Bioluminescence Resonance Energy Transfer (BRET) Based Nanostructured Biosensor for Tear Glucose Detection
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The monitoring and management of blood glucose levels are key elements for people with diabetes to maintain their health.Here, we describe a bioluminescence resonance energy transfer (BRET) sensor for non-invasively detecting glucose molecules.The sensor is assembled by conjugating quantum dots CdTe (PL≈ 565nm), which is used as the acceptor, with a recombinant protein containing the bacterial glucose binding protein (GBP), at the N-terminal and a bioluminescent protein Renilla luciferase (Rluc), used as the donor, which is at the C-terminal.The distance between the BRET pair is initially far.In the presence of glucose, GBP binds glucose in a highly specific manner and the conformational change of resultant GBP brings a closer distance between the Rluc and QDs, results an increasing of the emission intensity of the QDs.The bioluminescence intensity of both around 470nm and 565nm are observed.The ratio of the acceptor (QDs) and the donor (Rluc) are also observed to increase with the increasing of the glucose concentration.This study laid a technical foundation for further exploration of non-invasive measurement systems for tear glucose.Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.
Pseudomonas putida
Charge-coupled device
Luminescent Measurements
Bioluminescence imaging
Luciferin
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Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.
Pseudomonas putida
Charge-coupled device
Luminescent Measurements
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Abstract Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.
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Abstract Fluorescent biosensors enable to study cell physiology with spatiotemporal resolution, yet most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster Resonance Energy Transfer (FRET) pairs with near quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD + with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by either changing the fluorescent protein or the synthetic fluorophore, which enabled to monitor simultaneously free NAD + in different subcellular compartments upon genotoxic stress. Minimal modifications furthermore allow the readout of these biosensors to be switched to fluorescence intensity, lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors. Graphical abstract
Fluorescent protein
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FRET plays an important role in light-induced processes in life sciences, e.g. energy transfer in light harvesting complexes. We present a method to tune the energy transfer from donor to acceptor in an optical microresonator.
Acceptor
Resonant inductive coupling
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In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.
Bioluminescence imaging
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Many genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein-based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.
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This chapter contains sections titled: A Rosy Crystal Ball View of FRET Do Not Ask What FRET Can Do for You, Ask What You Can Do for FRET FRET: Future Research with an Exciting Technology Future of FRET Outlook on Single–Molecule FRET Outlook on FRET with fluorescent proteins Luminescent Nanoparticles: Scaffolds for Assembling "Smarter" FRET Probes
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Bioluminescence resonance energy transfer (BRET) is a useful technique for visualizing cellular functions and responses to stimuli.To construct efficient biosensing protein probes using BRET, novel luminescent and red fluorescent protein pairs, which have separate peaks of luminescence and fluorescence and can cause energy transfer efficiently, were screened.The red fluorescent protein, mScarletI, used as an acceptor, induced a highly efficient BRET signal from green or blue luminescent proteins [Emerald Luc (ELuc) or NanoLuc (NLuc)].Novel pairs of luminescent and red fluorescent protein (mScarletI) could be applied to the analysis of calcium ions (Ca 2+ ).The BRET-based biosensing protein pair of mScarletI and NLuc showed an increased intensity of the BRET signal, depending on the concentration of Ca 2+ (0-4 μM).Intracellular Ca 2+ influx was monitored in HEK293A cells stimulated with 50 mM KCl and 15 mM arginine using the BRET-based biosensing protein probe with the novel protein pair.This pair of proteins was particularly suited to cellular imaging in vitro and even in vivo.Therefore, it could be useful for various BRET-based analyses of cell and tissue samples.
Resonant inductive coupling
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Bioluminescence System has been mostly studied in reporter technology. There are forty different bioluminescent systems occurred in nature while there are only seven of these light emitting systems and their biochemical reactions have been studied however the pathway of only two biochemical systems has been understood yet. Here, we have provided an overview for these bioluminescent systems as a tool for researchers working on bioluminescence for better future applications of bioluminescent.
Luciferases
Bioluminescence imaging
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