PKlc8Q@<@<refs.MYD 5ough the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjug Ia Chip83TOTAL ANALYSIS SYSTEMS NUCLEIC-ACID HYBRIDIZATION ESCHERICHIA-COLI MICROFLUIDIC SYSTEMS BIOFILM FORMATION IMMOBILIZED ENZYMES CHITOSAN POLY(DIMETHYLSILOXANE) PROTEIN CHIP_We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, thr@>7.^Luo, X. L. Lewandowski, A. T. Yi, H. M. Payne, G. F. Ghodssi, R. Bentley, W. E. Rubloff, G. W.2008]Programmable assembly of a metabolic pathway enzyme in a pre-packaged reusable bioMEMS device420-430 Lab on 8ation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.://000254424300009VISI Document Delivery No.: 280JY Times Cited: 0 Cited Reference Count: 58 Cited References: ANDERSSON H, 2003, SENSOR ACTUAT B-CHEM, V92, P315, DOI 10.1016/S0925-4005(03)00266-1 ANDERSSON H, 2006, LAB CHIP, V6, P467, DOI 10.1039/b602048h ATENCIA J, 2005, NATURE, V437, P648, DOI 10.1038/nature04163 AUROUX PA, 2002, ANAL CHEM, V74, P2637 BALESTRINO D, 2005, J BACTERIOL, V187, P2870, DOI 10.1128/JB.187.8.2870-2880.2005 BARRIOS AFG, 2006, J BACTERIOL, V188, P305 BEEBE DJ, 2002, ANNU REV BIOMED ENG, V4, P261, DOI 10.1146/annurev.bioeng.4.112601.125916 BILITEWSKI U, 2003, ANAL BIOANAL CHEM, V377, P556, DOI 10.1007/s00216-003-2179-4 CAO LQ, 2005, CURR OPIN CHEM BIOL, V9, P217, DOI 10.1016/j.cbpa.2005.02.014 CHAKI NK, 2002, BIOSENS BIOELECTRON, V17, P1 CHANG WJ, 2003, BIOMED MICRODEVICES, V5, P281 DENG JL, 2006, PROG CHEM, V18, P1397 DITTRICH PS, 2006, ANAL CHEM, V78, P3887, DOI 10.1021/ac0605602 DUERRE JA, 1962, J BIOL CHEM, V237, P3737 DUFFY DC, 1998, ANAL CHEM, V70, P4974 FEDERLE MJ, 2003, J CLIN INVEST, V112, P1291, DOI 10.1172/JCI200320195 FERNANDES R, 2004, LANGMUIR, V20, P906, DOI 10.1021/la0357312 FERNANDES R, 2007, METAB ENG, V9, P228, DOI 10.1016/j.ymben.2006.11.004 FERRO AJ, 1976, BIOCHIM BIOPHYS ACTA, V438, P487 HOLDEN MA, 2004, ANAL CHEM, V76, P1838, DOI 10.1021/ac035234q HONG JW, 2006, J PHYS-CONDENS MAT, V18, S691, DOI 10.1088/0953-8984/18/18/S14 JO BH, 2000, J MICROELECTROMECH S, V9, P76 JUNG GY, 2004, SCIENCE, V304, P428 KISAILUS D, 2006, P NATL ACAD SCI USA, V103, P5652, DOI 10.1073/pnas.0508488103 KOEV ST, 2007, LAB CHIP, V7, P103, DOI 10.1039/b609149k KU BS, 2006, BIOTECHNOL PROGR, V22, P1102, DOI 10.1021/bp050413+ LEE CS, 2006, BIOTECHNOL BIOPROC E, V11, P146 LEWANDOWSKI AT, 2006, BIOTECHNOL BIOENG, V93, P1207, DOI 10.1002/bit.20840 LEWANDOWSKI AT, 2008, BIOTECHNOL BIOENG, V99, P499, DOI 10.1002/bit.21580 LHOSTIS E, 2000, SENSOR ACTUAT B-CHEM, V64, P156 LI YG, 1999, ANAL CHIM ACTA, V382, P277 LIU Y, 2006, CHEM-EUR J, V12, P6585, DOI 10.1002/chem.200501622 MALPASS CA, 2002, J BIOMED MATER RES, V63, P822, DOI 10.1002/jbm.10410 MAO HB, 2002, ANAL CHEM, V74, P379 MCDONALD JC, 2000, ELECTROPHORESIS, V21, P27 NIEMEYER CM, 2003, NUCL ACIDS RES, V31 PARK JJ, 2006, LAB CHIP, V6, P1315, DOI 10.1039/b603101c PSALTIS D, 2006, NATURE, V442, P381, DOI 10.1038/nature05060 QUIST AP, 2005, ANAL BIOANAL CHEM, V381, P591, DOI 10.1007/s00216-004-2847-z RAGIONE ED, 1985, BIOCHEM J, V232, P335 RANDALL GC, 2005, P NATL ACAD SCI USA, V102, P10813, DOI 10.1073/pnas.0503287102 RASMUSSEN TB, 2005, J BACTERIOL, V187, P1799, DOI 10.1128/JB.187.5.1799-1814.2005 RASMUSSEN TB, 2005, MICROBIOL-SGM 5, V151, P1325, DOI 10.1099/mic.0.27715-0 REN DC, 2001, ENVIRON MICROBIOL, V3, P731 REYES DR, 2002, ANAL CHEM, V74, P2623 SHIM J, 2003, CURR TOP MED CHEM, V3, P687 SPERANDIO V, 2001, J BACTERIOL, V183, P5187 SPERANDIO V, 2002, MOL MICROBIOL, V43, P809 UNGER MA, 2000, SCIENCE, V288, P113 URBAN PL, 2006, BIOTECHNOL ADV, V24, P42, DOI 10.1016/j.biotechadv.2005.06.001 VILKNER T, 2004, ANAL CHEM, V76, P3373, DOI 10.1021/ac040063q WU LQ, 2002, LANGMUIR, V18, P8620, DOI 10.1021/la020381p WU LQ, 2003, LANGMUIR, V19, P519, DOI 10.1021/la026518t YI HM, 2004, ANAL CHEM, V76, P365, DOI 10.1021/ac034662t YI HM, 2005, BIOMACROMOLECULES, V6, P2881, DOI 10.1021/bm0504101 YI HM, 2005, LANGMUIR, V21, P2104, DOI 10.1021/la047529k YI HM, 2005, NANO LETT, V5, P1931, DOI 10.1021/nl051254r ZHU J, 2002, P NATL ACAD SCI USA, V99, P3129 Luo, Xiaolong Lewandowski, Angela T. Yi, Hyunmin Payne, Gregory F. Ghodssi, Reza Bentley, William E. Rubloff, Gary W.Lab ChipISI:000254424300009Univ Maryland, Dept Mat Sci & Engn, College Pk, MD 20742 USA. Univ Maryland, Fischell Dept Bioengn, College Pk, MD 20742 USA. Univ Maryland, Dept Chem & Biomol Engn, College Pk, MD 20742 USA. Univ Maryland, UMBI, College Pk, MD 20742 USA. Univ Maryland, Dept Elect & Comp Engn, College Pk, MD 20742 USA. Univ Maryland, ISR, College Pk, MD 20742 USA. Tufts Univ, Dept Chem & Biol Engn, Medford, MA 02155 USA. Rubloff, GW, Univ Maryland, Dept Mat Sci & Engn, College Pk, MD 20742 USA. rubloff@umd.eduEnglish>7/^Lewandowski, A. T. Yi, H. M. Luo, X. L. Payne, G. F. Ghodssi, R. Rubloff, G. W. Bentley, W. E.2008fProtein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag499-507 Biotechnology and Bioengineering993pro-tag protein assembly bioMEMS biofabrication HYDROGEN-PEROXIDE BIOSENSOR MORI SILK FIBROIN IMMOBILIZATION MATRIX GLUCOSE BIOSENSOR CHITOSAN SURFACE FILM ELECTRODEPOSITION BIOFABRICATION MONOLAYERSFebHWe report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface- assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices.://000252511800002 ISI Document Delivery No.: 253IL Times Cited: 1 Cited Reference Count: 40 Cited References: CAELEN I, 2002, LANGMUIR, V18, P2463 CHEN TH, 2002, BIOPOLYMERS, V64, P292 CHEN TH, 2003, LANGMUIR, V19, P9382, DOI 10.1021/la0347098 CHEN X, 2003, ELECTROANAL, V15, P608 CHOI JW, 2002, LAB CHIP, V2, P27, DOI 10.1039/b1077540n DIAO JP, 2005, ANAL BIOCHEM, V343, P322, DOI 10.1016/j.ab.2005.05.010 FODOR SPA, 1991, SCIENCE, V251, P767 FREDDI G, 2006, J BIOTECHNOL, V125, P281, DOI 10.1016/j.jbiotec.2006.03.003 GRAYSON ACR, 2004, P IEEE, V92, P6, DOI 10.1109/JPROC.2003.820534 HYUN J, 2001, LANGMUIR, V17, P6358 JUNG GY, 2004, SCIENCE, V304, P428 KANE RS, 1999, BIOMATERIALS, V20, P2363 KARTALOV EP, 2006, BIOTECHNIQUES, V40, P85, DOI 10.2144/000112071 KIM YD, 2001, BIOTECHNOL BIOENG, V73, P331 LEE CS, 2003, BIOSENS BIOELECTRON, V18, P437 LEE MY, 2005, P NATL ACAD SCI USA, V102, P983, DOI 10.1073/pnas.0406755102 LEWANDOWSKI AT, 2006, BIOTECHNOL BIOENG, V93, P1207, DOI 10.1002/bit.20840 LI C, 2004, ANAL BIOCHEM, V333, P381, DOI 10.1016/j.ab.2004.06.007 LUO XL, 2004, ANAL BIOCHEM, V334, P284, DOI 10.1016/j.ab.2004.07.005 LUO XL, 2005, BIOSENS BIOELECTRON, V21, P190, DOI 10.1016/j.bios.2004.07.029 LUO XL, 2005, CHEM COMMUN, P2169, DOI 10.1039/b419197h MIAO Y, 2001, ANAL CHIM ACTA, V437, P87 PANG X, 2005, MATER CHEM PHYS, V94, P245, DOI 10.1016/j.matchemphys.2005.04.040 PARK JJ, 2006, LAB CHIP, V6, P1315, DOI 10.1039/b603101c SAMPAIO S, 2005, J BIOTECHNOL, V116, P21, DOI 10.1016/j.jbiotec.2004.10.003 SEBRA RP, 2006, ANAL CHEM, V78, P3144, DOI 10.1021/ac052246y SUH KY, 2004, BIOMATERIALS, V25, P557, DOI 10.1016/S0142-9612(03)00543-X TENDER LM, 1996, LANGMUIR, V12, P5515 VEISEH M, 2002, LANGMUIR, V18, P6671, DOI 10.1021/la025529j WANG G, 2003, BIOSENS BIOELECTRON, V18, P335 WANG J, 1999, ELECTROANAL, V11, P866 WILLIAMS RA, 1994, BIOSENS BIOELECTRON, V9, P159 WU LQ, 2002, LANGMUIR, V18, P8620, DOI 10.1021/la020381p WU LQ, 2003, LANGMUIR, V19, P519, DOI 10.1021/la026518t WU LQ, 2004, TRENDS BIOTECHNOL, V22, P593, DOI 10.1016/j.tibtech.2004.09.008 YANG MH, 2004, ANAL BIOCHEM, V334, P127, DOI 10.1016/j.ab.2004.07.009 YI HM, 2004, ANAL CHEM, V76, P365, DOI 10.1021/ac034662t YI HM, 2005, BIOMACROMOLECULES, V6, P2881, DOI 10.1021/bm0504101 YI HM, 2005, LANGMUIR, V21, P2104, DOI 10.1021/la047529k ZHEN GL, 2004, LANGMUIR, V20, P10464, DOI 10.1021/la0482812 Lewandowski, Angela T. Yi, Hyunmin Luo, Xiaolong Payne, Gregory F. Ghodssi, Reza Rubloff, Gary W. Bentley, William E.Biotechnol. Bioeng.ISI:000252511800002Univ Maryland, Inst Biotechnol, Ctr Biosyst Res, College Pk, MD 20742 USA. Univ Maryland, Dept Chem & Biomol Engn, College Pk, MD 20742 USA. Univ Maryland, Dept Mat Sci & Engn, College Pk, MD 20742 USA. Univ Maryland, Fischell Dept Bioengn, College Pk, MD 20742 USA. Univ Maryland, Syst Res Inst, College Pk, MD 20742 USA. Univ Maryland, Dept Elect & Comp Engn, College Pk, MD 20742 USA. Bentley, WE, Univ Maryland, Inst Biotechnol, Ctr Biosyst Res, College Pk, MD 20742 USA. bentley@eng.umd.eduEnglish]>70hPark, J. J. Luo, X. L. Yi, H. M. Valentine, T. M. Payne, G. F. Bentley, W. E. Ghodssi, R. Rubloff, G. W.2006ZChitosan-mediated in situ biomolecule assembly in completely packaged microfluidic devices 1315-1321 Lab on a Chip610-CHIP FABRICATION BIOMEMS SURFACE SYSTEM GLASSOctWe report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.://000241731200015$ISI Document Delivery No.: 101HF Times Cited: 6 Cited Reference Count: 30 Cited References: AHN CH, 2004, P IEEE, V92, P154, DOI 10.1109/JPROC.2003.820548 BAIN CD, 1989, J AM CHEM SOC, V111, P321 BECKER H, 2002, TALANTA, V56, P267 BEEBE DJ, 2002, ANNU REV BIOMED ENG, V4, P261, DOI 10.1146/annurev.bioeng.4.112601.125916 BLANCHARD AP, 1996, BIOSENS BIOELECTRON, V11, P687 BOUSSE L, 1994, IEEE ENG MED BIOL, V13, P396 BRUIN GJM, 2000, ELECTROPHORESIS, V21, P3931 CARLIER J, 2004, J MICROMECH MICROENG, V14, P619, DOI 10.1088/0960-1317/14/4/024 CHOI JW, 2002, LAB CHIP, V2, P27, DOI 10.1039/b1077540n FODOR SPA, 1991, SCIENCE, V251, P767 FUJII T, 2002, MICROELECTRON ENG, V61, P907 HARRISON DJ, 1993, SCIENCE, V261, P895 HIRATSUKA A, 2004, BIOSENS BIOELECTRON, V19, P1667, DOI 10.1016/j.bios.2004.01.003 KEIPER B, 2000, J LASER APPL, V12, P189 KIM D, 2004, P SOC PHOTO-OPT INS, V5321, P309, DOI 10.1117/12.523693 LANGE SA, 2004, ANAL CHEM, V76, P1641 LI XG, 2002, J MICROELECTROMECH S, V11, P625, DOI 10.1109/JMENS.2002.805211 LIU J, 2003, ANAL CHEM, V75, P4718, DOI 10.1021/ac0346407 POWERS MA, 2005, LAB CHIP, V5, P583, DOI 10.1039/b503112e SANDERS GHW, 2000, TRAC-TREND ANAL CHEM, V19, P364 SENTURIA SD, 2002, MICROSYSTEM DESIGN, P50 TAN W, 2003, BIOMED MICRODEVICES, V5, P235 WILLIAMS KR, 2003, J MICROELECTROMECH S, V12, P761, DOI 10.1109/JMEMS.2003.820936 WU LQ, 2002, LANGMUIR, V18, P8620, DOI 10.1021/la020381p WU LQ, 2003, LANGMUIR, V19, P519, DOI 10.1021/la026518t XIA YN, 1998, ANNU REV MATER SCI, V28, P153 YI HM, 2004, ANAL CHEM, V76, P365, DOI 10.1021/ac034662t YI HM, 2005, BIOMACROMOLECULES, V6, P2881, DOI 10.1021/bm0504101 YI HM, 2005, LANGMUIR, V21, P2104, DOI 10.1021/la047529k ZHANG ZL, 2005, MICROELECTRON ENG, V78, P556, DOI 10.1016/j.mee.2004.12.071 Park, Jung Jin Luo, Xiaolong Yi, Hyunmin Valentine, Theresa M. Payne, Gregory F. Bentley, William E. Ghodssi, Reza Rubloff, Gary W.Lab ChipISI:000241731200015Univ Maryland, Syst Res Inst, College Pk, MD 20742 USA. Univ Maryland, Dept Mat Sci & Engn, College Pk, MD 20742 USA. Univ Maryland, Bioengn Grad Program, College Pk, MD 20742 USA. Univ Maryland, Dept Elect & Comp Engn, College Pk, MD 20742 USA. Univ Maryland, Inst Biotechnol, Ctr Biosyst Res, College Pk, MD 20742 USA. Univ Maryland, Inst Res Elect & Appl Phys, College Pk, MD 20742 USA. Rubloff, GW, Univ Maryland, Syst Res Inst, College Pk, MD 20742 USA. rubloff@umd.eduEnglishPKb8I/**refs.FRM 0B< !// !HPRIMARYyearIndex 6ByP/) idreference_type text_stylesauthoryear title pages secondary_title volume numbernumber_of_volumessecondary_authorplace_published publishersubsidiary_authoredition keywords type_of_workdate2)  abstractlabelurltertiary_titletertiary_author notes isbn custom_1 custom_2 custom_3 custom_4alternate_titleaccession_number call_number short_title custom_5 custom_6sectionoriginal_publicationH) reprint_editionreviewed_itemauthor_addressimagecaption custom_7 electronic_resource_number link_to_pdf translated_author translated_titlename_of_databasedatabase_providerresearch_notes language access_datelast_modified_date !! H!H!H! (H! 3H! >H! IH! TH!_H!jH!uH! H!H!H! H! H!H! H!H!H!H!H! H! H! H! H! %H! 0H!;H!FH! QH! \H! gH! rH!}H!H!H!H!H!H!H! H! H! H! H! H!H! H!H! "H! -H!8H!idreference_typetext_stylesauthoryeartitlepagessecondary_titlevolumenumbernumber_of_volumessecondary_authorplace_publishedpublishersubsidiary_authoreditionkeywordstype_of_workdateabstractlabelurltertiary_titletertiary_authornotesisbncustom_1custom_2custom_3custom_4alternate_titleaccession_numbercall_numbershort_titlecustom_5custom_6sectionoriginal_publicationreprint_editionreviewed_itemauthor_addressimagecaptioncustom_7electronic_resource_numberlink_to_pdftranslated_authortranslated_titlename_of_databasedatabase_providerresearch_noteslanguageaccess_datelast_modified_datePKlc8Q@<@<refs.MYDPKb8I/**f<refs.FRMPKldg