Acidification of the Oxygen Scavenging System in Single-Molecule Fluorescence Studies: In Situ Sensing with a Ratiometric Dual-Emission Probe
For most of the single-molecule fluorescence studies to date, biomolecules of interest are labeled with small organic dyes which suffer from their limited photostability evidenced by blinking and photobleaching. An enzymatic oxygen scavenging system of glucose oxidase and catalase is widely used to...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2010-07, Vol.82 (14), p.6132-6138 |
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| creator | Shi, Xinghua Lim, John Ha, Taekjip |
| description | For most of the single-molecule fluorescence studies to date, biomolecules of interest are labeled with small organic dyes which suffer from their limited photostability evidenced by blinking and photobleaching. An enzymatic oxygen scavenging system of glucose oxidase and catalase is widely used to improve the dye photostability but with the unfavorable side effect of producing gluconic acid. It is known that accumulation of this byproduct in solution can lead to considerable acidification, but the uncertainty in its severity under experimentally relevant conditions has been a long-standing area of concern due to the lack of a suitable assay. In this paper we report a fluorescence-based analytical assay for quantitatively assessing the acidification of oxygen scavenging systems in situ. By using a ratiometric, dual-emission dye, SNARF-1, we observed the presence and, for the first time, measured the severity of solution acidification due to the oxygen scavenging system for a number of conditions relevant to single-molecule studies. On the basis of the quantitative analysis of the acidification profile under these conditions, practical guidelines for optimizing the oxygen scavenging system are provided. This in situ assay should be applicable to a large variety of future single-molecule fluorescence studies. |
| doi_str_mv | 10.1021/ac1008749 |
| format | Article |
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An enzymatic oxygen scavenging system of glucose oxidase and catalase is widely used to improve the dye photostability but with the unfavorable side effect of producing gluconic acid. It is known that accumulation of this byproduct in solution can lead to considerable acidification, but the uncertainty in its severity under experimentally relevant conditions has been a long-standing area of concern due to the lack of a suitable assay. In this paper we report a fluorescence-based analytical assay for quantitatively assessing the acidification of oxygen scavenging systems in situ. By using a ratiometric, dual-emission dye, SNARF-1, we observed the presence and, for the first time, measured the severity of solution acidification due to the oxygen scavenging system for a number of conditions relevant to single-molecule studies. On the basis of the quantitative analysis of the acidification profile under these conditions, practical guidelines for optimizing the oxygen scavenging system are provided. 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Chem</addtitle><description>For most of the single-molecule fluorescence studies to date, biomolecules of interest are labeled with small organic dyes which suffer from their limited photostability evidenced by blinking and photobleaching. An enzymatic oxygen scavenging system of glucose oxidase and catalase is widely used to improve the dye photostability but with the unfavorable side effect of producing gluconic acid. It is known that accumulation of this byproduct in solution can lead to considerable acidification, but the uncertainty in its severity under experimentally relevant conditions has been a long-standing area of concern due to the lack of a suitable assay. In this paper we report a fluorescence-based analytical assay for quantitatively assessing the acidification of oxygen scavenging systems in situ. By using a ratiometric, dual-emission dye, SNARF-1, we observed the presence and, for the first time, measured the severity of solution acidification due to the oxygen scavenging system for a number of conditions relevant to single-molecule studies. On the basis of the quantitative analysis of the acidification profile under these conditions, practical guidelines for optimizing the oxygen scavenging system are provided. 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Lim, John ; Ha, Taekjip</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a461t-8219fe1289bd68dda20baf7f8a3569eef734c99b6009059d41437cff1fb28f923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Analytical chemistry</topic><topic>Benzopyrans - chemistry</topic><topic>Catalase - metabolism</topic><topic>Chemistry</topic><topic>Enzymes</topic><topic>Exact sciences and technology</topic><topic>Fluorescence</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Gluconates - metabolism</topic><topic>Glucose</topic><topic>Glucose Oxidase - metabolism</topic><topic>Molecules</topic><topic>Naphthols - chemistry</topic><topic>Oxygen</topic><topic>Oxygen - chemistry</topic><topic>Photobleaching</topic><topic>Rhodamines - chemistry</topic><topic>Spectrometric and optical methods</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Uncertainty</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shi, Xinghua</creatorcontrib><creatorcontrib>Lim, John</creatorcontrib><creatorcontrib>Ha, Taekjip</creatorcontrib><collection>American Chemical Society (ACS) Open Access</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shi, Xinghua</au><au>Lim, John</au><au>Ha, Taekjip</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Acidification of the Oxygen Scavenging System in Single-Molecule Fluorescence Studies: In Situ Sensing with a Ratiometric Dual-Emission Probe</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. 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| ispartof | Analytical chemistry (Washington), 2010-07, Vol.82 (14), p.6132-6138 |
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| source | MEDLINE; ACS Publications |
| subjects | Analytical chemistry Benzopyrans - chemistry Catalase - metabolism Chemistry Enzymes Exact sciences and technology Fluorescence Fluorescent Dyes - chemistry Gluconates - metabolism Glucose Glucose Oxidase - metabolism Molecules Naphthols - chemistry Oxygen Oxygen - chemistry Photobleaching Rhodamines - chemistry Spectrometric and optical methods Spectrometry, Fluorescence - methods Uncertainty |
| title | Acidification of the Oxygen Scavenging System in Single-Molecule Fluorescence Studies: In Situ Sensing with a Ratiometric Dual-Emission Probe |
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