Mark A. Klein*, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt and Rachel Friedman Ohana*,
{"title":"荧光素酶校准物可实现生物发光功率的绝对定量","authors":"Mark A. Klein*, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt and Rachel Friedman Ohana*, ","doi":"10.1021/acsmeasuresciau.3c00036","DOIUrl":null,"url":null,"abstract":"<p >Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10<sup>–18</sup> and 7.25 × 10<sup>–20</sup> watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2–5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.</p>","PeriodicalId":29800,"journal":{"name":"ACS Measurement Science Au","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2023-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsmeasuresciau.3c00036","citationCount":"0","resultStr":"{\"title\":\"Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power\",\"authors\":\"Mark A. Klein*, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt and Rachel Friedman Ohana*, \",\"doi\":\"10.1021/acsmeasuresciau.3c00036\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10<sup>–18</sup> and 7.25 × 10<sup>–20</sup> watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2–5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.</p>\",\"PeriodicalId\":29800,\"journal\":{\"name\":\"ACS Measurement Science Au\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2023-10-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.acs.org/doi/epdf/10.1021/acsmeasuresciau.3c00036\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Measurement Science Au\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acsmeasuresciau.3c00036\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Measurement Science Au","FirstCategoryId":"1085","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acsmeasuresciau.3c00036","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power
Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10–18 and 7.25 × 10–20 watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2–5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.
期刊介绍:
ACS Measurement Science Au is an open access journal that publishes experimental computational or theoretical research in all areas of chemical measurement science. Short letters comprehensive articles reviews and perspectives are welcome on topics that report on any phase of analytical operations including sampling measurement and data analysis. This includes:Chemical Reactions and SelectivityChemometrics and Data ProcessingElectrochemistryElemental and Molecular CharacterizationImagingInstrumentationMass SpectrometryMicroscale and Nanoscale systemsOmics (Genomics Proteomics Metabonomics Metabolomics and Bioinformatics)Sensors and Sensing (Biosensors Chemical Sensors Gas Sensors Intracellular Sensors Single-Molecule Sensors Cell Chips Arrays Microfluidic Devices)SeparationsSpectroscopySurface analysisPapers dealing with established methods need to offer a significantly improved original application of the method.