Fluorescent Indicators for Live-Cell and in Vitro Detection of Inorganic Cadmium Dynamics

2021 ◽  
Author(s):  
Shulin Hu ◽  
Jun Yang ◽  
Anqi Liao ◽  
Ying Lin ◽  
Shuli Liang
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Cadmium Concentration ◽  
High Dynamic Range ◽  
Ion Concentration ◽  
Cadmium Ion ◽  
Fluorescent Indicators ◽  
Cadmium Detection ◽  
Time Readout

Abstract Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. Under optimal conditions, the fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

scholarly journals Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness

Sensors ◽  
2019 ◽  
Vol 19 (13) ◽  
pp. 2982 ◽  
Author(s):  
Liubov A. Kost ◽  
Violetta O. Ivanova ◽  
Pavel M. Balaban ◽  
Konstantin A. Lukyanov ◽  
Evgeny S. Nikitin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Response Speed ◽  
Red Fluorescent Protein ◽  
Positive Slope ◽  
Reporter Protein ◽  
Fluorescent Indicators ◽  
Linker Length ◽  
Final Performance ◽  
Voltage Indicator

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188—a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn’t show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

scholarly journals A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chihiro Nakamoto ◽  
Yuhei Goto ◽  
Yoko Tomizawa ◽  
Yuko Fukata ◽  
Masaki Fukata ◽  
...  
Keyword(s):  
Hela Cells ◽  
Hippocampal Neurons ◽  
High Selectivity ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Receptor Activation ◽  
Spatiotemporal Dynamics ◽  
Intracellular Loop ◽  
Brain Functions

AbstractDopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

scholarly journals FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

2020 ◽  
Vol 21 (8) ◽  
pp. 3012 ◽  
Author(s):  
Natalia V. Barykina ◽  
Vladimir P. Sotskov ◽  
Anna M. Gruzdeva ◽  
You Kure Wu ◽  
Ruben Portugues ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Neuronal Cultures ◽  
Two Phase ◽  
Intracellular Environment

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

scholarly journals Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

2014 ◽  
Vol 81 (2) ◽  
pp. 736-744 ◽  
Author(s):  
Alexandra Jung ◽  
Sabrina Eisheuer ◽  
Emöke Cserti ◽  
Oliver Leicht ◽  
Wolfgang Strobel ◽  
...  
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Genetic Manipulation ◽  
Expression System ◽  
Genetic System ◽  
High Dynamic Range ◽  
Content Type ◽  
Depth Analysis ◽  
Inducible Synthesis

ABSTRACTThe alphaproteobacteriumHyphomonas neptuniumproliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies twoH. neptuniumpromoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions inH. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

Long-wavelength Fluorophores for Voltage Sensing

2019 ◽  
Author(s):  
Gloria Ortiz ◽  
Pei Liu ◽  
Su Naing ◽  
Vikram Muller ◽  
Evan Miller
Keyword(s):  
Fluorescent Protein ◽  
Voltage Sensitivity ◽  
Porcine Liver ◽  
Design Synthesis ◽  
Fluorescent Indicators ◽  
Voltage Imaging ◽  
Chemical Genetic ◽  
Green Fluorescent ◽  
Oxygen Substitution

<p>We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p<i>K</i><sub>a</sub> of carbofluorescein dyes, coupled with their tendency to cyclize to non-fluorescent configurations precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p<i>K</i>a by 2 units to 5.2 and sulfonation to prevent cyclization to the non-absorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p<i>K</i><sub>a</sub> values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive Fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles >560 nm, displays high voltage sensitivity (>30% ΔF/F per 100 mV), and can be used in the presence of other blue-excited fluorophores like green fluorescent protein (GFP). Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase (PLE) both <i>in vitro</i> and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.</p>

Long-wavelength Fluorophores for Voltage Sensing

2019 ◽  
Author(s):  
Gloria Ortiz ◽  
Pei Liu ◽  
Su Naing ◽  
Vikram Muller ◽  
Evan Miller
Keyword(s):  
Fluorescent Protein ◽  
Voltage Sensitivity ◽  
Porcine Liver ◽  
Design Synthesis ◽  
Fluorescent Indicators ◽  
Voltage Imaging ◽  
Chemical Genetic ◽  
Green Fluorescent ◽  
Oxygen Substitution

<p>We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p<i>K</i><sub>a</sub> of carbofluorescein dyes, coupled with their tendency to cyclize to non-fluorescent configurations precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p<i>K</i>a by 2 units to 5.2 and sulfonation to prevent cyclization to the non-absorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p<i>K</i><sub>a</sub> values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive Fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles >560 nm, displays high voltage sensitivity (>30% ΔF/F per 100 mV), and can be used in the presence of other blue-excited fluorophores like green fluorescent protein (GFP). Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase (PLE) both <i>in vitro</i> and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.</p>

Long-wavelength Fluorophores for Voltage Sensing

2019 ◽  
Author(s):  
Gloria Ortiz ◽  
Pei Liu ◽  
Su Naing ◽  
Vikram Muller ◽  
Evan Miller
Keyword(s):  
Fluorescent Protein ◽  
Voltage Sensitivity ◽  
Porcine Liver ◽  
Design Synthesis ◽  
Fluorescent Indicators ◽  
Voltage Imaging ◽  
Chemical Genetic ◽  
Green Fluorescent ◽  
Oxygen Substitution

<p>We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p<i>K</i><sub>a</sub> of carbofluorescein dyes, coupled with their tendency to cyclize to non-fluorescent configurations precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p<i>K</i>a by 2 units to 5.2 and sulfonation to prevent cyclization to the non-absorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p<i>K</i><sub>a</sub> values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive Fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles >560 nm, displays high voltage sensitivity (>30% ΔF/F per 100 mV), and can be used in the presence of other blue-excited fluorophores like green fluorescent protein (GFP). Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase (PLE) both <i>in vitro</i> and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.</p>

scholarly journals Genetically Encoded Fluorescent Indicators for Imaging Brain Chemistry

Biosensors ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 116
Author(s):  
Xiaoke Bi ◽  
Connor Beck ◽  
Yiyang Gong
Keyword(s):  
Dynamic Range ◽  
Signal To Noise Ratio ◽  
Spatial Scales ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Brain Chemistry ◽  
Fluorescent Indicators ◽  
Recent Developments

Genetically encoded fluorescent indicators, combined with optical imaging, enable the detection of physiologically or behaviorally relevant neural activity with high spatiotemporal resolution. Recent developments in protein engineering and screening strategies have improved the dynamic range, kinetics, and spectral properties of genetically encoded fluorescence indicators of brain chemistry. Such indicators have detected neurotransmitter and calcium dynamics with high signal-to-noise ratio at multiple temporal and spatial scales in vitro and in vivo. This review summarizes the current trends in these genetically encoded fluorescent indicators of neurotransmitters and calcium, focusing on their key metrics and in vivo applications.

scholarly journals Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome

2019 ◽  
Vol 20 (14) ◽  
pp. 3488 ◽  
Author(s):  
Oksana M. Subach ◽  
Natalia V. Barykina ◽  
Konstantin V. Anokhin ◽  
Kiryl D. Piatkevich ◽  
Fedor V. Subach
Keyword(s):  
Calcium Ions ◽  
Mammalian Cells ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Cultured Cells ◽  
Infrared Region ◽  
Calcium Indicator

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.

scholarly journals Optimisation and validation of a sensitive bioanalytical method for niclosamide

2021 ◽  
Author(s):  
Usman Arshad ◽  
Henry Pertinez ◽  
Helen Box ◽  
Lee Tatham ◽  
Rajith KR Rajoli ◽  
...  
Keyword(s):  
Dynamic Range ◽  
High Dynamic Range ◽  
Exposure Response ◽  
Accuracy And Precision ◽  
New Chemical Entities ◽  
Liquid Chromatography Tandem Mass ◽  
High Dynamic ◽  
Bioanalytical Assays ◽  
Low Dynamic Range

AbstractThe SARS-CoV-2 pandemic has spread at an unprecedented rate, and repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Niclosamide has been proposed as a candidate for repurposing for SARS-CoV-2 based upon the observation that it is amongst the most potent antiviral molecules evaluated in vitro. To investigate the pharmacokinetics of niclosamide, reliable, reproducible and sensitive bioanalytical assays are required. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 31.25-2000 ng/mL (high dynamic range) and 0.78-100 ng/mL (low dynamic range). Accuracy and precision ranged between 97.2% and 112.5%, 100.4% and 110.0%, respectively. The presented assay should have utility in preclinical evaluation of the exposure-response relationship and may be adapted for later evaluation of niclosamide in clinical trials.

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