Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research
We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerizatio...
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| Veröffentlicht in: | Analytical and bioanalytical chemistry 2018-10, Vol.410 (25), p.6677-6687 |
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| creator | Sathyanarayanan, Gowtham Haapala, Markus Kiiski, Iiro Sikanen, Tiina |
| description | We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (
n
= 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro. |
| doi_str_mv | 10.1007/s00216-018-1280-7 |
| format | Article |
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n
= 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.</description><identifier>ISSN: 1618-2642</identifier><identifier>EISSN: 1618-2650</identifier><identifier>DOI: 10.1007/s00216-018-1280-7</identifier><identifier>PMID: 30073515</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Analytical Chemistry ; Biochemistry ; Characterization and Evaluation of Materials ; Chemical properties ; Chemistry ; Chemistry and Materials Science ; Cytochrome ; Cytochrome P-450 ; Cytochrome P-450 Enzyme System - chemistry ; Cytochrome P450 ; Cytochromes P450 ; Detoxification ; Drug metabolism ; Fluorescence ; Food Science ; Isoforms ; Lab-On-A-Chip Devices ; Laboratory Medicine ; Metabolism ; Microfluidics ; Monitoring/Environmental Analysis ; Organic chemistry ; Pharmaceutical research ; Photopolymerization ; Plugs ; Polymers ; Printing ; Reactors ; Reproducibility ; Reproducibility of Results ; Research Paper ; Resorufin ; Statistical analysis ; Temperature effects ; Turnover rate ; Xenobiotics</subject><ispartof>Analytical and bioanalytical chemistry, 2018-10, Vol.410 (25), p.6677-6687</ispartof><rights>The Author(s) 2018</rights><rights>COPYRIGHT 2018 Springer</rights><rights>Analytical and Bioanalytical Chemistry is a copyright of Springer, (2018). All Rights Reserved. © 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c574t-358cae4fbd063f8e168331f42a05e81aa3b80e32874cee46d362b975be02865b3</citedby><cites>FETCH-LOGICAL-c574t-358cae4fbd063f8e168331f42a05e81aa3b80e32874cee46d362b975be02865b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00216-018-1280-7$$EPDF$$P50$$Gspringer$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00216-018-1280-7$$EHTML$$P50$$Gspringer$$Hfree_for_read</linktohtml><link.rule.ids>230,314,780,784,885,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30073515$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sathyanarayanan, Gowtham</creatorcontrib><creatorcontrib>Haapala, Markus</creatorcontrib><creatorcontrib>Kiiski, Iiro</creatorcontrib><creatorcontrib>Sikanen, Tiina</creatorcontrib><title>Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research</title><title>Analytical and bioanalytical chemistry</title><addtitle>Anal Bioanal Chem</addtitle><addtitle>Anal Bioanal Chem</addtitle><description>We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (
n
= 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.</description><subject>Analytical Chemistry</subject><subject>Biochemistry</subject><subject>Characterization and Evaluation of Materials</subject><subject>Chemical properties</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cytochrome</subject><subject>Cytochrome P-450</subject><subject>Cytochrome P-450 Enzyme System - chemistry</subject><subject>Cytochrome P450</subject><subject>Cytochromes P450</subject><subject>Detoxification</subject><subject>Drug metabolism</subject><subject>Fluorescence</subject><subject>Food Science</subject><subject>Isoforms</subject><subject>Lab-On-A-Chip Devices</subject><subject>Laboratory Medicine</subject><subject>Metabolism</subject><subject>Microfluidics</subject><subject>Monitoring/Environmental Analysis</subject><subject>Organic chemistry</subject><subject>Pharmaceutical research</subject><subject>Photopolymerization</subject><subject>Plugs</subject><subject>Polymers</subject><subject>Printing</subject><subject>Reactors</subject><subject>Reproducibility</subject><subject>Reproducibility of Results</subject><subject>Research Paper</subject><subject>Resorufin</subject><subject>Statistical analysis</subject><subject>Temperature effects</subject><subject>Turnover 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Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sathyanarayanan, Gowtham</au><au>Haapala, Markus</au><au>Kiiski, Iiro</au><au>Sikanen, Tiina</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research</atitle><jtitle>Analytical and bioanalytical chemistry</jtitle><stitle>Anal Bioanal Chem</stitle><addtitle>Anal Bioanal Chem</addtitle><date>2018-10-01</date><risdate>2018</risdate><volume>410</volume><issue>25</issue><spage>6677</spage><epage>6687</epage><pages>6677-6687</pages><issn>1618-2642</issn><eissn>1618-2650</eissn><abstract>We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (
n
= 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>30073515</pmid><doi>10.1007/s00216-018-1280-7</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical Chemistry Biochemistry Characterization and Evaluation of Materials Chemical properties Chemistry Chemistry and Materials Science Cytochrome Cytochrome P-450 Cytochrome P-450 Enzyme System - chemistry Cytochrome P450 Cytochromes P450 Detoxification Drug metabolism Fluorescence Food Science Isoforms Lab-On-A-Chip Devices Laboratory Medicine Metabolism Microfluidics Monitoring/Environmental Analysis Organic chemistry Pharmaceutical research Photopolymerization Plugs Polymers Printing Reactors Reproducibility Reproducibility of Results Research Paper Resorufin Statistical analysis Temperature effects Turnover rate Xenobiotics |
| title | Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research |
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