00305a
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Determination of zinc using carbonic anhydrase-based fluorescence biosensors.

    Bozym, Rebecca / Hurst, Tamiika K / Westerberg, Nissa / Stoddard, Andrea / Fierke, Carol A / Frederickson, Christopher J / Thompson, Richard B

    Methods in enzymology

    2009  Volume 450, Page(s) 287–309

    Abstract: This chapter summarizes the use of carbonic anhydrase (CA)-based fluorescent indicators to determine free zinc in solution, in cells, and in subcellular organelles. Expression (both in situ and in vitro) and preparation of CA-based indicators are ... ...

    Abstract This chapter summarizes the use of carbonic anhydrase (CA)-based fluorescent indicators to determine free zinc in solution, in cells, and in subcellular organelles. Expression (both in situ and in vitro) and preparation of CA-based indicators are described, together with techniques of their use, and procedures to minimize contamination. Recipes for zinc buffers are supplied.
    MeSH term(s) Biosensing Techniques ; Carbonic Anhydrases/chemistry ; Spectrometry, Fluorescence/methods ; Zinc/analysis
    Chemical Substances Carbonic Anhydrases (EC 4.2.1.1) ; Zinc (J41CSQ7QDS)
    Language English
    Publishing date 2009-01-06
    Publishing country United States
    Document type Journal Article
    ISSN 1557-7988
    ISSN (online) 1557-7988
    DOI 10.1016/S0076-6879(08)03414-9
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article: DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions.

    Sumner, James P / Westerberg, Nissa M / Stoddard, Andrea K / Hurst, Tamiika K / Cramer, Michele / Thompson, Richard B / Fierke, Carol A / Kopelman, Raoul

    Biosensors & bioelectronics

    2005  Volume 21, Issue 7, Page(s) 1302–1308

    Abstract: ... of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are ...

    Abstract The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.
    MeSH term(s) Biosensing Techniques/instrumentation ; Biosensing Techniques/methods ; Copper/analysis ; Copper/chemistry ; Ions ; Luminescent Proteins/analysis ; Luminescent Proteins/chemistry ; Spectrometry, Fluorescence/instrumentation ; Spectrometry, Fluorescence/methods
    Chemical Substances Ions ; Luminescent Proteins ; fluorescent protein 583 ; Copper (789U1901C5)
    Language English
    Publishing date 2005-07-18
    Publishing country England
    Document type Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1011023-9
    ISSN 1873-4235 ; 0956-5663
    ISSN (online) 1873-4235
    ISSN 0956-5663
    DOI 10.1016/j.bios.2005.04.023
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 2275: Show issues Location:
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  7. Article ; Online: Combining Active Carbonic Anhydrase with Nanogels

    Si D / Nie G / Hurst TK / Fierke CA / Kopelman R

    International Journal of Nanomedicine, Vol Volume 16, Pp 6645-

    Enzyme Protection and Zinc Sensing

    2021  Volume 6660

    Abstract: Di Si,1 Guochao Nie,2– 4 Tamiika K Hurst,1 Carol A Fierke,1 Raoul Kopelman1 1Department ...

    Abstract Di Si,1 Guochao Nie,2– 4 Tamiika K Hurst,1 Carol A Fierke,1 Raoul Kopelman1 1Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; 2School of Physics and Telecommunication Engineering, Yulin Normal University, Yulin, People’s Republic of China; 3China-Ukraine Joint Research Center for Nano Carbon Black, Yulin, People’s Republic of China; 4Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin, People’s Republic of ChinaCorrespondence: Guochao NieSchool of Physics and Telecommunication Engineering, Yulin Normal University, Yulin, People’s Republic of ChinaEmail bccu518@ylu.edu.cnRaoul KopelmanDepartment of Chemistry, University of Michigan, 930 N University Avenue, Ann Arbor, MI, USAEmail kopelman@umich.eduBackground: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo.Methods and Results: Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al2O3 adsorptive filtration purification procedure. In the surface conjugation method, blank PAAm nanogels were activated by N-hydroxysuccinimide and the CA was cross-linked to the nanogels. The amount of active CA immobilized in the nanoparticles was ...
    Keywords paam hydrogel ; nanogel ; nanoparticles ; carbonic anhydrase ; encapsulation ; conjugation ; zn2+ ; Medicine (General) ; R5-920
    Subject code 540
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher Dove Medical Press
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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