Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power

IF 4.6 Q1 CHEMISTRY, ANALYTICAL
Mark A. Klein*, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt and Rachel Friedman Ohana*, 
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引用次数: 0

Abstract

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.

Abstract Image

Abstract Image

荧光素酶校准物可实现生物发光功率的绝对定量
由荧光素酶催化氧化荧光素而发出的生物发光已被广泛用于报告生物事件,主要是通过光输出的相对变化。蛋白质工程和合成化学领域的最新进展已经产生了亮度和生物利用率显著提高的生物发光系统。这些发展不仅使生物事件的检测表达水平大大降低,也为利用生物发光驱动细胞内的光化学提供了新的机会。无论是哪种应用,生物发光分析在很大程度上都依赖于使用光度计来测量系统的光输出。目前的光度计以相对单位报告样品的光输出,限制了仪器间数据比较的能力,也无法量化生物发光系统的绝对功率。由荧光酶及其同源荧光素组成的荧光溶液校准液具有绝对光输出的特征,可以校准任何特定的发光仪进行绝对光子计数。为此,我们定制了一种光检测仪器,并将其与波长匹配的 LED 光源(波长分别为 450 纳米和 561 纳米)一起使用,分别鉴定了一系列 NanoLuc 和萤火虫荧光素酶溶液的绝对功率。这种方法显示,这两种常见的荧光素酶产生的功率分别为 3.72 × 10-18 瓦特/分子和 7.25 × 10-20 瓦特/分子。这些发光溶液校准液的成分可在市场上买到,并能在 2-5 分钟内产生稳定的生物发光信号,因此任何发光仪都能对这两种荧光素酶发出的生物发光进行功率测量校准,单位为瓦特或光子/秒。
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来源期刊
ACS Measurement Science Au
ACS Measurement Science Au 化学计量学-
CiteScore
5.20
自引率
0.00%
发文量
0
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.
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